Assessing ballast water treatments: Evaluation of viability methods for ...

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Author's personal copy Journal of Great Lakes Research 36 (2010) 540–547

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Journal of Great Lakes Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j g l r

Assessing ballast water treatments: Evaluation of viability methods for ambient freshwater microplankton assemblages Euan D. Reavie a,⁎, Allegra A. Cangelosi b,1, Lisa E. Allinger a,2 a b

Natural Resources Research Institute, University of Minnesota Duluth, 1900 East Camp Street, Ely, MN 55731, USA Northeast-Midwest Institute, 50 F St., NW, Suite 950, Washington, DC 20001, USA

a r t i c l e

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Article history: Received 29 January 2010 Accepted 5 May 2010 Communicated by Marley Waiser Index words: Ballast water Invasive species Phytoplankton Fluorescein diacetate Algae Viability

a b s t r a c t For decades the Great Lakes have been subject to invasive species introductions through the discharge of ships' ballast water. Several treatment technologies involving physical, chemical, and biological processes have been developed to remove or inactivate organisms in this discharge. Assessing the efficacy of these technologies involves estimating the number of viable propagules in treated discharge relative to untreated controls. For organisms in the 10–50 µm size range, for example, the International Maritime Organization (IMO) mandates that fewer than 10 viable organisms per milliliter may be discharged. To date, however, there is no standard method to assess viability of natural assemblages of organisms in this size group (largely phytoplankton and protozoans) in freshwater environments. We report here on a process of assemblage concentration, staining with fluorescein diacetate (FDA), and microscopic observation as a reliable and efficient method to assess densities of viable freshwater organisms in this size category in ballast discharge. A number of other methods, including digestion with enzymes, flow cytometry, and a variety of vital and mortal stains, were tested and discarded during this vetting process due to inconsistent or ambiguous results. © 2010 Elsevier B.V. All rights reserved.

Introduction Commercial vessels are the primary vector for transport of nonnative aquatic species to the Great Lakes–St. Lawrence River system (Mills et al., 1993; Ricciardi and MacIsaac, 2000). More than 180 aquatic invasive species have been confirmed as introduced and established in the Great Lakes, and ballast water is likely responsible for approximately 70% of these species (Grigorovich et al., 2003). These organisms threaten not only ecological stability and diversity and abundance of native taxa but commercial activities as well. Over the last two decades, researchers have described the vectors, ecology, and impacts of invasive species such as zooplankters (including the spiny water flea [Bythotrephes] and the zebra mussel [Dreissenia polymorpha]; Garton et al., 1993), fish (e.g., the round goby [Neogobius melanostomus]; Jude, 1997), and more recently, pathogens (e.g., Johengen et al., 2005 ; Knight et al., 1999). While vertebrate and large invertebrate invaders garner the most public attention, several other worrisome microscopic taxa have been observed in ballast tanks of vessels entering the Great Lakes. Johengen et al. (2005) recorded phytoplankton that are known to form harmful algal blooms, including the dinoflagellate Pfiesteria piscida (Steidinger et al., 1996) ⁎ Corresponding author. Tel.: +1 218 235 2184. E-mail address: [email protected] (E.D. Reavie). 1 Tel.: +1 202 464 4007. 2 Tel.: +1 218 235 2157. 0380-1330/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jglr.2010.05.007

and the golden-brown alga Aureococcus anophagefferens (Bricelj and Lonsdale, 1997). They also observed protozoans known to be dangerous intestinal parasites, including Giardia lamblia and Encephalitozoon intestinalis. Although currently not confirmed as an established invasive species in the Great Lakes, there is concern (Whitton et al., 2009) that the mat-forming diatom Didymosphenia geminata could become established in the Great Lakes nearshore ecosystems if spread of invasive microscopic organisms is not prevented. The National Aquatic Nuisance Prevention and Control Act of 1990 required the first federal measures to prevent ballast-related invasive species introductions into US waters. Initially focused on the Great Lakes, Congress expanded the scope of this program to a national level in 1996 through the National Invasive Species Act. Since 1997, the International Maritime Organization (IMO) has called for voluntary ballast management, and in 2004 agreed to mandatory ballast water exchange. Ballast water exchange, a management practice requiring no new hardware, has been shown to be somewhat effective but not feasible under all scenarios (Minton et al., 2005) and is difficult to enforce (Cangelosi and Mays, 2006). The IMO's “International Convention for the Control and Management of Ships' Ballast Water and Sediments” specifies deadlines for the imposition of discharge limits for viable organisms by size categories for ballast water (IMO, 2004). The IMO Convention sets discharge limits on densities of live organisms by size class of organism. A ship may neither discharge 10 or more live organisms greater than 50 µm in minimum dimension

Author's personal copy E.D. Reavie et al. / Journal of Great Lakes Research 36 (2010) 540–547

