## 4 Discussion  
While earlier shipboard tests of ballast water treatment [7] demonstrated the need for a seasonal testing regime, there is also a clear requirement for information on the seasonal and temporal variability of the phytoplankton flora in the Great Lakes, and the effect of seasonal and temporal changes on the size/taxonomic profile of these organisms. From the perspective of ballast water management, questions include how different locations and seasonal changes in taxonomic groups may affect the size profile of the phytoplankton fraction, with consequent changes to the challenge water characteristics. For example, a seasonal bloom of potentially harmful dinoflagellates or bacteria may provide a much greater challenge to treatment than at other times. Therefore, there is clear need for as much information as possible on the make-up of both bacterial and phytoplankton flora likely to be encountered by Great Lakes vessels playing their trade over a large spatial and temporal range. Microscopic phytoplankton results reported here show the numbers and types of phytoplankton that freighters will encounter in a normal year-long season in all of the locations that the ships visit. In a parallel study of harbor water and ballast water from coastal China released in the ports of Shanghai, China; Durban, S. Africa; Los Angeles, CA, U.S.A. and Singapore. HTS was used to identify bacterial biomarkers primarily used to assess water of origin [17]. Whole organism (i.e., non-molecular) counts differed from the current study in that, unlike the microscopic phytoplankton counts performed here, IDEX Colilert(r) and Enteroler(r) MPN assays were used for counts of indicator bacteria [17]. An HTS analysis of ballast water arriving at east coast, west coast and Gulf coast ports in the U.S. was able to differentiate among protists arriving at different ports according to source water with specific reference to toxic dinoflagellates [16]. Although all of these studies clearly illustrate the potential for HTS to characterize the biome according to season and origin, the value of this technique as a tool for ballast water management is less clear. While a major goal of this project was to examine the potential for nucleic acid sequencing to identify key taxonomic groups in ballast water from different locations and seasons, at least qualitatively, the potential for this technique as an indicative test for the effectiveness of ballast water treatment technology remains speculative. It must be emphasized that the results reported from this and other molecular surveys of ballast water were from untreated water. As such they are have the potential for tracking the presence and movement of potentially harmful species. However, in order to demonstrate the feasibility of this approach as an indicative tool for ballast water management it would be necessary to identify characteristic nucleic acid profiles for key taxonomic groups, and to demonstrate changes in nucleic acid sequences that correlate with diminished phytoplankton viability and/or reproductive potential following ballast water treatment. Nucleic acid sequencing has yet to be coupled with demonstration of ballast water treatment, and would require field trials of treatment including a comparison of nucleic acid sequencing with current compliance assessment methods and other indicative technologies currently in development [28]. HTS has the potential to provide a comprehensive picture of the planktonic flora at any particular time or location without resorting to highly specialized microscopy. Comparison with conventional microscopy can provide important information on the utility of molecular techniques in identifying potentially harmful taxonomic groups in freshwater and marine environments. In the context of identifying potentially harmful invasive organisms, however, it is clear that substantial further refinement would be needed for molecular techniques such as HTS to be universally applicable. Clear differences between microscopy and HTS remain.