Flow cytometry, microscopy, and DNA analysis as complementary phytoplankton screening methods in ballast water treatment studies

Ballast water is the main vector for marine invasions. To minimize the spread of invasive species, the International Maritime Organization (IMO) has adopted the Ballast Water Management Convention which requires the installation of shipboard ballast water treatment systems (BWTS). During BWTS tests, the phytoplankton abundance and species composition were followed after treatment with both filtration and ultraviolet radiation. Although the installation fulfilled the IMO criteria after a 5-day holding time in a model ballast tank, the ultimate effectiveness of the treatment was further tested in long-term (20 days) incubation experiments under optimal phytoplankton growth conditions. Application of flow cytometry, microscopy, and DNA sequencing to these incubation samples gave an indication of the phytoplankton species that might be introduced by ballast water discharge—despite treatment. Phytoplankton was reliably quantified using flow cytometry, while fast identification was best done using microscopy. Some groups that contained potentially toxic species could not be identified at species level using microscopy; for these species, identification using genetic techniques was necessary. It is concluded that if long-term incubation experiments are used as an additional tool in testing BWTS effectiveness, a combination of phytoplankton screening methods can be applied depending on the detail of information that is required.     

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.     

Recent mass invasion of the North American Great Lakes by Ponto-Caspian species

The North American Great Lakes have been invaded and dramatically altered by more than 145 alien species. Many invasions have occurred during the past few decades because of the release of Eurasian ballast water from transoceanic ships. Current regulations require ships to exchange foreign ballast with highly saline water before entering the Great Lakes; this procedure should prevent colonization by strictly freshwater species, but species with broad salinity tolerance might survive transport in exchanged water. A recent series of invasions by euryhaline organisms from the Black and Caspian Seas region signals a new phase in the transformation of the Great Lakes - one that supports the concept of an 'invasional meltdown'.     

Relative Invasion Risk for Plankton across Marine and Freshwater Systems: Examining Efficacy of Proposed International Ballast Water Discharge Standards

Understanding the implications of different management strategies is necessary to identify best conservation trajectories for ecosystems exposed to anthropogenic stressors. For example, science-based risk assessments at large scales are needed to understand efficacy of different vector management approaches aimed at preventing biological invasions associated with commercial shipping. We conducted a landscape-scale analysis to examine the relative invasion risk of ballast water discharges among different shipping pathways (e.g., Transoceanic, Coastal or Domestic), ecosystems (e.g., freshwater, brackish and marine), and timescales (annual and per discharge event) under current and future management regimes. The arrival and survival potential of nonindigenous species (NIS) was estimated based on directional shipping networks and their associated propagule pressure, environmental similarity between donor-recipient ecosystems (based on salinity and temperature), and effects of current and future management strategies (i.e., ballast water exchange and treatment to meet proposed international biological discharge standards). Our findings show that current requirements for ballast water exchange effectively reduce invasion risk to freshwater ecosystems but are less protective of marine ecosystems because of greater environmental mismatch between source (oceanic) and recipient (freshwater) ecoregions. Future requirements for ballast water treatment are expected to reduce risk of zooplankton NIS introductions across ecosystem types but are expected to be less effective in reducing risk of phytoplankton NIS. This large-scale risk assessment across heterogeneous ecosystems represents a major step towards understanding the likelihood of invasion in relation to shipping networks, the relative efficacy of different invasion management regimes and seizing opportunities to reduce the ecological and economic implications of biological invasions.     

Viability of invertebrate diapausing eggs exposed to saltwater: implications for Great Lakes’ ship ballast management

