Comparison of three techniques to evaluate the number of viable phytoplankton cells in ballast water after ultraviolet irradiation treatment

The unintentional release of aquatic nonindigenous species (NIS) via ballast water has long been recognized as a primary vector of biological invasions. To reduce the risk of ballast-mediated invasions, the International Maritime Organization (IMO) will direct ships to meet standards regarding the maximum number of viable organisms discharged in ballast water, with most ships expected to install ballast water management systems (BWMSs). Currently, filtration?+?ultraviolet (UV) irradiation is utilized as a common BWMS. There are issues, however, with enumerating viable phytoplankton after treatment at the low UV doses used onboard ships because the physiological effect occurs at the DNA level—organisms are reproductively sterilized but may remain alive for hours or days after treatment. The objective of this study is to examine three techniques to measure the number of viable phytoplankton cells following filtration?+?UV treatment: pulse amplitude modulation (PAM) fluorometry, epifluorescence microscopy using fluorescein diacetate (FDA) stain, and the serial dilution culture most probable number (MPN) method. PAM and staining techniques demonstrated similar patterns of phytoplankton reduction after UV irradiation. After 14 days, the MPN method confirmed viability of treated samples in enriched culture medium incubations and may be used to indicate potential recovery of damaged cells (i.e., “re-growth”). All cells that survived treatment and showed detectable growth after 14 days of incubation were smaller than 10 µm, as determined by microscopy. Combinations of direct and/or indirect measurements and culture-based methods might be the best solution to improve detection strategies and eliminate nonindigenous species.     

Critical review of the IMO international convention on the management of ships’ ballast water and sediments

The International Maritime Organization (IMO), the United Nations body which administers the international regulatory regime for shipping, noted the negative impact of non-indigenous organisms transported in the ballast water of ships already in the early 1970s. Consequently, measures were taken with the aim to minimize ballast water mediated species invasions through IMO Marine Environmental Protection Committee (MEPC) Resolutions. As a result of long-term IMO efforts, it was determined that an international convention would best meet the needs of the global community, hence the International Convention for the Control and Management of Ships’ Ballast Water and Sediments was adopted in a Diplomatic Conference in 2004 and is now open for signature by IMO Member States. This very complex (and by no means “simple”) Convention aims to reduce the transfer and subsequent impact of aquatic organisms in the ballast water and sediment of ships by acting to reduce the load of these organisms in discharged ballast water. A set of 15 guidelines provides technical guidance for the implementation of the Convention principles. This review considers critical aspects of this Convention and selected guidelines seen from perspectives of biological, shipping and regulatory concerns.     

The efficiency of regional ballast water exchange: Changes in phytoplankton abundance and diversity

Ballast water was sampled on 12 occasions before and after an exchange process carried out in regional seas in order to assess the efficiency of this type of ballast water management at reducing the abundance and diversity of phytoplankton. Although there was an overall reduction in the average abundance and number of taxa after exchange this was not consistent between tanks and voyages. On some occasions there were changes in species composition after exchange and, in some cases, there were increases in potentially harmful species after the exchange process. Factors such as the depth of the water during the exchange process, the season and the method of exchange influenced the efficacy of the exchange process. The variability in the results after exchange mean that this is unlikely to be a ballast water management method that would give consistent results and careful consideration would have to be given to the suitability of using this method in regional seas as a means of reducing the risk of introducing non-native species.     