per cubic meter of water nor 10 or more organisms greater than 10 µm and less than 50 µm in minimum dimension per milliliter of water. Currently, the US Coast Guard is proposing the same limits be applied through US regulation, with a stricter criterion of 1000-fold the IMO discharge limits as a second phase standard. Treatment options for ballast water include: physical processes (filtration, cavitation, sonification, heating; Cangelosi et al., 2007; Kato, 2003; Holm et al., 2008; Rigby et al., 2004); chemical processes (applied or generated biocides such as chlorine or peroxide; Gregg and Hallegraeff, 2007; Kuzirian et al., 2001), and biological processes (microbial deoxygenation; McCollin et al., 2007). Assessing treatment effectiveness against numeric discharge limits necessitates detecting and enumerating viable organisms in treated ballast discharge water, but significant scientific questions persist regarding the best methods of doing so. In particular, there are no standard methods for readily and reliably discerning live and dead organisms less than 50 µm and greater than or equal to 10 µm in minimum dimension, herein referred to as the “10–50 µm group.” These organisms are largely phytoplankton and protozoans. Cangelosi and Mays (2005), in a survey of analytical methods available for ballast treatment performance assessments, determined that no method was currently available to reliably discern live and dead phytoplankton across ecosystem types. No such analysis has been strictly associated with freshwater ambient assemblages of organisms in the 10–50 µm group. A reliable method for counting and sizing these organisms is also an open question. Microscopy is a well-vetted method, but broad taxonomic expertise is required to effectively assess considerable diversity, including free-living cells, multicellular colonies, and filamentous forms. In addition, unlike for most zooplankton species, many phytoplankton cells in the 10–50 µm size range are non-motile in situ and under microscopic assessments. Flow cytometry can provide a rapid automated assessment of phytoplankton assemblages (Phinney and Cucci, 1989), especially since several recent advances have mitigated mechanical problems, like clogging, that have hampered its use in phytoplankton research in the past (Veldhuis and Kraay, 2000). The ability of flow cytometry to accurately distinguish live/dead status across protist taxonomic groups remains uncertain. As a result of issues with known methods, estimates of potential ballast treatment systems have been limited largely to enumeration of live zooplankton in discharge. Estimates of ballast treatment effectiveness within the 10–50 µm group, especially in freshwater assemblages, have been absent or based on responses of standard test organisms in the laboratory, land-based experiments, or bulk algal biomass evaluations. Evaluations using surrogate organisms provide only limited treatment performance predictions. Bulk assessments are not reliable indicators of viability and cannot enumerate live organisms consistent with the proposed discharge standards for ballast water. Laboratory studies are limited by the numbers and types of organisms evaluated, whereas natural assemblages are diverse and offer a wide range of organism types for treatment performance evaluation, including several analogs of potentially invasive taxa. Furthermore, compared to standard organism cultures, ambient assemblages are fully adapted to the natural water medium and are likely better fit to resist treatment. It is well known that organisms in the 10–50 µm size class can pose severe risk to receiving freshwater systems (e.g., Dobbs and Rogerson, 2005). As the prospect of mandatory ballast treatment by ships becomes a reality, the need for rigorous, reliable, and efficient means of assessing treatment effectiveness relative to these taxa and absolute densities of viable propagules in ballast discharge becomes more urgent. Without such a tool, effective development and vetting of treatment candidates for regulatory approval will be insufficient to prevent the environmental risk associated with ballast discharge.

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For the current investigation, a range of methods for live/dead analysis of this challenging group of organisms is reviewed for their relevance in assessing ballast treatment effectiveness for ambient freshwater systems. Candidate methods are tested and an effective method is identified and described. Methods Review and selection of candidate methods Using available literature, possible methods for quantitatively discerning living and dead organism densities in the 10–50 µm range were reviewed against a set of criteria for their potential applicability to mixed unknown assemblages associated with freshwater ballast discharge. The criteria for evaluation of each method were: 1. Signal is quantitatively precise such as that necessary to assess organism condition upon discharge from a ship's ballast system (lack of false negatives or positives at the time of analysis); 2. Signal is consistent across taxa in ambient freshwater assemblages; 3. Signal is consistent across treatment types; 4. Method is practical for rapid, high throughput, on-site assessments. Methods considered were (1) natural autofluorescence from chlorophyll to identify living cells (e.g., Booth, 1987; Pouneva, 1997); (2) vital, metabolic, and mortal stains such as those typically associated with specific standard test organisms including propidium iodide (Franqueira et al., 2000), TO-PRO-1 (Okochi et al., 1999), fluorescein diacetate (Invitrogen product code F1303; FDA; Berglund et al., 1987; Buggé and Allam, 2005; Franklin et al., 2001; Gilbert et al., 1992; Murphy & Cowles, 1997; Saga et al., 1987; Schumann et al., 2003), Sytox® Green (Invitrogen product code S7020; Buttino et al., 2004; Timmermans et al., 2007; Veldhuis et al., 2001; Veldhuis et al., 2006), and several stain combinations supplied by Molecular Probes® [LIVE/DEAD® Kit #1 (Invitrogen product code L-7013; SYTO 10 + DEAD Red), LIVE/DEAD® Violet Viability Kit (Invitrogen product code L34958; CellTrace™ calcein violet+ aqua-fluorescent dye), and LIVE/DEAD® Cell Vitality Assay Kit (Invitrogen product code L34951; C12-Resazurin + Sytox Green)]; (3) cell digestion methods, particularly DNAse (New England BioLabs® product code M0303S) + trypsin (Cellgro® product code 25-054-Cl) digestion (Agustí and Sánchez, 2002; Darzynkiewicz et al., 1994); and (4) automated cell enumeration methods including standard flow cytometry and FlowCAM® (Fluid Imaging Technologies; e.g., Sieracki et al., 1998) combined with use of vital and mortal stains. Empirical validation of candidate analytical methods The subset of methods that, based on available literature, were determined to potentially meet criteria outlined above was empirically validated against the same criteria using ambient freshwater assemblages of organisms. Six viability analysis methods (five epifluorescent staining methods and one cell digestion method) and one flow cytometry method for automated counting and characterization of prepared samples were investigated. All quantitative measures were maintained throughout evaluations, including volumes filtered, backwashed and strewn on counting chambers, total lengths and widths of microscopic transects, and cell and entity counts. Sample collection Whole-water samples were collected from several pelagic freshwater sources during the ice-free periods of 2006, 2007, and 2008. These sites included Shagawa Lake (47°54′48″N, 91°52′50″W), Miners Lake (47°54′33″N, 91°51′26″W), and Burntside River (47°54′53″N, 91°57′01″W) located near the city of Ely in northeastern Minnesota. Several test samples were also collected during validation of the Great