International shipping has been the dominant vector of nonindigenous species introductions to the Laurentian Great Lakes over the past century. Apparent ballast-mediated invasions have been recorded in recent years, despite the implementation of voluntary ballast water exchange regulations in 1989. Since unregulated no-ballast-on-board vessels currently dominate inbound traffic to the Great Lakes, it has been proposed that live or dormant organisms contained in residual ballast of these vessels may be partially responsible for recent invasions. Alternatively, euryhaline species may pose a significant invasion threat because they can potentially survive ballast exchange. In this study, we explored whether exposure to open-ocean water (32) reduced the viability of invertebrate diapausing eggs in ballast sediments. Sediments collected from three transoceanic ships and from three freshwater habitats were exposed to open-ocean seawater. Egg viability, assessed as the abundance of taxa hatched between exposed and unexposed sediments, was not affected by saltwater exposure in any experiment. Species richness of hatched diapausing eggs was reduced by saltwater exposure in only one of seven trials. Our results indicate that oligostenohaline zooplankton may pose an invasion risk because their diapausing eggs are largely resistant to exposure to open-ocean saltwater.     

Dynamics and short-term survival of toxic cyanobacteria species in ballast water from NOBOB vessels transiting the Great Lakes—implications for HAB invasions

We measured the presence, viability and potential toxicity of cyanobacteria in ships’ ballast tanks during three domestic voyages through the North American Great Lakes. Using molecular methods, the toxin-producing forms of Microcystis and Anabaena were monitored in ballast water after ships’ ballast tanks were filled at their first port of call, and at subsequent ports as ships transited the Great Lakes. Microcystis was detected in ballast water at intermediate and final ports of call in all three experiments, but the presence of Anabaena was more variable, suggesting low abundance or patchy distribution in ballast tanks. Both species were detected in ballast water up to 11 days old. Detection of the microcystin synthetase gene, mcyE, in ballast tanks indicated entrained cells were capable of producing microcystin, and further analyses of RNA indicated the toxin was being expressed by Microcystis, even after 11 days in dark transit. These data demonstrate within-basin transport and delivery of planktonic harmful algal bloom (HAB) species to distant ports in the world's largest freshwater reservoir, with potential implications for drinking water quality. These implications are discussed with respect to management of microbial invasions and the fate of introduced phytoplankton in their receiving environment.     

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 response of zooplankton and phytoplankton from the North American Great Lakes to filtration

Filtration of ballast water was investigated as a means of minimizing the introduction of nonindigenous zooplankton and phytoplankton by ships visiting the North American Great Lakes-St. Lawrence Seaway system (GLSLSS). An automatic backwash screen filtration (ABSF) system with nominal filtration options of 25, 50 or 100 μm was mounted on the deck of an operating Seaway-sized dry bulk carrier, the MV Algonorth. Water was pumped through the ABSF with a deck mounted pump at 341 m³ hr⁻¹ during routine ship operations in the GLSLSS, and effectiveness of the various screen pore sizes at removing taxonomic categories of zooplankton and phytoplankton was measured using matched treatment and control ballast tanks. The smallest pore sizes (25 and 50 μm) performed better than the 100 μm pore size at removing biological material. There was no difference in the filtration efficiency of the 25 and 50 μm screens relative to macro- or microzooplankton in these tests, but this result was probably due to low densities of macrozooplankton, and soft-bodied (aloricate) characteristics of the microzooplankton present. The 25 and 50 μm pore sizes were subjected to more controlled tests on board a stationary barge platform equipped with triplicate 700 L catchment bins moored in Duluth Harbor of Lake Superior. In these tests, filter pore size, organism size and rigidity influenced zooplankton removal efficiency by the ABSF. The 25 μm screen reduced both macrozooplankton and microzooplankton significantly more than the 50 μm screen. Zooplankton width was more determinative of filtration performance than length, and both filters removed loricate species of rotifers significantly more efficiently than aloricate species of the same length and width size classes. The 25 and 50 μm ABSF also significantly reduced algal densities, with the exception of colonial and filamentous green algae (50 μm only). Filter efficiency relative to algal particles was influenced by filter pore size, organism morphology and structure, and intake density, while algal particle size was not determinative. This research provides compelling evidence that 25 or 50 μm filtration is a potentially powerful means of reducing densities of organisms discharged by ships operating in the Great Lakes but an additional treatment step would be necessary to effectively minimize risk and meet the International Maritime Organization's discharge standards associated with organisms of all sizes in the water column.     