In vitro isolation of nanobodies for selective Alexandrium minutum recognition: A model for convenient development of dedicated immuno-reagents to study and diagnostic toxic unicellular algae

At the present, the identification of planktonic species in coastal water is still a time intensive process performed by highly trained personnel that relies either on qPCR or on light microscopy observation and in vitro culturing. Furthermore, the increasing danger represented by Harmful Algal Blooms (HABs) inside phytoplankton community and the recent implementation of the legislation on ballast water management to prevent the introduction of HABs and NIS (Non Indigenous Species) urge the development of faster and reliable diagnostic methods. Immuno-based approaches could fulfil this need provided that the costs for antibody selection and production will be reduced.In this work it is demonstrated for the first time the feasibility to recover nanobodies (VHHs) selective for native surface epitopes of Alexandrium minutum by direct whole cell bio-panning using a pre-immune phage display library. The recombinant nature of VHHs enabled their rapid engineering into eGFP fluorescent reagents (fluobodies) that were produced recombinantly in bacteria and are directly suitable for fluorescence microscopy and flow cytometry. Immune-detection identified also cysts and anti-Alexandrium fluobodies showed no cross-reactivity with indigenous not-toxic phytoplankton microalgae belonging to different geni. The fluobodies were able to bind selectively to the target cells in both fixed and fresh samples with minimal processing.     

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.     

Reflections on the ballast water dispersal—harmful algal bloom paradigm

The ballast water dispersal—HAB paradigm, increasingly invoked circumstantially to explain puzzling and unaccountable HAB species outbreaks when lacking the multiple tests of confirmation recommended by Bolch and de Salas (2007), is evaluated. The types and examples of natural dispersions and taxon cycles are compared to exotic species bloom behavior linked to ballast water vectoring. The regional spreading, bloom behavior and disjunct distributions of the brown tide pelagophyte Aureococcus anophagefferens and the toxic dinoflagellate Gymnodinium catenatum, attributed to ballast water vectoring, are used as representative examples to evaluate the general application of the ballast water—HAB paradigm and associated interpretative problems. Human-aided emigration has a seeding and colonization ecology that differs from bloom ecology. For self-sustaining blooms to occur, these two ecologies must be accommodated by habitat growth conditions. The three stages that a non-native species must pass through (pioneering, persistence, community entry) to achieve colonization, community maintenance, and to bloom, and the niche-related factors and role of habitat disturbance are discussed. The relevance of cryptic occurrences, cyst deposits, dormancy periods and bloom rhythms of HAB species to their blooms attributed to ballast water-assisted introductions is also sketched. The different forms of HAB species rarity, their impact on the ballast water dispersal—HAB paradigm, and the dispersion and blooms of specialist and generalist HAB species are discussed. The remarkable novel and, often, monospecific blooms of dinoflagellate HAB species are being paralleled by similar eruptive bloom behavior cutting across phylogenetic lines, and being found also in raphidophytes, haptophytes, diatoms, silicoflagellates, etc. These blooms cannot be explained only as seeding events. An ecological release of ‘old barriers’ appears to be occurring generally at coastal bloom sites, i.e. something significant is happening ecologically and embedded within the ballast water—HAB paradigm. There may be a relationship between Life Form type [Smayda, T.J., Reynolds, C.S., 2001. Community assembly in marine phytoplankton: application of recent models to harmful dinoflagellate blooms. J. Plankton Res. 23, 447–461] and mode of expatriation; HAB dinoflagellate species commonly reported to produce ballast water-assisted toxic blooms invariably are members of cyst-producing Life Forms IV, V, VI. Ballast water vectoring of Life Forms I, II, III is rarely reported, even though many produce cysts, and where their novel introductions do occur they are more likely to be ichthyotoxic and vectored in shellfish stock consignments. The relevance of, and need to distinguish between morphospecies and their geographic/ribotype clades are discussed based on the Alexandrium tamarense/catenella/fundyense complex. Morphospecies-level ballast water dispersions are probably minor compared to the dispersal of the different ribotypes (toxic/non-toxic clades) making up HAB morphospecies; the redistribution and admixture of genotypes should be the focus. Ballast water-assisted expatriations impact the global occurrence of HABs through the direct transfer of previously absent species or introduction of genetic strains from the donor habitat that are ecologically favored over resident strains. The hybridization of species may be of potentially greater impact, resulting from the (1) mating of individuals from the donor and recipient habitats, or (2) through the interbreeding of strains introduced from two different donor sites into the recipient site, and whose progeny have greater ecological fitness than indigenous strains. Exceptional ecological changes of some sort appear to be occurring globally which, in combination with the genetically altered ecophysiological behavior of HAB species linked to ballast water dispersion and admixture, underpins the global HAB phenomenon. The impact of ballast water and shellfish transplantation on HABs and phytoplankton community ecology, generally, is considerably greater than the current focus on HAB species distributions, vectoring, and blooms. The methodological, investigative and conceptual potential of the ballast water—HAB paradigm should be exploited by developing a GEOHAB type intiative to advance quantification of global HAB ecology.     

Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5‐chloromethylfluorescein diacetate

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5‐chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat‐killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per‐cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live‐dead classification was essentially error free. But overlap between the frequency distributions of living and heat‐killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat‐killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8–10 of 24 species (i.e., 33%–42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone.     

Transport of the harmful bloom alga Aureococcus anophagefferens by oceangoing ships and coastal boats

It is well established that cyst-forming phytoplankton species are transported in ships' ballast tanks. However, there is increasing evidence that other phytoplankton species which do not encyst are also capable of surviving ballast transit. These species have alternative modes of nutrition (hetero- or mixotrophy) and/or are able to survive long-term darkness. In our studies of no-ballast-on-board vessels arriving in the Great Lakes, we tested for the presence of the harmful algal bloom species Aureococcus anophagefferens (brown tide) in residual (i.e., unpumpable) ballast water using methods based on the PCR. During 2001, the brown tide organism was detected in 7 of 18 ballast water tanks in commercial ships following transit from foreign ports. Furthermore, it was detected after 10 days of ballast tank confinement during a vessel transit in the Great Lakes, a significant result given the large disparity between the salinity tolerance for active growth of Aureococcus (>22 ppt) and the low salinity of the residual ballast water (approximately 2 ppt). We also investigated the potential for smaller, recreational vessels to transport and distribute Aureococcus. During the summer of 2002, 11 trailered boats from the inland bays of Delaware and coastal bays of Maryland were sampled. Brown tide was detected in the bilge water in the bottoms of eight boats, as well as in one live-well sample. Commercial ships and small recreational boats are therefore implicated as potential vectors for long-distance transport and local-scale dispersal of Aureococcus.     

Inactivation of Amphidinium sp. in ballast waters using UV/Ag-TiO2+O3 advanced oxidation treatment

Ballast water poses a biological threat to the world’s waterways by transferring aquatic species from one body of water to another. This study investigates the use of combined ultraviolet (UV)/Ag-TiO₂ + ozone (O₃) processes for treating ballast water using Amphidinium sp. as an indicator microorganism. Sufficient Amphidinium sp. cells in ballast waters can be inactivated using O₃ alone, UV irradiation alone (with or without an Ag-TiO₂ coating), and combined treatments. For the low inactivation ratio (<40%) regime, the effects of ozonation and photocatalysis were observed to be cumulative. The combined UV/Ag-TiO₂ + O₃ treatment produced excess hydroxyl radicals and total residual oxidants (TROs), and readily damaged cell membranes to release intracellular substances. The comparison tests revealed that the combined treatments synergistically inactivate Escherichia coli in ballast waters. However, the combined process did not synergistically inactivate Amphidinium sp. cells. Inactivating different aqua species in ballast waters needs distinct treatment methods and dosages.     

