Toxic Cyanoprokaryotes in resource waters : monitoring of their occurrence and toxin detection

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1 Toxic Cyanoprokaryotes in resource waters : monitoring of their occurrence and toxin detection Bouaïcha, N. 1, Via-Ordorika, L. 1, Vandevelde, T. 2, Fauchon, N. 2, Puiseux-Dao, S. 1 1 : CEMATMA, Cryptogamie, Muséum National d Histoire Naturelle, Paris; 2 : Compagnie Générale des Eaux, Paris Introduction Marine algal toxins are known to cause seafood poisoning. In freshwaters blue-green-algae (also Cyanoprokaryotes or Cyanobacteria) can be toxic. They have been involved in human and animal intoxications (Carmichaël, 1989; Codd et al., 1989; Pearl, 1996). Two main types of toxins have been described : hepatotoxins (Falconer, 1994; Falconer and Humpage, 1996; Honkanan et al., 1994; Huynh- Delerme and Puiseux-Dao,1998; Ohtani et al., 1992; Puiseux-Dao et al., in press) - microcystins and nodularins: potent inhibitors of protein phosphatases and liver tumor promoters: MCYST-LR : LD50 : 50 µg/kg (ip in mice); - cylindrospermopsin: inhibits protein synthesis: LD50 : 2mg/kg (ip in mice). neurotoxins (Rappala et al., 1993; Sivonen et al., 1989) - saxitoxin and neosaxitoxin: sodium channel blockers: LD50 :10-20 µg/kg (ip in mice); - anatoxin-a and homoanatoxin-a: postsynaptic depolarizing agents: LD50 : µg/kg (ip in mice); - anatoxin-a(s): anticholinesterase activity: LD50 : µg/kg (ip in mice). Figures 1 and 2 show the most frequently occurring microalgae and some of the toxins they produce (see appendix A and B). Our aim was to define a methodology for the surveillance of the water quality with respect to algal toxins. Selection of methods Three different sites were selected for our study. In these sites the Compagnie Générale des Eaux is pumping water for the city of Paris and the suburbs. They are located on the three rivers Seine (Choisy-le-Roi), Marne (Neuilly-sur-Marne) and Oise (Méry-sur-Oise). Every month water samples were taken for toxin detection. At the same time microalgae were collected with a plankton net and fixed by adding neutral formaldehyde (5%), in order to relate microalgae and toxicity. The microalgae were then identified under the light microscope. Three types of methods are assayed for the toxin detection and quantification: Biological methods in vivo bioassays: the mouse bioassay is the only bioassay recognized by WHO until now; in vitro bioassays: e.g. tissue culture tests (Erikson et al., 1987). biochemical methods -ELISA (Enzyme-Linked ImmunoSorbent Assay) (An and Carmichaël, 1994); -Serine-Threonine protein phosphatase inhibition assay (Tubaro et al., 1996, modified by Bouaïcha et al., unpublished); Bouaïcha, N. et al. 1

2 -acetylcholinesterase inhibition assay (Henriksen et al.,1997, modified by Bouaïcha et al., unpublished). chemical methods -High-performance Liquid Chromatography (HPLC) (Lawton et al., 1994); -Gas Chromatography (GC) (Bumke-Vogt et al., 1996); -Capillary Electrophoresis (CE) (Bouaïcha et al., 1993). Table 1: Dominant Cyanobacteria in Paris aera rivers SEINE MARNE OISE Oscillatoria spp. Oscillatoria spp. Oscillatoria spp. Chroococcus sp. Chroococcus sp. Chroococcus sp. Aug Anabaena sp. Synechocystis sp. Microcystis sp. Oscillatoria spp. Oscillatoria spp. Oscillatoria spp. Sep Chroococcus sp. Chroococcus sp. Synechocystis sp. Merismopedia sp. Oscillatoria spp. Oscillatoria spp. Oscillatoria spp. Oct Synechocystis sp. Oscillatoria spp. Oscillatoria spp. Oscillatoria spp. Jan Synechocystis sp. Anabaena sp. Feb Oscillatoria sp. Anabaena sp. Oscillatoria sp. Oscillatoria sp. Mar Oscillatoria spp. Oscillatoria spp. Oscillatoria spp. Apr _ Oscillatoria sp. Oscillatoria sp. Oscillatoria spp. Anabaena sp. May 1998 Chroococcus sp. Oscillatoria sp. Oscillatoria sp. Oscillatoria sp. Chroococcus sp. Jun Synechocystis sp. Merismopedia sp. Microcystis sp. Bouaïcha, N. et al. 2