Post-exchange zooplankton in ballast water of ships entering the San Francisco Estuary

The San Francisco Estuary in California (CA), USA, has beenheavily altered by invasions of nonnative zooplankton and benthicorganisms, presumably by the discharge of ships’ ballastwater. Since 2000, ships entering CA have been required to exchangeballast water with oceanic water during the voyage to decreasethe number of organisms discharged into the Estuary that hadpreviously been taken aboard at foreign ports. We examined abundanceof zooplankton in ballast water of 18 container ships and 48bulk carriers. Asia dominated the sources of ballast water,which contained multiple nonnative zooplankton including speciesthat have invaded and since become common residents in the Estuary.The abundance of zooplankton was significantly lower in ballastwater that had been emptied and refilled with oceanic waterthan those that had continuously been flushed with oceanic water(about three times the volume of ballast water), suggestingthat empty–refill is more effective in removing exoticzooplankton.     

Towards minimizing transport of aquatic nuisance species in ballast water: Do organisms in different size classes respond uniformly to biocidal treatment?

To reduce the transport and delivery of aquatic nuisance species in ships’ ballast water and comply with standards for the number of living organisms that may be discharged, biocidal agents and processes, such as chemical dosing, have been repurposed to treat ballast water. We evaluated whether marine planktonic organisms—the typical targets of these biocides—respond in unison to simulated treatment. Organisms were concentrated from seawater, which was amended with dissolved and particulate matter and cultured microalgae, and then treated by chlorination, ultraviolet radiation, or deoxygenation. Living organisms in three size classes (≥50, ≥10 and <50, and <10 µm [represented by culturable, heterotrophic bacteria]) were counted prior to and periodically after treatment. Regardless of whether the differences in concentrations between the control and treatments were significant or insignificant, in general, organisms across the size classes reacted comparably to treatments, with some exceptions in the <10 µm size class. The parallel responses of organisms to treatment—if shown to generalize to other water conditions, assemblages of organisms, and scales of treatment—may justify using a single size class to predict the responses of organisms across the broad size spectra. Notably, because most ballast water management systems employ a filtration step to remove organisms ≥50 µm, if organisms in the ≥10 and <50 µm size class were assessed to determine a vessel’s compliance with the discharge standard, it would be imperative that any filters would be evaluated to ensure they were functioning properly and removed organisms as designed.     

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.     

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.     

Transoceanic ships as vectors for nonindigenous freshwater bryozoans

Aim The transport of organisms in ships’ ballast tanks is a dominant vector for aquatic invasions worldwide. Until recently, efforts to manage this vector have overlooked the potential transport of invertebrate resting stages in the residual waters and sediments within emptied ballast tanks, i.e. NOBOB (‘No Ballast On Board’) tanks. The resting stages (statoblasts) of freshwater bryozoans are often buoyant and locally abundant and thus can be taken up easily during ballasting operations. They are also resistant to extreme environmental conditions and can generate new colonies after being dormant for decades; as such, they would likely remain viable propagules after lengthy transport in ship ballast tanks. This study quantified the occurrence of freshwater bryozoan statoblasts in ballast tank sediments of transoceanic ships. Location North American Great Lakes. Methods We quantified the frequency of occurrence, abundance and diversity of bryozoans (as statoblasts) in residual sediment samples taken from 51 NOBOB tanks of 33 transoceanic ships visiting the Great Lakes from 2000 to 2002. Results Our study identified 11 species, comprising nearly 12% of the total number of freshwater bryozoans known worldwide. These include two exotic species unrecorded in the Great Lakes (Fredericella sultana and Lophopus crystallinus), an exotic species already established in the region (Lophopodella carteri) and three cosmopolitan species (Plumatella casmiana, P. fungosa and P. repens). Our estimates suggest that a ship with NOBOB tanks may carry up to 10⁶ statoblasts. Main conclusions The discovery of species unrecorded in the Great Lakes and the potentially large numbers of statoblasts being transported in ship ballast tanks indicate a significant risk of new species introductions. Furthermore, the presence of cosmopolitan species and an exotic species already established in the Great Lakes suggests the strong possibility of cryptic invasions via the introduction of exotic genotypes.     