Transport of the Harmful Bloom Alga Aureococcus anophagefferens by Oceangoing Ships and Coastal Boats

It is well established that cyst-forming phytoplankton species are transported in ships' ballast tanks. However, there is increasing evidence that other phytoplankton species which do not encyst are also capable of surviving ballast transit. These species have alternative modes of nutrition (hetero- or mixotrophy) and/or are able to survive long-term darkness. In our studies of no-ballast-on-board vessels arriving in the Great Lakes, we tested for the presence of the harmful algal bloom species Aureococcus anophagefferens (brown tide) in residual (i.e., unpumpable) ballast water using methods based on the PCR. During 2001, the brown tide organism was detected in 7 of 18 ballast water tanks in commercial ships following transit from foreign ports. Furthermore, it was detected after 10 days of ballast tank confinement during a vessel transit in the Great Lakes, a significant result given the large disparity between the salinity tolerance for active growth of Aureococcus (>22 ppt) and the low salinity of the residual ballast water (~2 ppt). We also investigated the potential for smaller, recreational vessels to transport and distribute Aureococcus. During the summer of 2002, 11 trailered boats from the inland bays of Delaware and coastal bays of Maryland were sampled. Brown tide was detected in the bilge water in the bottoms of eight boats, as well as in one live-well sample. Commercial ships and small recreational boats are therefore implicated as potential vectors for long-distance transport and local-scale dispersal of Aureococcus.     

Coastal and Port Environments: International Legal and Policy Responses to Reduce Ballast Water Introductions of Potentially Invasive Species

Marine transportation moves the vast majority of cargo volume in international trade. Ballast water loaded in one port to stabilize ships for a safe voyage contains local aquatic species that are later discharged into other ports, where they are potentially invasive and can cause ecological, socioeconomic, and human health consequences. This article discusses the new Global Ballast Water Convention, what the Convention suggests about the International Maritime Organization (IMO), and its relationship to the United Nations Convention on the Law of the Sea. The article also considers implementation of the Ballast Water Convention by presenting a decision support model that allows regulators to explore tradeoffs between costs and benefits of new technologies and derive optimal reductions of ballast-water-borne biological pollutants.     

Species Richness and Invasion Vectors: Sampling Techniques and Biases

During a European Union Concerted Action study on species introductions, an intercalibration workshop on ship ballast water sampling techniques considered various phytoplankton and zooplankton sampling methods. For the first time, all the techniques presently in use worldwide were compared using a plankton tower as a model ballast tank spiked with the brine shrimp and oyster larvae while phytoplankton samples were taken simultaneously in the field (Helgoland Harbour, Germany). Three cone-shaped and 11 non-cone shaped plankton nets of different sizes and designs were employed. Net lengths varied from 50 to 300 cm, diameters 9.7–50 cm, and mesh sizes 10–100 μm. Three pumps, a Ruttner sampler, and a bucket previously used in ballast water sampling studies were also compared. This first assessment indicates that for sampling ballast water a wide range of techniques may be needed. Each method showed different results in efficiency and it is unlikely that any of the methods will sample all taxa. Although several methods proved to be valid elements of a hypothetical `tool box' of effective ship sampling techniques. The Ruttner water sampler and the pump P30 provide suitable means for the quantitative phytoplankton sampling, whereas other pumps prevailed during the qualitative trial. Pump P15 and cone-shaped nets were the best methods used for quantitative zooplankton sampling. It is recommended that a further exercise involving a wider range of taxa be examined in a larger series of mesocosms in conjunction with promising treatment measures for managing ballast water. This revised version was published online in July 2006 with corrections to the Cover Date.     

Flow cytometry: instrumentation and application in phytoplankton research

In flow cytometry, light scattering and fluorescence of individual particles in suspension is measured at high speed. When applied to planktonic particles, the light scattering and (auto-)fluorescence properties of algal cells can be used for cell identification and counting. Analysis of the wide size spectrum of phytoplankton species, generally present in eutrophic inland and coastal waters, requires flow cytometers specially designed for this purpose. This paper compares the performance in phytoplankton research of a commercial flow cytometer to a purpose built instrument. It reports on the identification of phytoplankton and indicates an area where flow cytometry may supersede more conventional techniques: the analysis of morphological and physiological characteristics of subpopulations in phytoplankton samples.     