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Ships Initiative (GSI) land-based facility (Duluth-Superior Harbor, 46°42′42″N, 92°02′52″W) throughout the summers of 2007 and 2008. Harbor samples were collected via in-line sampling pitots on the intake stream (i.e., during filling of the onshore simulation ballast tanks) and on the discharge streams from treatment and control tanks following 18-h or 5-day incubations. The variations in seasons, locations, and types of samples allowed for a variety of samples for methods testing. Within 30 min of collection, water samples were concentrated to facilitate microscopy. Samples were concentrated using a Wildco® chlorophyll filtering kit (Fig. 1a). In place of the glass fiber filters that would typically be used for chlorophyll collections, a 7-cm square piece of 10-µm gauge Nitex® plankton netting was used. (Earlier attempts to use 5-µm gauge netting resulted in rapid clogging of the mesh, and so a larger gauge was used.) To prevent cell damage, vacuum pressure did not exceed 200 mm mercury (Hg) and exposure of the concentrated organisms to the air was minimized. Water was concentrated in 100-ml increments until pressure began to rise. The netting containing the organisms was removed and placed, concentrate side up, on a “washing tube” (a 7-cm length of 4-cm internal diameter polyvinyl chloride piping; Fig. 1b). A short flange (a 1-cm length of 4.2-cm internal diameter piping) was pushed over the edges of the netting to hold it firmly in place. The tube with netting attached was inverted so that the organism concentrate faced down into a 210ml CoorsTek® casserole dish. Between 15 and 20 ml of filtrate was collected from the vacuum flask and added to the washing tube to backwash the organisms into the casserole dish. A rubber pipette bulb, sealed around the lip of the washing tube, was helpful to force filtrate water through the mesh and dislodge organisms.

Sample treatment Three methods of killing organisms were applied to 2-ml subsamples of the concentrated samples: heating to 100 °C for 5 min, freezing overnight and thawing, and adding 200 µl of 37% formalin (final concentration in samples = 3.4% formalin). Each viability assessment method, unless it was rejected early due to obvious performance issues, was applied to sets of at least five replicate subsamples subjected to each of the killing methods, and one set of five replicate untreated subsamples (controls). For comparison to freeze-killed assemblages, untreated samples were retained overnight at 5 °C. Assemblages were observed microscopically within 2 h following collection, concentration, and treatment to minimize cell degradation. Analyses of samples for freeze-killing comparisons were observed within 24 h.

Sample analysis Method 1: FDA epifluorescent stain A subsample of 2 ml of the concentrated sample was added to a 5ml sample bottle and stained with 5 µl of FDA stock solution [5 mg FDA powder dissolved in 1 ml dimethyl sulfoxide (DMSO)]. The sample was mixed by inverting five times and allowed to incubate in the dark at room temperature for 5 min. After incubation a stained subsample of 1.1 ml was added to a Sedgwick-Rafter cell. Organisms in the rafter cell were allowed to settle for 2 min before beginning microscopic observation. Counting was performed using an Olympus CKX-41 inverted microscope configured for easy transition between bright field and epifluorescence for taxonomic identification and viability assessment, respectively. Fluorescence specifications were excitation wavelength of 460–490 nm (blue) using a 50-W mercury lamp and observation filtered wavelength of 520–700 nm to observe simultaneous green and red fluorescence. Specimens were counted and identified along horizontal transects using 200× magnification. Closer examination at 400× allowed for better taxonomy of some specimens. Complexity of this assessment was variable due to variations in species assemblages, and we aimed to count at least 100 entities (i.e., unicellular organisms, colonies, and/or filaments) within 1 h. If time permitted, specimens were counted until 1 h was reached; counts frequently exceeded 300 entities and 1000 cells. Single cell entities and cells comprising colonial and filamentous entities were characterized as follows using FDA-stained samples as an example: • Alive: cells showing obvious green fluorescence from cell contents; • Dead: cells showing no, or very little, evidence of green fluorescence from cell contents. For entities containing multiple cells, all cells must be confirmed as dead to fulfill this category; • Ambiguous: entities that cannot be clearly identified as alive or dead, or which show incongruous activity such as a moving flagellated cell with no fluorescence.

Fig. 1. Apparatus used for concentration of organism assemblages in whole-water samples.

Counting and measuring of entities followed a standard procedure for individuals (length and width), colonies (e.g., number of cells, cell length and width), and filaments (e.g., number of cells, cell length and width, or total filament length if cells could not be discerned). Entities less than 10 µm in all visible dimensions were not counted. Entities with cells or organisms (e.g., zooplankton) greater than 50 µm in minimum dimension were not counted as they were assessed separately by GSI zooplankton experts. Entities that were long dead, typically identified as empty diatom frustules or otherwise significantly degraded, were not counted. All remaining concentrated samples of organisms were archived using Lugol's solution.


Methods 2–5: Sytox Green and LIVE/DEAD combination kits Sample preparation using Sytox Green and the three LIVE/DEAD combination kits (see Results) was similar to that for FDA. Volumes used, incubation methods, and emission spectra varied slightly, and the reader is referred to their respective standard methods (www. invitrogen.com). Method 6: Cell digestion Preparation for DNAse + trypsin digestion followed Agustí (2004). We added 200 µl of 80 µg/l DNAse solution to a 2-ml concentrated subsample of organisms and the sample was incubated at 37 °C for 15 min. Trypsin was added to the sample [200 µl of 2% trypsin in Hank's balanced salt solution (HBSS)] and the sample was incubated for 30 min at 37 °C. To halt digestion, 0.6 ml of 5.8 mg/ml trypsin inhibitor was added to the sample. Samples were observed as above using bright field microscopy instead of epifluorescence. Living cells were assumed to be those with intact cytoplasmic contents. Quantification through flow cytometry To determine the applicability of flow cytometry to our freshwater assessments, a hands-on workshop with Fluid Imaging Technologies was held in July 2007. Water samples from Duluth-Superior Harbor were assessed using a benchtop-model FlowCAM and a selection of vital and mortal stains. Since the workshop, discussions with Fluid Imaging Technologies have continued to determine if new developments will be helpful in our viability assessments for the 10–50 µm group. Results Review of candidate methods The majority of viability assessment methods reported in the literature involve the use of fluorescent probes and observation using microscopy or flow cytometry. These applications date back to the early 1970s (Crippen and Perrier, 1974). In our literature review, few of these methods clearly met all of our criteria for potential applicability to ballast treatment testing and monitoring in the context of natural freshwater assemblages, either because they clearly did not meet the criteria or because the literature was not adequate to support a determination. Methods that clearly did not met the criteria included chlorophyll autofluorescence and some epifluorescent staining applications. Autofluorescence methods were precluded because they would likely fail the first review criterion considering chlorophyll autofluorescence can be maintained for many weeks in dead cells (Moss, 1968). This method would therefore deliver false positives. In addition, autofluorescence would not be effective with protozoans and other organisms with no natural autofluorescing pigments, thus violating the second review criterion. Some fluorescent stains also failed to meet the first two criteria. For instance, the red fluorescent signal in dead cells caused by the stain propidium iodide (Darzynkiewicz et al., 1994) would likely conflict with red autofluorescence from chlorophyll in both living and dead algal cells. Several stain combinations were likewise precluded because the excitation and emission requirements for many stain pairs interfere with simultaneous observation of epifluorescent results, in conflict with our fourth criterion. Methods for which the literature was not conclusive either way included some epifluorescent staining and cell digestion methods. For example, the literature shows that FDA (available from multiple vendors) has been successfully applied to viability assessments following ballast water treatments. FDA stains cells with respiratory