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.     

Efficacy of ‘saltwater flushing’ in protecting the Great Lakes from biological invasions by invertebrate eggs in ships’ ballast sediment

1. Mid‐ocean exchange and saltwater flushing were implemented as management practices to reduce the likelihood of new biological invasions in the Laurentian Great Lakes associated with ships’ ballast water and sediments. Despite this, there has been no formal assessment of the efficacy of these procedures. Here, we conduct a comparative analysis of community composition of dormant taxa transported by ballast sediment before and after regulations came into effect in 2006. 2. Ballast sediment samples were collected from 17 ships during the post‐regulation interval of 2007 and 2008. Invertebrate eggs were counted, hatched and species identified in the laboratory. Results were compared to similar samples collected from 39 ships between 2000 and 2002, prior to implementation of saltwater flushing regulations. 3. The estimated amount of residual ballast sediment transported by vessels was significantly lower during the post‐regulation period, ranging from <1 to 45 tonnes per ship, with an average of 5 tonnes. Mean density and number of dormant viable eggs per ship declined 91 and 81%, respectively. 4. Community composition also changed through time, with Rotifera accounting for 78% of taxa transported prior to regulation, whereas Cladocera and Copepoda each accounted for 38% of abundance post‐regulation. Although the number of non‐indigenous species (NIS) declined 73% per ship after 2006, the reduction was not statistically significant; however, the number of freshwater NIS – which pose the greatest risk of invasion for the Great Lakes – was significantly lowered. 5. Our comparative analysis suggests that ballast management regulations enacted in 2006 markedly reduced the probability of introduction of NIS via dormant eggs carried in ballast sediments.     

Invasion Pressure to a Ballast-flooded Estuary and an Assessment of Inoculant Survival

The relationships between invasion pressure, post-transport inoculant survival, and regional susceptibility to invasion are poorly understood. In marine ecosystems, the movement and release of ballast water from ocean-going ships provides a model system by which to examine the interplay among these factors. One of the largest estuaries in North America, the Chesapeake Bay, receives tremendous amounts of foreign ballast water annually and thus should be at high invasion risk. To date, however, few introductions in Chesapeake Bay have been attributed to ballast release. To understand better the dynamics of this invasion process, we (1) characterized and quantified the biota arriving to Chesapeake Bay in foreign ballast water, (2) compared temperatures and salinities of ballast water and harbor water in upper Chesapeake Bay, and (3) tested experimentally survival of organisms collected from ballast water in temperatures and salinities characteristic of the region. From 1993 to 1994, we sampled planktonic and benthic organisms from 60 foreign vessels arriving to Chesapeake Bay. Our data show that the estuary is being inoculated by a diverse assemblage of aquatic organisms from around the world. Furthermore, the short transit time (15d) for most vessels ensured that substantial numbers of larval and post-larval organisms were being deballasted alive. Most of the ballast water discharged into the upper Chesapeake Bay, however, was significantly higher in salinity (>20) than that of the receiving harbor. In laboratory tolerance experiments, ballast water organisms perished under such conditions. Thus, a mismatch in physical conditions between donor and receiver regions may explain the dearth of invasions in the upper Bay. It is likely that the lower Chesapeake Bay, which is more saline, remains at higher risk to ballast water invasion. Recognition of such intraregional differences should allow more focused predictions for monitoring and management.     

Patterns of invasion in the Laurentian Great Lakes in relation to changes in vector activity

