Non-native invasive species are increasingly evident in marine and estuarine environments, largely because of the intake and release of ballast water from sea vessels. Innovative methods are needed to quickly and accurately detect and speciate non-native and/or harmful phytoplankton in ballast water. Recent advances in ionization techniques such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have allowed detection of intact biomolecules within ballast samples. Statistically-based algorithms are used to discern "fingerprints" of phytoplankton species and to discern individual species from mixtures. MALDI-MS is particularly attractive for field applications because of the speed of analysis, minimal liquids/consumables required, and femtomole (10^-15) sensitivity.

 The objective of this project was to develop a rapid assay technique that was less time-consuming than more traditional methods of microorganism species identification in ballast water samples. Specific objectives included: (1) modifying MALDI-MS bacterial identification techniques for the analysis of phytoplankton, (2) determining the uniqueness of phytoplankton MALDI-MS fingerprints with a limited subset of phytoplankton, and (3) initiating evaluations of known phytoplankton identification in spiked environmental/ballast water samples.

For this project, MALDI-MS bacterial identification techniques developed at Pacific Northwest National Laboratory were modified for phytoplankton analysis. Using a pure strain of a representative phytoplankton species, an appropriate MALDI matrix material was determined and found to be compatible with the analyte sample. Mass spectral "fingerprints" of this species were examined for reproducibility using statistically-based algorithms. "Fingerprints" of several representative diatoms and dinoflagellates were developed, and the reliability of MALDI-MS to identify the target phytoplankton species in ballast water of Navy vessels was evaluated. Ballast water was spiked with the target phytoplankton.

Results from this study demonstrated that differentiation, and therefore identification, is possible between phytoplankton species and with minimal sample preparation. Recent advances in MALDI-MS instrumentation and tandem mass spectometry capabilities can provide further structural ion identification information. Overall, this study provided the first indication that identification and screening of phytoplankton is feasible with MALDI-MS and that the development of such techniques will ultimately provide a streamlined, cost-effective approach to assess and manage microorganism transport in ballast water. (Project Completed - 2007)