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activity and the method has been successfully applied to singlespecies cultures of diatoms (Berglund et al., 1987; Murphy and Cowles, 1997), marine macroalgae (Saga et al., 1987), unicellular green algae (Franklin et al., 2001), dinoflagellates (Gilbert et al., 1992), parasitic protists (Buggé and Allam, 2005), and marine bacteria (Schumann et al., 2003). Some studies, however, have suggested that application of FDA to natural assemblages provides ambiguous results. For instance, the intensity of the fluorescent signal (i.e., green fluorescence from excitation by blue light) varies among several marine species of algae (Garvey et al., 2007). With regard to Sytox Green, this stain passes through compromised cell membranes of dead or dying cells and subsequently emits fluorescence upon binding to DNA. As Sytox does not pass through living cell membranes, unstained cells are assumed to be alive. Sytox Green has been used to assess viability in select marine species of copepods (Buttino et al., 2004) and algae (dinoflagellates and diatoms; Buttino et al., 2004; Timmermans et al., 2007; Veldhuis et al., 2001). Veldhuis et al. (2006) used Sytox Green to assess viability in ambient marine assemblages in a model ballast water system. Their assessments were based on flow cytometry with non-viable cells identified by an arbitrary cut-off of brightness from fluorescing entities. The authors concluded that their assessment was useful in determining effectiveness of PERACLEAN® Ocean disinfectant but recognized that dealing with colonial algae is problematic. Another candidate, LIVE/DEAD Kit #1 (SYTO 10 + DEAD Red; Molecular Probes), is a combination of fluorescent dyes intended to provide independent staining of living and dead cells. Like Sytox, SYTO 10 penetrates all cell membranes and binds DNA while DEAD Red also binds to DNA but only penetrates dead cell membranes. Cells are identified as dead if their nuclear materials fluoresce yellow (a combination of fluorescence from both stains) and living if they only express green fluorescence. Based on current literature this kit has been used in several mammalian studies, particularly humans (e.g., Matsuo, 2001) but has not been tested in microscopic aquatic assemblages. LIVE/DEAD Violet Viability Kit (CellTrace™ calcein violet + aquafluorescent dye; Molecular Probes) also uses a combination of two stains for simultaneous live and dead cell determination. These probes measure plasma membrane integrity (aqua-fluorescent amine-reactive dye) and intracellular esterase activity (calcein violet) as a measure of cell vitality. Live cells fluoresce violet, whereas dead cells are blue-green. We found no published literature regarding the use of this kit for staining microscopic aquatic assemblages. LIVE/DEAD Cell Vitality Assay Kit (C12-Resazurin + Sytox Green; Molecular Probes) uses a combination of Sytox Green (for staining the nuclear material of dead cells) and C12-Resazurin, a metabolic stain that is reduced to red-fluorescing C12-resorufin in the presence of metabolic enzymes in live cells. Dead cells exhibit green fluorescence of nuclear material, whereas live cells fluoresce red. Although we anticipated that the red signal from Resazurin might conflict with algal autofluorescence, we also surmised that the signals could be distinguishable. A final method of interest, DNAse+ trypsin digestion, involves dead cell digestion. This assay was originally developed by Darzynkiewicz et al. (1994) and was modified for phytoplankton by Agustí and Sánchez (2002). Two enzymes (trypsin and DNAse I), which pass through the cell membranes of dead or dying cells, are added to cell mixtures. Upon entering cells these enzymes should fully digest the cell contents, whereas morphology and function of living cells remain unchanged. Cell counts can then be performed by assuming that all cells with cytoplasmic contents are living. Unlike most of the viability assessments using stains, the digestion method requires no epifluorescence equipment and allows digested samples to be preserved for intact cell assessment at a later date. This digestion method has been successfully applied to marine cyanobacterial assemblages (Agustí, 2004) and natural algal assemblages from Florida lakes (Agustí et al., 2006).




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Table 1 Array of results for various treatments and viability assessment methods for natural assemblages in the 10–50 µm size group. Proportions of live, dead, and ambiguous cells for each treatment are based on the average of at least five sample assessments. Significance of comparisons are based on at least five assemblage counts for each treatment using a nonparametric Student's t-test between numbers of (apparently) living cells in treated and untreated (control) assemblages. Killing treatment

FDA

Sytox® Green LIVE/DEAD® Kit #1 LIVE/DEAD® Violet Viability Kit LIVE/DEAD® Cell Vitality Assay Kit Trypsin + DNAse I digestion

Heat

0 100 Trace Yes 0 100 Trace Yes 0 100 Trace Yes

10 83 7 Yes 6 81 13 Yes 10 76 14 Yes

% Live % Dead % Ambiguous Sig. different? Freeze/thaw % Live % Dead % Ambiguous Sig. different? Formalin % Live % Dead % Ambiguous Sig. different?