A Spatial Modeling Approach to Predicting the Secondary Spread of Invasive Species Due to Ballast Water Discharge

Ballast water in ships is an important contributor to the secondary spread of invasive species in the Laurentian Great Lakes. Here, we use a model previously created to determine the role ballast water management has played in the secondary spread of viral hemorrhagic septicemia virus (VHSV) to identify the future spread of one current and two potential invasive species in the Great Lakes, the Eurasian Ruffe (Gymnocephalus cernuus), killer shrimp (Dikerogammarus villosus), and golden mussel (Limnoperna fortunei), respectively. Model predictions for Eurasian Ruffe have been used to direct surveillance efforts within the Great Lakes and DNA evidence of ruffe presence was recently reported from one of three high risk port localities identified by our model. Predictions made for killer shrimp and golden mussel suggest that these two species have the potential to become rapidly widespread if introduced to the Great Lakes, reinforcing the need for proactive ballast water management. The model used here is flexible enough to be applied to any species capable of being spread by ballast water in marine or freshwater ecosystems.     

Relative risk assessment for ballast-mediated invasions at Canadian Arctic ports

Vector-based risk assessment is a powerful and efficient management approach for nonindigenous species (NIS). By managing a vector, an entire assemblage of associated NIS is simultaneously considered. The majority of current risk assessment frameworks have been conducted for a single, or selected few, target species and thus are not useful for managing vectors transporting a large number of potentially unknown species. Here we develop a predictive framework to assess relative invasion risk for a vector (ballast water) transporting an unknown species assemblage, using the Canadian Arctic as a case study. Ballast water discharge is a known high-risk vector globally, but its magnitude in the Arctic has not been well characterized. Our framework determined relative invasion risks between different transit pathways by quantifying the probability of NIS successfully transiting all stages of the invasion process and the magnitude of consequences of introduction to those ports. Churchill, Manitoba was ranked at ‘higher’ invasion risk via ballast water discharged by international merchant vessels than any other recipient port studied. The overall pattern of ballast water discharge suggests that water originating from coastal domestic sources carried by international merchant vessels may be important for dispersal of NIS. In addition, ballast-mediated NIS are more likely to arrive to the Hudson Bay region during summer months. These results can be useful for developing prevention and early detection programs for the region. Our risk assessment framework is not limited to ballast water and could be applied to other vectors for effective management of NIS.     

Holoplankton of ship ballast water in the Port of Vladivostok

Ballast water samples were taken from three ships that arrived at the Port of Vladivostok (Peter the Great Bay, Sea of Japan) from ports of Japan (Sea of Japan and Pacific Ocean) and China (Yellow Sea and Yangtze River). The holoplankton in samples was presented by seven taxonomic groups, among which copepods (subclass Copepoda, 33 species) and cladocerans (subclass Cladocera, 5 species) dominated. In the samples, eight nonindigenous copepod species were revealed. The information contained in this paper may be of importance for the assessment of the risk of subsequent invasion of new species and the development of techniques for monitoring the qualitative and quantitative structures of ballast water.     

Comparison of techniques used to count single-celled viable phytoplankton

Four methods commonly used to count phytoplankton were evaluated based upon the precision of concentration estimates: Sedgewick Rafter and membrane filter direct counts, flow cytometry, and flow-based imaging cytometry (FlowCAM). Counting methods were all able to estimate the cell concentrations, categorize cells into size classes, and determine cell viability using fluorescent probes. These criteria are essential to determine whether discharged ballast water complies with international standards that limit the concentration of viable planktonic organisms based on size class. Samples containing unknown concentrations of live and UV-inactivated phytoflagellates (Tetraselmis impellucida) were formulated to have low concentrations (<100?mL^?1) of viable phytoplankton. All count methods used chlorophyll a fluorescence to detect cells and SYTOX fluorescence to detect nonviable cells. With the exception of one sample, the methods generated live and nonviable cell counts that were significantly different from each other, although estimates were generally within 100% of the ensemble mean of all subsamples from all methods. Overall, percent coefficient of variation (CV) among sample replicates was lowest in membrane filtration sample replicates, and CVs for all four counting methods were usually lower than 30% (although instances of ~60% were observed). Since all four methods were generally appropriate for monitoring discharged ballast water, ancillary considerations (e.g., ease of analysis, sample processing rate, sample size, etc.) become critical factors for choosing the optimal phytoplankton counting method.     

