Isolation and identi®cation of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria) R. Li a , W.W. Carmichael a, * , S. Brittain a , G.K. Eaglesham b , G.R. Shaw c , A. Mahakhant d , N. Noparatnaraporn e , W. Yongmanitchai e , K. Kaya f , M.M. Watanabe f a Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA b Queensland Health Scienti®c Services, 39 Kessels Road, Coopers Plains, Queensland 4108, Australia c National Research Center for Environmental Toxicology, 39 Kessels Road, Coopers Plains, Queensland 4108, Australia d Thailand Institute of Scienti®c and Technological Research, Bangkok 10900, Thailand e Faculty of Science, Kasetsart University, Bangkok 10900, Thailand f National Institute for Environmental Studies, Onogawa 16-2, Tsukuba, Ibaraki 305-0053, Japan Received 31 July 2000; accepted 24 October 2000 Abstract A strain of Cylindrospermopsis (Cyanobacteria) isolated from a ®shpond in Thailand was examined for its taxonomy based upon morphology and 16S rRNA gene sequence. It was also examined for production of the hepatotoxic cyanotoxin called cylindrospermopsin (CYN) and deoxycylindrospermopsin (deoxy-CYN). The strain (CY-Thai) was identi®ed as C. raciborskii (Woloszynska) Seenaya and Subba Raju based upon morphological examination which was con®rmed by 16S rRNA gene sequences and phylogenetic comparisons based upon its 16S rRNA gene. The alkaloid heptatotoxin CYN was con®rmed using mouse bioassay, HPLC and HPLC-MS/MS while deoxy-CYN was con®rmed using HPLC-MS/MS. The mouse bioassay gave a minimum lethal dose at 250 mg dry weight cells/kg body weight within 24 h and 125 mg/kg at 72 h, with signs of poisoning the same as in literature reports for CYN. HPLC chromatographic comparison of the CY-Thai toxin with standard CYN gave the same retention time and an absorbance maximum at 262 nm. HPLC-MS/MS con®rmed the presence of CYN (M 1 H 416) and deoxy-CYN (M 1 H 400). The CYN content in strain CY-Thai was estimated at 1.02 mg/g and approximately 1/10 of this amount for deoxy-CYN. This is the ®rst report from Asia of a CYN, deoxy-CYN producing Cylindrospermopsis raciborskii. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cyanotoxin; Cylindrospermopsin; Deoxy-cylindrospermopsin; Cyanobacteria; Toxin; Cylindrospermopsis 1. Introduction Increasingly, harmful algal blooms (HABs) are being reported worldwide due to several factors, primarily-eutrophi- cation, climate change and more scienti®c investigation (Hallegraeff et al., 1995). All but cyanobacteria toxin poison- ings (CTPs) are mainly a marine occurrence (Carmichael, 1997; Chorus and Bartram, 1999). CTPs occur in fresh (lakes, ponds, rivers and reservoirs) and brackish (seas, estu- aries and lakes) waters throughout the world. Organisms responsible include an estimated 40 genera but the main ones are Anabaena, Aphanizomenon, Lyngbya, Microcystis, Nostoc and Oscillatoria (Planktothrix). Cyanobacteria toxins (cyanotoxins) include cytotoxins and biotoxins with biotoxins being responsible for acute lethal, acute, chronic and sub- chronic poisonings of wild/domestic animals and humans. The biotoxins include the neurotoxins; anatoxin-a, anatoxin- a(s) and saxitoxins plus the hepatotoxins; microcystins and nodularins. Con®rmation of human deaths, from cyanotoxins, Toxicon 39 (2001) 973±980 0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S0041-0101(00)00236-1 www.elsevier.com/locate/toxicon * Corresponding author. Tel.: 11-937-775-3173; fax: 11-937- 775-3320. E-mail address: wayne.carmichael@wright.edu (W.W. Carmichael).