10 84 6 Yes 9 85 6 Yes 8 82 10 Yes

12 83 5 Yes 12 80 8 Yes 9 82 9 Yes

With regard to automated live cell counting in mixed assemblages, the literature neither confirmed nor precluded the possible use of flow cytometry through the use of a FlowCAM® (Fluid Imaging Technologies), a continuous imaging flow cytometer designed to characterize microscopic particles in water. Numerous articles (e.g., Buskey and Hyatt, 2006) discuss the value of the FlowCAM to eliminate microscopy tedium, allowing for rapid examination of more cells for greater counting precision. Specialized sensors have also allowed detection of viability signals in planktonic organisms during analysis. Empirical analysis of candidate methods Epifluorescent probes As a viability assessment method, FDA-stained living cells were clearly apparent via the green glow emanating from cytoplasmic contents. The fluorescein signal was detectable across taxa, although there were taxon-specific variations in signal brightness (Fig. 2). Dead cells in all killed treatments were consistently unstained (Table 1) indicating that the method is highly reliable for identifying dead cells across lethal treatments. Occasional entities in killed assemblages exhibited green autofluorescence, but this autofluorescence was easily distinguished from that coming from fluorescein (Fig. 2a). Autofluorescence generally occurred in the cell walls of select species, and so was easily distinguished from a viable cytoplasmic fluorescein signal. Fluorescein fading occurred relatively rapidly; most specimens faded completely within a minute under persistent blue light excitation. The impact of fading on assessments was minimized by the microscopist's ability to block the path of the excitation light using an opaque filter. The antifade agent Slowfade® Gold was added to select samples and though it increased fade time to about 2 min, it did not improve assessments due to the speed of enumeration and identification and our ability to block excitation. Ambiguous signals, generally attributed to cells with weak signs of life, occurred in fewer than 0.5% of specimens (e.g., Fig. 2t). Sytox Green was able to correctly mark 76% or more of dead specimens in killed freshwater assemblages (Table 1). Although there were significantly more cells identified as dead in killed samples, Sytox provided incorrect or ambiguous responses for certain taxa. For

10 84 6 Yes 11 80 9 Yes 7 84 9 Yes

90 10 0 No 84 16 0 No 91 9 0 No

instance, Sytox was unable to stain nuclei of Scenedesmus (small green algal species) in killed samples, and so dead cells may be falsely identified as alive. In contrast, in living assemblages we occasionally observed motile naviculoid diatoms with stained nuclei. Ambiguous signals were also obtained from some cyanobacteria such as Anabaena; the cell contents of some species in dead assemblages stained faintly, and so confirmation of mortality was difficult. Due to assemblage composition variation, the amount of mischaracterized entities varied similarly. The proportion incorrectly identified as either alive or dead ranged from 5% to 25% in any given sample. The various LIVE/DEAD kits also showed some ability to track living and dead cells using epifluorescence (Table 1), but like Sytox, speciesspecific issues hampered assessments. The SYTO 10 component of Kit #1 appeared to stain the nuclear material of most taxa (whether dead or alive) but the combined yellow (dead) signal provided by DEAD Red occurred in only ∼50% of taxa and 82–85% of cells encountered in killed assemblages. The Violet Viability Kit falsely marked many species in killed assemblages as alive, and 10–20% of taxa did not stain at all even after higher concentrations of stain were added. Due to the inclusion of Sytox Green in the Cell Vitality Assay Kit this kit had similar problems as described for Sytox above. The C12-Resazurin component of the kit provided obvious red fluorescence of living cell contents, but for some smaller algae species we could not determine if the red fluorescence was due to staining or chlorophyll autofluorescence. Cell digestion We did not observe cell digestion in killed assemblages as described by Agustí (2004) and related articles (Table 1). No significant difference was observed in the numbers of intact cells in living and killed assemblages treated with digestive enzymes. Several attempts were made to facilitate digestion with higher concentrations of enzymes (as much as five-fold higher), longer incubation times (up to 2 h), and warmer incubation temperatures (up to 60 °C), but these modifications made little difference. With higher enzyme concentration, some atrophying of large diatoms (e.g., Stephanodiscus niagarae) was observed, but full digestion of the dead cells was never observed. Replacement with freshly purchased batches of enzyme solutions was ineffective.

Fig. 2. Paired bright field/epifluorescent micrographs of select specimens from freshwater samples. Each micrograph pair shows a photo (left or top) taken using phase contrast at 200× magnification and a photo (right or bottom) taken using blue light excitation on samples stained with FDA. (a) A mixture of concentrated phytoplankton from Duluth/Superior harbor containing 95% freeze-killed and 5% living organisms. One living diatom filament shows clear fluorescence from FDA, while other algae show faint or no fluorescence. Green algae: (b) Closterium (alive); (c) Pandorina (alive); (d) Crucigenia (alive); (e) cf. Westella (alive); (f) Gloeocystis (alive); (g) Cosmarium (alive); (h) Staurastrum (alive); (i) Euglena (alive); (j) Microspora (alive). Dinoflagellates: (k) Glenodinium (alive); (l) cf. Gymnodinium (alive); (m) a dead filament of the diatom Melosira, a dead dinoflagellate Glenodinium, and a living filament of the diatom Aulacoseira. Diatoms: (n) Synedra (dividing, alive); (o) Aulacoseira granulata (mixture of living and dead cells); (p) Cymbella (alive); (q) Gyrosigma (alive); (r) Navicula (alive); (s) Encyonema (alive); (t) diatom filament (mixture of living and dead cells, showing variations in brightness from FDA); (u) Cyclotella (dead); (v) Fragilaria (mixture of living and dead cells); (w) a dead colony of the diatom Asterionella colonized by living sporangia of living Rhizophydium; (x) the ciliate Vorticella (alive); and (y) a filament of the cyanobacterium Anabaena (alive).