3 Since microcystins are the most often implied phytotoxins and because of their carcinogenetic potential, we have been dealing with the phosphatase 2A inhibition in priority. We used CE for the chemical identification; one must keep in mind that only three standards of microcystins are commercially available: MCYST-LR, -RR, and -YR. Results Table 1 shows the Cyanoprokaryote genus which have been identified. In all three rivers and at all times Oscillatoria was observed and found to be the most abundant genus. During the months of August, September and October Cyanoprokaryote proliferation was at a maximum. It appears that a microcystin-type activity was detectable (Fig. 3), when examining the results of microcystin detection in water samples with the phosphatase 2A assay and MCYST-LR as control. In almost all cases the highest level was in the Seine. As it was to be expected, the months of August, September and October were the months with the most relevant toxicity. The concentration, however, never reached 1µg/L, which is the tolerated limit of WHO Equiv. MCYST-LR (pg/l) Seine Marne Oise N.T. : Not tested 0 N. N.T Aug. Oct. Nov. Jan. Feb. Apr. May Figure 3: Equivalent Microcystin-LR in Paris area rivers (phosphatase 2A inhibition assay) Another fact can be seen in Figure 3: during the first half of 1998 the weather showed a peculiar evolution. Colder grey periods and very sunny warm periods succeeded each other. During a warm period in February a small bloom of Oscillatoria alone was observed. The water temperature around 10 C was apparently too low for diatoms or chlorophytes, but the cyanobacterium could grow. A toxic peak of phosphatase inhibition was detected in parallel in the water samples. In April a similar situation occurred. Heavy rains at the time of collection prevented us from obtaining observable algal samples. However, a high toxic potential was detected in the Seine water with the phosphatase assay. The strong water current suggests that the toxicity could come from upstream. The enzymatic assay indicates the presence of toxic substance(s). To confirm the presence of microcystins in water, capillary electrophoresis was used, but with the problem that only three Bouaïcha, N. et al. 3

4 standards of microcystins are available. After sample preconcentration, microcystins -LR and possibly -YR were detected applying the UV spectrum analysis of the peaks. Figure 4: Electropherograms and UV Spectra: A - Standard, 1 MCYST-LR. 2 MCYST-YR; B - Water sample (Seine, April 1998); C - UV spectrum MCYST-LR; D - UV spectrum peak 1 of B Bouaïcha, N. et al. 4

5 At the same time 50 strains among the collected Cyanobacteria were purified under the direction of R. Rippka (Pasteur Institute) in order to obtain molecular probes for algal identification. The toxicity of some of the samples was determined with enzymatic assays, the phosphatase 2A and an acetylcholinesterase assaytable 2 shows some of the data: hepatotoxic as well as neurotoxic Cyanoprokaryotes were found; one Oscillatoria strain even showed both toxicities. Table 2: Toxicity assay of purified Cyanoprokaryote strains Seine Marne Oise Gloeocapsa PCC9638 Gloeocapsa* Gloeocapsa* Chamaesiphon PCC9629 Microcystis PCC9624 Synechococcus PCC9627 Svnechococcus PCC9630 Svnechococcus PCC9620 Svnechococcus PCC9636 Svnechococcus PCC97O7 Synechocystis PCC9626 Synechocystis PCC9634 Synechocystis PCC9616 Synechocystis PCC9706 Synechocystis PCC9617 Xenococcus PCC9703 Leptolyngbya PCC9628 Leptolyngbya* Oscillatoria PCC9625 Oscillatoria PCC9631 1) ( * ) Genera carrying an asterisk are still in the process of purification 2) Underlined Protein phosphatase inhibition Acetylcholinesterase inhibition Both inhibitions Conclusion A survey of Cyanoprokaryotes in water in parallel with the collection of water samples allows a correlation of the toxic potency of the water to phycotoxins. The water toxicity can be followed by screening assays. The phosphatase 2A assay provides a good indication of microcystins even at very low concentrations of less than 1ng/L. However, the chemical identification of toxins is still necessary as a control. Convenient techniques seem to be preconcentration and the capillary electrophoresis, which requires small samples only. A similar method could be applied for neurotoxins with specific bioassays. Studies are now performed on purified Cyanoprokaryotes from the three rivers to obtain molecular probes in order to detect the toxic algal strains in blooms. Bouaïcha, N. et al. 5