The Laurentian Great Lakes basin has been invaded by at least 182 non-indigenous species. A new invader is discovered every 28 weeks, which is the highest rate recorded for a freshwater ecosystem. Over the past century, invasions have occurred in phases linked to changes in the dominant vectors. The number of ship-vectored invaders recorded per decade is correlated with the intensity of vessel traffic within the basin. Ballast water release from ocean vessels is the putative vector for 65% of all invasions recorded since the opening of the St. Lawrence Seaway in 1959. As a preventive measure, ocean vessels have been required since 1993 to exchange their freshwater or estuarine ballast with highly saline ocean water prior to entering the Great Lakes. However, this procedure has not prevented ship-vectored species introductions. Most ships visiting the Great Lakes declare 'no ballast on board' (NOBOB) and are exempt from the regulation, even though they carry residual water that is discharged into the Great Lakes during their activities of off-loading inbound cargo and loading outbound cargo. Recently introduced species consist predominantly of benthic invertebrates with broad salinity tolerance. Such species are most likely to survive in a ballast tank following ballast water exchange, as well as transport in the residual water and tank sediments of NOBOB ships. Thus, the Great Lakes remain at risk of being invaded by dozens of euryhaline invertebrates that have spread into Eurasian ports from whence originates the bulk of foreign ships visiting the basin.     

Life after treatment: detecting living microorganisms following exposure to UV light and chlorine dioxide

Rapid analytical methods are needed to quantify living microorganisms to determine if ships’ discharged ballast water is in compliance with national and international standards. Traditionally, regrowth assays and microscope counts of stained organisms—which are time-consuming, require expensive equipment, and require extensive staff training—are used to assess microorganisms. The goal of this study was to evaluate other approaches. Both ambient microorganisms from an oligotrophic marine environment and laboratory cultures of marine algae were evaluated following exposure to two types of ballast water treatment: ultraviolet (UV) light and chlorine dioxide (ClO₂). Microorganisms in two size classes (<10 and ≥10 to <50 μm) were quantified using regrowth assays and vital staining, and samples were evaluated using two rapid approaches: (1) chlorophyll a fluorescence and photochemical yield were measured using a pulse amplitude modulated fluorometer and (2) the concentration of adenosine triphosphate (ATP) was measured with a handheld luminometer. The response of microorganisms to UV and ClO₂ was evident in measurements of photochemical yield, as photochemical yield decreased at high doses. However, initial values of photochemical yield were variable and species-specific. Oddly, in some trials, initial fluorescence increased at intermediate UV doses; this phenomenon could lead to overestimation of total biomass. In samples treated with UV light, ATP was not significantly different among any of the doses used; however, concentrations of ATP were significantly lower at the highest dose of ClO₂ than control samples. These results demonstrate that approaches used for ballast water testing can be treatment-specific, and compliance approaches should be validated to determine their utility with the appropriate treatments.     

Supply-side invasion ecology: characterizing propagule pressure in coastal ecosystems

The observed rates and deleterious impacts of biological invasions have caused significant alarm in recent years, driving efforts to reduce the risk (establishment) of new introductions. Characterizing the supply of propagules is key to understanding invasion risk and developing effective management strategies. In coastal ecosystems, ships' ballast water is an important transfer mechanism (vector) for marine and freshwater species. Commercial ships exhibit a high degree of variation in ballast water operations that affect both the quantity and quality of propagule supply, and thereby invasion risk. The per-ship inoculation size from ballast water depends upon both the volume discharged and the organism density. Moreover, propagule quality will vary among source regions (ports) and voyage routes, due to differences in species composition and transport conditions, respectively. We show that significant differences exist in (i) the frequency and volume of ballast water discharge among vessel types, (ii) the frequency of vessel types and routes (source regions) among recipient ports, and (iii) the transit success (survivorship) of zooplankton in ballast tanks among voyage routes. Thus, propagule supply is not a simple function of total ship arrivals. For ships, as well as other vectors, variation in propagule quantity and quality must be explicitly considered to estimate invasion risk and advance predictive ability.     

Verifying Compliance with Ballast Water Discharge Regulations

U.S. and international rules have been proposed to reduce the risks associated with invasive aquatic organisms by requiring that ships’ ballast water be treated to kill or remove living organisms and achieve certain standards before being discharged. Enforcing these rules requires verifying when a discharge violates these standards. A preliminary comparison of verification systems indicates that mandatory reporting and inspecting treatment equipment do not provide an acceptable level of confidence and that sampling and analyzing enough ballast water to achieve acceptable confidence is prohibitively costly. The most cost-effective alternative that achieves an acceptable level of confidence involves indirect measures of ballast water using sensors that indicate whether discharge standards are met.