Enumeration methods Flow cytometry was rendered unreliable by species-specific affinity for stains, usually characterized as variations in fluorescent intensity; automated cytometric sensors had trouble distinguishing living from dead cells. Foremost were problems dealing with multicellular entities. IMO standards are based on densities of viable cells in water being released, but while current flow cytometry is capable of accurate particle counts, counting cells in complex colonies and filaments is not possible without significant time spent in postanalysis of specimen photographs. Furthermore, we observed many cases of multicellular entities with mixtures of living and dead cells (Fig. 2j, o, t, v). In these cases, sensors could only identify cells as being “alive” or “dead” but not a mixture. The inability to distinguish live from dead is problematic especially when using stains such as Sytox. Colonial microorganisms would likely be characterized as dead if they contained dead cells, regardless of viable cell presence. Mixtures of living and dead cells were frequently observed in several freshwater entities, such as cyanobacteria (e.g., Microcystis) and filamentous diatoms (e.g., Aulacoseira). Microscopy overcame automation problems, but a smaller sample volume was practicable given the time required for sample analysis before samples were affected by age. At the time of these analyses the IMO criterion necessitated the analysis of at least 1 ml of sample water containing pre-treatment concentrations of at least 1000 cells/ml. Although microscopic sample enumeration for 1 h met this challenge, any significant increase in sample volume requirement would lead to problems with microscopic assessment. Discussion This study reviewed and tested several methods in order to choose the best one for determining viability of freshwater microorganism assemblages occurring in treated ballast water. From these analyses, microscopic examination of FDA-stained samples appears to be the best approach currently available for determining viability, while fluorescence microscopy remains the best method for live cell enumeration. Autofluorescence gives frequent false positives and therefore has been discounted as a viability determination method in previous studies. Based on our empirical investigations of candidate stains, we recommend caution when using Sytox and the LIVE/DEAD kits to perform viability assessments in natural freshwater assemblages. Our research indicated clear discrepancies between the reported ability to discern living from dead organisms and the actual method performance in laboratory tests. We believe this discrepancy arose because methods were originally designed for application to (and tested with) standard laboratory test organisms, and not natural assemblages. Also of note is the fact that stains rejected by this study were not adequately quantitative for this application, in general. While most staining methods can categorically distinguish living from dead cells, in an application in which the precise number of live is sought in a regulatory context, the proportion of incorrect or ambiguous results precludes their use. This observation, however, does not preclude their use for other types of ballast treatment research. For example, Sytox Green accurately measures mortality in some monocultures (Timmermans et al., 2007), and we found it to be a highly reliable method for bench-scale assessments using cultured Selenastrum capricornutum following treatment applications (unpublished data). We rejected the cell digestion method due to our inability to reliably digest dead cells. However, the problems we confronted might be specific to northern Minnesota and Lake Superior water conditions, rather than freshwater generally. The natural assemblages and water matrix tested in this study may be less susceptible to artificial lysis with DNAse and trypsin. This method has potential to provide rapid cost-effective viability assessments and should be

studied and developed for possible use across a wider variety of environments and assemblages. We found that assemblage concentration, staining with FDA, and microscopic observation met our evaluation criteria as a reliable method to discern live densities of viable freshwater organisms in the 10–50 µm size category in ballast discharge. Though FDA may be inappropriate for some environments and species assemblages (one researcher suggests that it does not adequately stain some diatoms from coastal marine environments; Mario Tamburri, Maritime Environmental Resource Center, Chesapeake Biological Laboratory, personal communication), we are confident that it is suitable to ballast water treatment assessment in Lake Superior. An initial phase of method evaluation is recommended for any testing program to validate its use in a particular subsystem within the Great Lakes. It should be noted that any staining method, even one that appears to have broad spectrum applicability and reliability across treatment processes (like FDA did in this study) may not discern living from viable cells. There are probably many cases where ballast water treatment systems render living cells no longer reproductively viable, and thereby eliminate any environmental risk associated with those cells. However, confirmation that living cells are viable would require subsequent grow-out experiments following treatment (e.g., Pertola et al., 2006). Grow-out is especially important if testing includes resting cysts, such as those from amoebae, chrysophytes, and dinoflagellates. To make conservative risk assessments, regulators are likely to assume that cells deemed to be living via a reliable stain are also able to propagate if introduced to an aquatic environment. With regard to characterizing numbers of live cells, we see no reliable substitute for microscopy. In particular, the ability of flow cytometry to automate counting and sizing of live cells in ambient assemblages is limited by the stain reliability as noted above. Accordingly, flow cytometry, including FlowCAM, appears ideally suited to assessing viability in monocultures and unicellular entities (Cid et al., 1996). Microscopy, however, may limit sample volume necessary to achieve statistical confidence if regulatory criteria designate analysis of larger sample volumes. Realistically, an approximate limit for one analyst using the microscopic method would be complete analysis of 3 ml of sample water at 1000 living cells/ml within 1 h, subject to the complexity of the species assemblage. The selected method appears capable of performing the task of live/dead protist enumeration in natural freshwater assemblages. However, ship discharge monitoring imposes additional requirements on analytical tools. Variable ballast water sources such as occur in ships greatly complicate the tasks, and there may be unanticipated influences by certain treatment methods on the effectiveness of FDA in vitality assessments. Nonetheless, we have eliminated several methods that are currently not suited to ballast assessments in the Great Lakes, and the selected method provides a strong foundation for future microorganism assessments in ballast water. Acknowledgements We thank Fluid Imaging Technologies for support during the flow cytometry workshop. Kathleen Kennedy helped with field collections. This work was supported by funds and in-kind efforts assembled from the private sector, federal grants, Congressional appropriations, foundations, and states, including contributions from Canadian and US Great Lakes ports, National Oceanic and Atmospheric Administration, St. Lawrence Seaway Development Corporation, St. Lawrence Seaway Management Corporation, US Maritime Administration, US Department of Transportation, Legislative-Citizen Commission on Minnesota Resources, University of Wisconsin Superior (Balcer M, TenEyck M), University of Minnesota Duluth (Hicks R, Branstrator D), Great Lakes carrier companies, and the Great Lakes Maritime Research Institute. This is contribution number 501 of the Center for Water and