6 References AN, J., W.W. CARMICHAEL (1994), Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study of microcystins and nodularins. Toxicon 32, pp BOUAICHA, N., C. RIVASSEAU, M.C. HENNION, P. SANDRA (1996), Detection of cyanobacterial toxins (microcystins) in cell extracts by micellar electrokinetic chromatography. Journal of Chromatography B 685, pp BUMKE-VOGT,C., W. MAILAHN, W. ROTARD, I. CHORUS (1996), A highly sensitive analytical method for the neurotoxin anatoxin-a, using GC-ECD, and first application to laboratory cultures. Phycologia 35, pp CARMICHAEL, W.W. (1989), Freshwater cyanobacteria toxins, in Natural toxins : Characterization, pharmacology and Therapeutics. Proceedings of the 9th World Congress on Animal Plant and Microbial toxins, Stillwater, Oklahoma. Owny C. L., Odell G. V. eds., Pergamon Press, Oxford, pp CODD, G.A., S.G. BELL, W.P. BROOKS (1989), Cyanobacterial toxins in water. Wat. Sci. Tech. 21, pp ERIKSSON, J.E., H. HAGERSTRAND, B. ISOMAA (1987), Cell selective cytotoxicity of a peptide toxin from the cyanobacterium Microcystis aeruginosa. Biochem. Biophys. Acta 930, FALCONER, I.R. (1994), Mechanism of toxicity of cyclic peptide toxins from blue-green algae. In algal Toxins in Seafood and Drinking Water,. Falconer I. ed., Acad. Press, London, pp FALCONER, I.R., A. HUMPAGE. (1996), Tumor promotion by cyanobacterial toxins. Phycologia 35, pp HENRIKSEN, P., W.W. CARMICHAEL, J. AN, O. MOESTRUP (1997) Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms of cyanobacteria/blue-green algae from danish lakes and in the stomach contents of poisoned birds. Toxicon 35, pp HONKANAN, R. E., B.A. CODISPOTI, K.TSE, A.L.BOYNTON (1994), Characterization of natural toxins with inhibitory activity against serine/threonine protein phosphatases. Toxicon 32, pp HUYNH-DELERME, C., S. PUISEUX-DAO (1998), Toxines d origine algale inhibitrices de sérine-thréonine phosphatases. C. R. Soc. Biol. 192, pp LAWTON, L.A., C. EDWARDS, G.A. CODD (1994), Extraction high-performance liquid chromatographic method for the determination of microcystins in raw and treated waters. Analyst. 119, pp OHTANI, I., R.E. MOORE, M.T.C. RUNNEGAR (1992), Cylindrospermopsin : a potent hepatotoxin from the blue-green alga Cylindrospermopsis raciborskii. Journal of the American Chemical Society 114, pp PEARL, H.W. (1996), A comparison of cyanobacterial bloom dynamics in freshwater, estuarine and marine environments. Phycologia 35 Suppl., pp PUISEUX-DAO, S., N. BOUAICHA, G. DIOGENE (in press), Maitotoxin, okadaic acid and microcystins : toxins disturbing signal transduction and phosphorylations. In Animal Bouaïcha, N. et al. 6

7 Toxins. Tools in Cell Biology. Rochat H. and Martin-Eauclaire M.F. eds. Chapman & Hill, Weinheim, Germany. RAPPALA, J., K. SIVONEN, R. LUKKAINEN, S.I. NIEMALA (1993), Anatoxin-a concentration in Anabaena and Aphanizomenon under different conditions and comparison of growth by toxic and non toxic Anabaena strains, a laboratory study. Journal of applied phycology 5, pp SIVONEN, K., K. HIMBERG, R. LUUKKAINEN, S.I. NIEMALA, G.K. POON, G.A. CODD (1989), Preliminary charaterization of neurotoxic cyanobacterian blooms and strains from Finland. Toxicity assessment : an international journal 4, pp TUBARO, A., C. FLORIO, E.LUXICH, S. SOSA, R. DELLA LOGGIA, T. YASUMOTO (1996), A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels. Toxicon 34, pp Bouaïcha, N. et al. 7

8 Appendix A Interlaken 98 Hepatotoxic Water Poisoning : HWP Nodularin R 1, R 2 = CH 3 [D-Asp 1 ] Nodularin R 1 = H, R 2 = CH 3 [DMAdda 3 ] Nodularin R 1 = CH 3, R 2 = H MCYST-YR; R 1 = Tyr, R 2 = CH 3 ; R3 = Arg; R 4 = CH 3 (MW = 1044) MCYST-LR; R 1 = Leu, R 2 = CH 3 ; R3 = Arg; R 4 = CH 3 (MW = 994) MCYST-RR; R 1 = Arg, R 2 = CH 3 ; R3 = Arg; R 4 = CH 3 (MW = 1037) Figure 1: Hepatotoxic compounds produced by Anabaena flos-aquae, Aphanizomenon flosaquae, Microcystis aeruginosa, Nodularia spumigena, Oscillatoria agardhii Bouaïcha, N. et al. 8

9 Appendix B Interlaken 98 Paralytic Water Poisoning : PWP Saxitoxin and derivatives Anatoxin - a Anatoxin - a(s) Figure 2: Paralytic compounds produced by Anabaena flos-aquae Anabaena circinanalis, Aphanizomenon flos-aquae, Oscillatoria sp., Cylindrospermum sp. Bouaïcha, N. et al. 9