Impact/Purpose:

The purpose of this research project is to develop a strong qualitative and quantitative "gold standard" laboratory reference analytical method for the simultaneous selective and sensitive identification and measurement of those cyanobacteria toxins that are of the highest priority to EPA and state and local drinking water authorities. These toxins are on the drinking water candidate contaminant list (CCL), but the specific toxins that are the most likely to occur, and which are the potentially most hazardous, are not defined on the CCL. Six toxins were recommended for highest consideration by an EPA workshop in May, 2001. These are the alkaloids anatoxin-a and cylindospermopsin, and the four microcystins abbreviated RR, LR, YR , and LA (these characters are the standard single-character designations for four natural aminoacids). There are no published analytical methods that have been demonstrated to have the ability to detect and reliably measure the concentrations of these six compounds in a single drinking or source water sample. The purpose of this research is to not only develop and demonstrate this capability with real environmental samples, but also to do it in a single economical laboratory analytical method that requires about one hour from initial sample preparation to obtaining the results. It is expected that some other toxins will be found in the real environmental samples tested, and perhaps even new toxins may be discovered in this work. The techniques employed in this work, have the capability of tentatively identifying known and unknown toxins even when standards are not available.

Description:

This project is part of a larger U. S. Environmental Protection Agency (EPA) effort, which includes the Office of Water, to investigate algal toxins in surface water supplies and drinking water. Toxins produced by cyanobacteria (blue-green algae) are among the most potent known and over 80 specific molecular structures have been discovered in just the last 15 years. These toxins are divided into neurotoxins (about 20), which are generally alkaloids, hepatotoxins, which are cyclic peptides often called microcystins (about 60), and lipopolysaccharide endotoxins (unknown number) which are compounds of fats and sugars. The neurotoxins and microcystins have the highest toxicities, are of most concern to EPA and local water authorities, and are the focus of this investigation. Published journal articles have presented analytical approaches for these toxins based on either immunochemical or instrumental techniques. However, all techniques published to date are mainly qualitative (few quantitative results have been reported), are limited to just a few neurotoxins or microcystins in separate analyses, and are not developed to contemporary standards of selectivity, sensitivity, quantitative accuracy, precision, speed, and efficiency. Furthermore no analytical method has been tested and documented to the extent required by EPA or any state or local water authority. In this project state-of-the-art high efficiency microbore liquid chromatography combined with on-line electrospray mass spectrometry is applied to the separation, detection, firm identification, and quantitative measurement of cyanobacteria toxins. The new or unique aspects of this project are: (1) the combination of the neurotoxins and microcystins into a single analytical method; (2) the development of a method which includes both very strong qualitative and quantitative analysis capabilities; and, (3) the application of time-of-flight mass spectrometry with sufficient resolving power to provide exact mass measurements to within about 10 parts-per-million (ppm) of observed molecular and fragment ions to ensure unequivocal identifications. The overall result will be the "gold standard" reference method against which all other methods claimed to be faster, cheaper, or simpler will be judged.

Record Details:

Record Type:PROJECT

Start Date:06/01/2000

Projected Completion Date:09/01/2005

Record ID: 18348

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Project Information:

Progress :An Oak Ridge Institute for Science and Education (ORISE) post-doctoral associate was recruited during the latter part of fiscal year (FY) 2000 and she began research in June of 2000. Her first effort was to develop laboratory skills with on-line high performance microbore liquid chromatography (LC) combined with electrospray time-of-flight mass spectrometry (ES/MS). Some of the training exercise work with pesticides was judged worthy of publication. A manuscript was prepared, peer reviewed, cleared, and published in the highest impact factor analytical chemistry journal in the world (Analytical Chemistry, 2001, 73, 5436-5430). An ORISE project plan and a quality assurance (QA) plan for the toxins research were completed and reviewed by the National Exposure Research Laboratory (NERL) QA staff. The project plan was also submitted to external peer review in April, 2001. Seven commercially available cyanobacteria toxins were acquired and a LC/ES/MS separation and unequivocal detection technique was developed. However, the available toxins did not include two priority toxins, cylindrospermopsin and microcystin-LA, which are not commercially available. Preliminary figures of merit, including estimated detection limits and extraction recoveries and precision, were obtained using fortified reagent water. A preliminary oral report was made by the post-doctoral associate at the Federation of Analytical Chemistry and Spectroscopy Societies (FACSS) annual conference in Detroit in early October, 2001. An invited seminar was presented by the principal investigator (PI) at the University of Cincinnati during April, 2002. Contributed posters were presented by the PI and the associate at the American Society for Mass Spectrometry's annual conference in Orlando, FL during June, 2002, and at the Oesper award symposium at the University of Cincinnati in October, 2002. These results were provided to James Sinclair, EPA Office of Ground Water and Drinking Water, during August, 2002 and were included in his summary presentation of many EPA projects on cyanobacteria toxins at a conference called by the State of Florida Department of Health. A brief summary was also presented by an academic colleague as part of an oral review of analytical methods at the November, 2002 Water Quality Technology Conference of The American Water Works Association in Seattle, WA. The post-doctoral associate resigned in March of 2003 to accept an excellent position in the pharmaceutical industry. Efforts were initiated to recruit a new post-doctoral associate. An updated contributed poster was presented by the PI at the American Society for Mass Spectrometry's annual conference in Montreal, CA during June, 2003. This poster included recoveries of toxins from natural river water and sewage treatment plant effluent samples. A manuscript for the research was prepared for publication in the journal Analytical Chemistry, and this manuscript was provided to two non-NERL scientists for peer-review and was submitted to the division office for formal clearance. A candidate for the post-doctoral position accepted an offer in October, 2003 and will report in December, 2003.

Relevance :This project is part of a larger EPA effort, which includes the Office of Water (OW), to investigate algal toxins in surface water supplies and drinking water. This is an important research project because the cyanobacteria toxins on the CCL list are among the most potent toxins known, and research is needed to develop an analytical method or methods to assess the potential for human and animal exposure to these toxins. Future potential applications include routine monitoring of unregulated or possibly regulated contaminants in drinking water supplies. This project will likely impact some aspect of the OW decision to regulate or require monitoring of these substances in drinking water supplies. It may also bear some relationship to the homeland security effort because of the diversity of highly toxic substances that can be produced merely by growing cyanobacteria on a body of nutrient-rich water.

Clients :Office of Ground Water and Drinking Water

Research Component :CCL (CHEMICAL)

Risk Paradigm :EXPOSURE

Project IDs:

ID Code :none

Project type :ORD-DW Plan

ID Code :9418

Project type :OMIS