the Environment, Natural Resources Research Institute, University of Minnesota Duluth. References Agustí, S., 2004. Viability and niche segregation of Prochlorococcus and Synechcoccus cells across the central Atlantic ocean. Aquat. Microb. Ecol. 36, 53–59. Agustí, S., Sánchez, C.M., 2002. Cell viability in natural phytoplankton communities quantified by a membrane permeability probe. Limnol. Oceanogr. 47, 818–828. Agustí, S., Alou, E., Hoyer, M.V., Frazer, T.K., Canfield, D.E., 2006. Cell death in lake phytoplankton communities. Freshw. Biol. 51, 1496–1506. Berglund, D.L., Taffs, R.E., Robertson, N.P., 1987. A rapid analytical technique for flow cytometric analysis of cell viability using calcofluor white m2r. Cytometry 8, 421–426. Booth, B.C., 1987. The use of autofluorescence for analyzing oceanic phytoplankton communities. Bot. Marina 30, 101–108. Bricelj, V.M., Lonsdale, D.J., 1997. Aureococcus anophagefferens: causes and ecological consequences of brown tides in U.S. Mid-Atlantic coastal waters. Limnol. Oceanogr. 42, 1023–1038. Buggé, D.M., Allam, B., 2005. A fluorometric technique for the in vitro measurement of growth and viability of quahog parasite unknown (qpx). J. Shellfish Res. 24, 1013–1018. Buskey, E.J., Hyatt, C.J., 2006. Use of the FlowCAM for semi-automated recognition and enumeration of red tide cells (Karenia brevis) in natural plankton samples. Harmful Algae 5, 685–692. Buttino, I., do Santo Espirito, M., Ianora, A., Miralto, A., 2004. Rapid assessment of copepod (Calanus helgolandicus) embryo viability using fluorescent probes. Mar. Biol. 145, 393–399. Cangelosi, A., Mays, N., 2005. Summary and Findings of the Ballast Discharge Monitoring Device Workshop, 2002 Aug. 12–16; U.S. Geological Survey's Western Fisheries Research Center's Marrowstone Island Field Station, WA. NortheastMidwest Institute, Washington, DC. 53 pp. (http://www.nemw.org/images/ stories/documents/marrowstonereport.pdf). Cangelosi, A., Mays, N., 2006. Great ships for the Great Lakes? Commercial vessels free of invasive species in the Great Lakes–St. Lawrence Seaway System. A scoping report for the Great Ships Initiative. Northeast-Midwest Institute, Washington D.C. 143 pp. (www.nemw.org). Cangelosi, A.A., Mays, N.L., Balcer, M.D., Reavie, E.D., Reid, D.M., Sturtevant, R., Gao, X., 2007. The response of zooplankton and phytoplankton from the North American Great Lakes to filtration. Harmful Algae 6, 547–566. Cid, A., Herrero, C., Abalde, J., 1996. Functional analysis of phytoplankton by flow cytometry: a study of the effect of copper on a marine diatom. Sci. Mar. 60, 303–308. Crippen, R.W., Perrier, J.L., 1974. The use of neutral red and Evans Blue for live–dead determination of marine phytoplankton. Stain Technol. 49, 97–104. Darzynkiewicz, Z., Li, W., Gong, J., 1994. Assays of cell viability: discrimination of cells dying by apoptosis. In: Darzynkiewicz, Z., Robinson, J.P., Crissman, J.A. (Eds.), Methods in Cells Cell Biology. Academic Press, New York, pp. 15–38. Dobbs, F.C., Rogerson, A., 2005. Ridding ships' ballast water of microorganisms. Environ. Sci. Technol. 39, 259–264. Franklin, N.M., Adams, M.S., Stauber, J.L., Lim, R.P., 2001. Development of an improved rapid response inhibition bioassay with marine and freshwater microalgae using flow cytometry. Arch. Environ. Contam. Toxicol. 20, 469–480. Franqueira, D., Orosa, M., Torres, E., Herrero, C., Cid, A., 2000. Potential use of flow cytometry in toxicity studies with microalgae. Sci. Total Environ. 247, 119–126. Garton, D.W., Berg, D.J., Stoeckmann, A.M., Hagg, W.R., 1993. Biology of recent invertebrate invading species in the Great Lakes: the spiny water flea, Bythotrephes cederstoemi, and the zebra mussel, Dreissena polymorpha. In: McKnight, B.N. (Ed.), Biological pollution: the control and impact of invasive exotic species. Indiana Academy of Science, Indianapolis, pp. 63–84. Garvey, M., Moriceau, B., Passow, U., 2007. Applicability of the FDA assay to determine the viability of marine phytoplankton under different environmental conditions. Mar. Ecol. Prog. Ser. 352, 17–26. Gilbert, F., Galgani, F., Cadiou, Y., 1992. Rapid assessment of metabolic activity in marine microalgae: application in ecotoxicological tests and evaluation of water quality. Mar. Biol. 112, 199–205. Gregg, M.D., Hallegraeff, G.M., 2007. Efficacy of three commercially available ballast water biocides against vegetative microalgae, dinoflagellate cysts and bacteria. Harmful Algae 6, 567–584. Grigorovich, I.A., Colautti, R.I., Mills, E.L., Holeck, K.T., Ballert, A.G., MacIsaac, H.J., 2003. Ballast-mediated animal introductions in the Laurentian Great Lakes: retrospective and prospective analyses. Can. J. Fish Aquat. Sci. 60, 740–756. Holm, E.R., Stamper, D.M., Brizzolara, R.A., Barnes, L., Deamer, N., Burkholder, J.M., 2008. Sonication of bacteria, phytoplankton and zooplankton: application to treatment of ballast water. Mar. Pollut. Bull. 56, 1201–1208.

