## 1 Introduction
[9] reported that the \(>\)50 \(\upmu\)M size class was dominated by _Certainum_ and _Protoperidinium_, which comprised 85% of individuals \(>\)50 \(\upmu\)M. Therefore, strictly speaking these taxonomic groups would not be counted in the 10-50 \(\upmu\)M (phytoplankton) regulated size class. In shipboard compliance testing off the Mexican coast, 12 out of 37 phytoplankton taxonomic groups (32.5%) were larger than 50 \(\upmu\)M in the smallest dimension and 13/37 taxa (35.2%) were smaller than 10 \(\upmu\)M in the smallest dimension. In terms of organism numbers, phytoplankton taxa \(<\)10 \(\upmu\)M and \(>\)50 \(\upmu\)M represented 14% and 27% of the total counts, i.e., only 59% of phytoplankton fell into the 10-50 \(\upmu\)M size category [10]. [PERSON] et al. [11] emphasize the omission of the \(<\)10 \(\upmu\)M size category as an important deficiency in current discharge standards. Results such as these call into serious question the strict reliance on standards based on numbers of live or viable organisms within published size categories. Additionally, it has been noted that smaller (non-regulated) species may be faster growing and more easily adaptable to new environments [11; 12; 13]. This problem is recognized in the current development of indicative tools for assessing compliance with the Ballast Water Convention. At the cellular level there is a need for new indicative tools for rapidly assessing the efficacy of ballast water treatment. Such tools, which may or may not be reliant on numeric standards include tagging with the vital stain Fluorescein Diacetate (FDA), chlorophyll fluorescence activity (CFA) and ATP determination using the luciferase enzyme. Indicators of cell viability may be partnered with flow cytometry to provide estimates of cell numbers. A recent, comprehensive shipboard study compared nine variants of these ballast water assessment technologies, five of which provided estimates of numbers of individuals [9]. Specific needs include the identification of 'high risk' organisms in ballast and receiving water and the development of rapid means of measuring the integrity of vital biological processes in treated organisms. Until recently nucleic acid sequencing had not been a focus for assessing cell viability in treated ballast water. While nucleic acid levels in aquatic samples have shown good correlation with phytoplankton cell count, the ability of DNA to persist outside live organisms can lead to overestimates of living material in discharged ballast water. Nevertheless, progress has been made in the use of DNA-based methods as a means of identifying the appearance of invasive species in aquatic environments [14] A significant advance in this respect has been the development of high throughput sequencing (HTS), which has expanded DNA barcoding, relating to an individual organism, into second generation sequencing (metabarcoding), capable of mapping whole populations of organisms in an environmental sample. The attractiveness of the technique is its potential for defining population shifts related to season and geography as well as subtle population changes related to invasive organisms. Several recent studies have advocated the use of molecular techniques in the analysis of ballast water [15; 16; 17]. [PERSON] et al. [16] used nucleic acid sequencing to characterize protist communities in ballast water derived from the east, west and gulf coasts of the United States. In a recent application of this technique to ballast water analysis, [PERSON] and [PERSON] [17] compared bacterial biomarkers identified by HTS and Most Probable Number counts in ballast water discharged at four major international marine ports in 2015-2016. The current study investigated the use of HTS to identify the effect of location and season on the phytoplankton and bacterial flora of ballast water encountered seasonally by Great Lakes freighters, together with the possibility of identifying potentially harmful taxonomic groups.