Phytoplankton and bacterial assemblages in ballast water of U.S. military ships as a function of port of origin, voyage time, and ocean exchange practices

We characterized the physical/chemical conditions and the algal and bacterial assemblages in ballast water from 62 ballast tanks aboard 28 ships operated by the U.S. Military Sealift Command and the Maritime Administration, sampled at 9 ports on the U.S. West Coast and 4 ports on the U.S. East Coast. The ballast tank waters had been held for 2–176 days, and 90% of the tanks had undergone ballast exchange with open ocean waters. Phytoplankton abundance was highly variable (grand mean for all tanks, 3.21 × 10⁴ viable cells m⁻³; median, 7.9 × 10³ cells m⁻³) and was unrelated to physical/chemical parameters, except for a positive relationship between centric diatom abundance and nitrate concentration. A total of 100 phytoplankton species were identified from the ballast tanks, including 23 potentially harmful taxa (e.g. Chaetoceros concavicornis, Dinophysis acuminata, Gambierdiscus toxicus, Heterosigma akashiwo, Karlodinium veneficum, Prorocentrum minimum, Pseudo-nitzschia multiseries). Assemblages were dominated by chain-forming diatoms and dinoflagellates, and viable organisms comprised about half of the total cells. Species richness was higher in ballast tanks with coastal water, and in tanks containing Atlantic or Pacific Ocean source waters rather than Indian Ocean water. Total and viable phytoplankton numbers decreased with age of water in the tanks. Diversity also generally decreased with water age, and tanks with ballast water age >33 days did not produce culturable phytoplankton. Abundance was significantly higher in tanks with recently added coastal water than in tanks without coastal sources, but highly variable in waters held less than 30 days. Bacterial abundance was significantly lower in ballast tanks with Atlantic than Pacific Ocean source water, but otherwise was surprisingly consistent among ballast tanks (overall mean across all tanks, 3.13 ± 1.27 × 10¹¹ cells m⁻³; median, 2.79 × 10¹¹ cells m⁻³) and was unrelated to vessel type, exchange status, age of water, environmental conditions measured, or phytoplankton abundance. At least one of four pathogenic eubacteria (Listeria monocytogenes, Escherichia coli, Mycobacterium spp., Pseudomonas aeruginosa) was detected in 48% of the ballast tanks, but toxigenic strains of Vibrio cholerae were not detected. For ships with tanks of similar ballasting history, the largest source of variation in phytoplankton and bacteria abundance was among ships; for ships with tanks of differing ballasting histories, and for all ships/tanks considered collectively, the largest source of variation was within ships. Significant differences in phytoplankton abundance, but not bacterial abundance, sometimes occurred between paired tanks with similar ballasting history; hence, for regulatory purposes phytoplankton abundance cannot be estimated from single tanks only. Most tanks (94%) had adequate records to determine the source locations and age of the ballast water and, as mentioned, 90% had had ballast exchange with open-ocean waters. Although additional data are needed from sediments that can accumulate at the bottom of ballast tanks, the data from this water-column study indicate that in general, U.S. Department of Defense (DoD) ships are well managed to minimize the risk for introduction of harmful microbiota. Nevertheless, abundances of viable phytoplankton with maximum dimension >50 μm exceeded proposed International Maritime Organization standards in 47% of the ballast tanks sampled. The data suggest that further treatment technologies and/or alternative management strategies will be necessary to enable DoD vessels to comply with proposed standards.     