547

International Convention for the Control and Management of Ship's Ballast Water and Sediments. International Maritime Organization (2004 Feb 13). London, UK (adopted 2004 Feb 16). (http://www.cfr.org/publication/20775/international_convention_for_the_control_and_management_of_ships_ballast_water_and_sediments.html). Johengen T, Reid D, Fahnenstiel G, MacIsaac H, Dobbs F, Doblin M, Ruiz G, Jenkins P. Assessment of Transoceanic NOBOB Vessels and Low-Salinity Ballast Water as Vectors for Non-indigenous Species Introductions to the Great Lakes. Final Report. Ann Arbor (MI): NOAA, Great Lakes Environmental Research Laboratory and University of Michigan, Cooperative Institute for Limnology and Ecosystems Research (US); 2005. 287 pp. Jude, D.J., 1997. Round gobies: cyberfish of the third millennium. Great Lakes Res. Rev. 3, 27–34. Kato H. Cavitation, as a tool of environmental protection. Proceedings of the 5th International Symposium on Cavitation; 2003 Nov 1-4; Oasaka, Japan [place unknown; publisher unknown]. 8 p. Knight I, Cangelosi AA, Colwell RR, Geller J, Huq A, MacGregor B, Reynolds K. Ballast water and pathogens: a focus on the Great Lakes. Report to the Great Lakes Protection Fund. 1999. 55 pp. Kuzirian, A.M., Terry, E.C.S., Bechtel, D.L., James, P.L., 2001. Hydrogen peroxide: an effective treatment for ballast water. Biol. Bull 201, 297–299. Matsuo, T., 2001. Trehalose protects corneal epithelial cells from death by drying. Br. J. Ophthalmol. 85, 610–612. McCollin, T., Quilez-Badia, G., Josefsen, K.D., Gill, M.E., Mesbahi, E., Frid, C.L.J., 2007. Ship board testing of a deoxygenation ballast water treatment. Mar. Pollut. Bull. 54, 1170–1178. Mills, E.L., Leach, J.H., Carlton, J.T., Secor, C.L., 1993. Exotic species in the Great Lakes: a history of biotic crises and anthropogenic introductions. J. Great Lakes Res. 19, 1–54. Minton, M.S., Verling, E., Miller, A.W., Ruiz, G.M., 2005. Reducing propagules supply and coastal invasions via ships: effects of emerging strategies. Front Ecol. Environ. 3, 304–308. Moss, B., 1968. Studies on the degradation of chlorophyll a and carotenoids in freshwaters. New Phytol. 67, 49–59. Murphy, A.M., Cowles, T.J., 1997. Effects of darkness on multi-excitation in vivo fluorescence and survival in a marine diatom. Limnol. Oceanogr. 42, 1444–1453. Okochi, M., Taguchi, T., Tsuboi, M., Nakamura, N., Matsunaga, T., 1999. Fluorometric observation of viable and dead adhering diatoms using to-pro-1 iodide and its application to the estimation of electrochemical treatment. Appl. Microbiol. Biotechnol. 51, 364–369. Pertola, S., Faust, M.A., Kuosa, H., 2006. Survey on germination and species composition of dinoflagellates from ballast water tanks and recent sediments in ports on the south coast of Finland, North-Eastern Baltic Sea. Mar. Pollut. Bull. 52, 900–911. Phinney, D.A., Cucci, T.L., 1989. Flow cytometry and phytoplankton. Cytometry 10, 511–521. Pouneva, I., 1997. Evaluation of algal culture viability and physiological state by fluorescent microscopic methods. Bulgaria J. Plant Physiol. 23, 67–76. Ricciardi, A., MacIsaac, H.J., 2000. Recent mass invasion of the North American Great Lakes by Ponto-Caspian species. TREE 15, 62–65. Rigby, G., Hallegraeff, G., Taylor, A., 2004. Ballast water heating offers a superior treatment option. J. Mar. Environ. Eng. 7, 217–230. Saga, N., Machiguchi, Y., Sanbonsuga, Y., 1987. Application of staining dyes for determining viability of cultured algal cells. Bull. Hokkaido Reg. Fish Res. Lab. 51, 39–44. Sieracki, C.K., Sieracki, M.E., Yentsch, C.S., 1998. An imaging-in-flow system for automated analysis of marine microplankton. Mar. Ecol. Prog. Ser. 168, 285–296. Schumann, R., Schiewer, U., Karsten, U., Rieling, T., 2003. Viability of bacteria from different aquatic habitats. II. Cellular fluorescent markers for membrane integrity and metabolic activity. Aquat. Microb. Ecol. 32, 137–150. Steidinger, K.A., Burkholder, J.M., Glasgow Jr, H.B., Hobbs, C.W., Garrett, J.K., Truby, E.W., Noga, E.J., Smith, S.A., 1996. Pfiesteria piscicida gen. et sp. nov. (pfiesteriaceae fam. nov.), a new toxic dinoflagellate with a complex life cycle and behavior. J. Phycol. 32, 157–164. Timmermans, K.R., Veldhuis, M.H.W., Brussaard, C.P.D., 2007. Cell death in three marine diatom species in response to different irradiance levels, silicate, or iron concentrations. Aquat. Microb. Ecol. 46, 253–261. Veldhuis, M.J.W., Kraay, G.W., 2000. Application of flow cytometry in marine phytoplankton research: current applications and future perspectives. Sci. Mar. 64, 121–134. Veldhuis, M.J.W., Kraay, G.W., Timmermans, K.R., 2001. Cell death in phytoplankton: correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth. Eur. J. Phycol. 36, 167–177. Veldhuis, M.J.W., Fuhr, F., Boon, J.P., Ten Hallers-Tjabbers, C.C., 2006. Treatment of blast water; how to test a system with a modular concept? Environ. Technol. 27, 909–921. Whitton, B.A., Ellwood, N.T.W., Kawecka, B., 2009. Biology of the freshwater diatom Didymosphenia: a review. Hydrobiologia 630, 1–37.