I niled Slides Hii\ ironnu'iK;il Pmicclion .\»cnc\ Office of Kesciirch iiikI Dexclopmcnl National Kxposure Research Laboratory Research Abstract Government Performance Results Act (GPRA) Goal #2 Annual Performance Measure #457 Significant Research Findings: A LC/MS Method for the Determination of Cyanobacteria Toxins in Water Scientific Several classes of very toxic compounds are produced by various genera, species, Problem and and strains of cyanobacteria. Cyanobacteria, which are also called blue-green Policy Issues algae, occur worldwide in fresh and marine waters and have likely been transported globally by natural and anthropogenic processes. Growth of cyanobacteria is stimulated by nitrogen and phosphorus containing nutrients, warm temperatures, stagnant water, and intense sunlight. The cyanobacteriaMicrocystis, Nodularia, Anabaena, Nostoc, and Oscillatoria produce cyclic peptide hepatotoxins that cause extensive liver damage by inhibition of the protein phosphatase 1 and 2A enzymes. Subacute exposures are thought to promote liver tumor formation. In addition, some species also produce neurotoxins, including anatoxin-a, cylindrospermopsin, and the paralytic shellfish saxitoxins. There is worldwide concern for the impacts of these toxins on humans, farm animals, pets, and wildlife through drinking and recreational water exposure. The toxicity and increased occurrence of cyanobacteria blooms led the U.S. Environmental Protection Agency (USEPA) to include cyanobacteria toxins on the drinking water Contaminant Candidate List for further study and future regulatory determination. During the spring of 2001, USEPA convened a panel of scientists to assist in identifying a target list of cyanobacteria toxins that are likely to pose a health risk in source and finished waters of drinking water utilities in the U.S. The toxins recommended for further research were the neurotoxins anatoxin-a and cylindrospermopsin and the cyclic heptapeptide microcystin hepatotoxins. To conduct large-scale occurrence surveys and human exposure studies, reliable, sensitive, and quantitative analytical methods are required for the priority toxins. All reported analytical methods have deficiencies that limit their applicability to large-scale occurrence surveys and human exposure studies. Analytical costs would be minimized if the priority toxins could be determined in a single efficient analytical method. Further benefits would accrue if the analytical method was capable of accurately recognizing other cyanobacteria toxins that may occur in unexpected frequencies and concentrations in water. ------- Research The goal of this research was to determine the feasibility of developing a Approach laboratory analytical method that would identify and measure selected cyanobacteria toxins in an environmental water sample in approximately one hour. To partition the toxins from a water sample, an extraction technique was developed that is based on a commercially available membrane filter containing finely divided and dispersed C-18 silica particles. Combined liquid chromatography/mass spectrometry (LC/MS) was selected as the only viable separation and determinative technique capable of providing high quality results that would meet the objectives of this study. Toxins were partitioned from water samples with extraction filter disks containing very fine C-18 silica particles enmeshed in a thin Teflon® matrix. The toxins were eluted from the filter disks with methanol, and the eluate was analyzed with LC/MS using positive ion electrospray ionization. A bench-top time-of-flight (TOF) mass spectrometer was employed with continuous full-spectrum data acquisition and real-time accurate mass-to-charge ratio (m/z) measurements. Results and Impact Excellent sensitivity was provided by the high duty cycle of the TOF analyzer, which ensures that few ions are lost or discarded in the ion pulsing region. Furthermore, no ions were deliberately discarded or discharged in the TOF analyzer, which is common in other high sensitivity MS techniques such as selected ion monitoring. Selectivity and confirmation of an ion's composition was supported by routine exact m/z measurements of all ions at a consistently available and resolving power of about 5,000. The cyanobacteria toxins anatoxin-a, microcystin-LR, microcystin-RR, microcystin-YR, and nodularin were separated in less than 30 minutes on several 1 mm x 15 cm reverse phase LC columns, and their electrospray mass spectra were measured using injections of 50 ng or less. Research Collaboration and Research Products New data from this work include recoveries of the five toxins from reagent water, river waters, and sewage treatment plant effluent samples. This method has the potential to provide a relatively simple and reasonable-cost sample preparation and LC/MS approach that provides the sensitivity, selectivity, reliability, and information content needed for source and drinking water occurrence and human exposure studies. Additional research (see Future Research) would be required to develop the complete analytical method needed for such applications. This research was supported in part by the appointment of Mila Maizels to the postgraduate research program administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and USEPA. Mila Maizels and William L. Budde. A LC/MS Method for the Determination of Cyanobacteria Toxins in Water. Anal. Chem. 2004, 76,1342-1351. Future Research The cyclic heptapeptide microcystin toxins produced by the B2666 strain of Microcystis aeruginosa that have not been investigated previously will be identified using the sample preparation and LC/MS technique developed in this research. This strain was selected by the USEPA as a test system to study treatment options for production of drinking water from cyanobacteria- contaminated source water. Additional research needed to make this method suitable for occurrence studies includes: (1) further testing with appropriate source and finished water samples to evaluate the impacts of matrix on the suppression and/or enhancement of ------- responses to the chemicals; (2) testing with additional analytes of the same general class; and (3) a determination of such parameters as the precision, accuracy, limits of detection, and ruggedness of the method. Questions and inquiries can be directed to: U.S. EPA, Office of Research and Development National Exposure Research Laboratory 26 W. Martin L. King Jr. Dr. Cincinnati, OH 45268 Phone:513/569-7309 E-mail: budde.william@epa.gov Funding for this project was through the U.S. EPA's Office of Research and Development, National Exposure Research Laboratory, and the work was conducted by the Microbiological and Chemical Exposure Assessment Research Division. Contacts for Additional Information William L. Budde, Ph.D. ------- |