Phytoplankton monitoring by high performance flow cytometry: a successful approach?

BACKGROUND: Regular phytoplankton monitoring in Dutch coastal waters is performed as an indicator of the ecological state of these waters. The monitoring program is focused on temporal and spatial changes of species composition and abundance. Flow cytometry has been introduced to provide additional information, to improve ecosystem understanding, and to increase the efficiency of analysis and reportage. METHODS: Phytoplankton community abundance and composition were routinely determined by flow cytometry and microscopy at six locations in the North Sea over three annual cycles between 2000 and 2003. Supplementary measurements were also made for fluorescence (chlorophyll-a and other pigments) and, in combination with flow cytometric and microscopic data, were used to determine phytoplankton abundance and composition as a function of their size distribution. Real-time imaging of species was also used to identify species on the basis of their flow cytometric optical characteristics. RESULTS: Flow cytometric analysis took 15 min on average. Analysis including data processing, and Web site reportage took less than 1 h. Phytoplankton concentrations (cells/ml), biomass (fluorescence/ml), and concentration of phycoerythrin- or phycocyanin-containing cells (cells/ml) as a function of their algal size were produced every 2 weeks on average. The phytoplankton integrated annual concentration and biomass were used as ecological indicators for overall phytoplankton status. Real-time imaging of cells in flow enabled the identification of dominant species and was applied as an early warning system for Phaeocystis spp. CONCLUSIONS: The reproducibility and count precision due to the large number of observations of the flow cytometric technique provided reliable data for monitoring long-term trends. Flow cytometrically based analyses extended the lower detection limit (<0.5 microm) of analysis beyond the capabilities of other techniques such as the relation between small and larger phytoplankton, the relation between cell counts and biomass as a function of cell size, but also the ability to monitor and report on blooms of harmful algae. A good correlation was found between concentrations (cells/ml) measured by flow cytometry and microscopy. In practice, flow cytometric analysis of a single marine sample took 15 min on average.     

Application of Ion Torrent Sequencing to the Assessment of the Effect of Alkali Ballast Water Treatment on Microbial Community Diversity

The impact of NaOH as a ballast water treatment (BWT) on microbial community diversity was assessed using the 16S rRNA gene based Ion Torrent sequencing with its new 400 base chemistry. Ballast water samples from a Great Lakes ship were collected from the intake and discharge of both control and NaOH (pH 12) treated tanks and were analyzed in duplicates. One set of duplicates was treated with the membrane-impermeable DNA cross-linking reagent propidium mono-azide (PMA) prior to PCR amplification to differentiate between live and dead microorganisms. Ion Torrent sequencing generated nearly 580,000 reads for 31 bar-coded samples and revealed alterations of the microbial community structure in ballast water that had been treated with NaOH. Rarefaction analysis of the Ion Torrent sequencing data showed that BWT using NaOH significantly decreased microbial community diversity relative to control discharge (p<0.001). UniFrac distance based principal coordinate analysis (PCoA) plots and UPGMA tree analysis revealed that NaOH-treated ballast water microbial communities differed from both intake communities and control discharge communities. After NaOH treatment, bacteria from the genus Alishewanella became dominant in the NaOH-treated samples, accounting for <0.5% of the total reads in intake samples but more than 50% of the reads in the treated discharge samples. The only apparent difference in microbial community structure between PMA-processed and non-PMA samples occurred in intake water samples, which exhibited a significantly higher amount of PMA-sensitive cyanobacteria/chloroplast 16S rRNA than their corresponding non-PMA total DNA samples. The community assembly obtained using Ion Torrent sequencing was comparable to that obtained from a subset of samples that were also subjected to 454 pyrosequencing. This study showed the efficacy of alkali ballast water treatment in reducing ballast water microbial diversity and demonstrated the application of new Ion Torrent sequencing techniques to microbial community studies.