Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water

There is enormous potential for global transfer of microorganisms, including pathogens, in ships' ballast water. We contend that a major advancement in the study of ballast-water microorganisms in particular, and of aquatic pathogens in general, will be expedited sample analysis, such as provided by the elegant technology of DNA microarrays. In order to use DNA microarrays, however, one must establish the appropriate conditions to bind target sequences in samples to multiple probes on the microarrays. We conducted proof-of-concept experiments to optimize simultaneous detection of multiple microorganisms using polymerase chain reaction (PCR) and Southern hybridization. We chose three target organisms, all potentially found in ballast water: a calicivirus, the bacterium Vibrio cholerae, and the photosynthetic protist Aureococcus anophagefferens. Here, we show simultaneous detection of multiple pathogens is possible, a result supporting the promising future use of microarrays for simultaneous detection of pathogens in ballast water.     

Potential microbial bioinvasions via ships' ballast water, sediment, and biofilm

A prominent vector of aquatic invasive species to coastal regions is the discharge of water, sediments, and biofilm from ships' ballast-water tanks. During eight years of studying ships arriving to the lower Chesapeake Bay, we developed an understanding of the mechanisms by which invasive microorganisms might arrive to the region via ships. Within a given ship, habitats included ballast water, unpumpable water and sediment (collectively known as residuals), and biofilms formed on internal surfaces of ballast-water tanks. We sampled 69 vessels arriving from foreign and domestic ports, largely from Western Europe, the Mediterranean region, and the US East and Gulf coasts. All habitats contained bacteria and viruses. By extrapolating the measured concentration of a microbial metric to the estimated volume of ballast water, biofilm, or residual sediment and water within an average vessel, we calculated the potential total number of microorganisms contained by each habitat, thus creating a hierarchy of risk of delivery. The estimated concentration of microorganisms was greatest in ballast water>sediment and water residuals>biofilms. From these results, it is clear microorganisms may be transported within ships in a variety of ways. Using temperature tolerance as a measure of survivability and the temperature difference between ballast-water samples and the water into which the ballast water was discharged, we estimated 56% of microorganisms could survive in the lower Bay. Extrapolated delivery and survival of microorganisms to the Port of Hampton Roads in lower Chesapeake Bay shows on the order of 10(20) microorganisms (6.8 x 10(19) viruses and 3.9 x 10(18) bacteria cells) are discharged annually to the region.     

Setting a size-exclusion limit to remove toxic dinoflagellate cysts from ships' ballast water

Dinoflagellate cysts are well-recognized biological constituents of ships' ballast tanks. They are present in ballast water, sediments and residual water in drained tanks, and in biofilms formed on interior tank surfaces. Therefore, cysts have the potential to be released during ballast discharge. The International Maritime Organization's (IMO) Ballast Water Management Convention (promulgated February 2004) stipulates a performance standard (Annex, Regulation D2) requiring discharged ballast water contain <10 viable organisms between 10 and 50 microm per ml and <10 viable organisms 50 microm per m3. The proposed size limit has potential to exclude both the smallest toxic and the largest toxic and non-toxic dinoflagellate (and other microalgal) cysts from discharged ballast water. Despite the appropriateness of size cutoffs however, ballast water containing predominantly small cysts (<50 microm) could be deemed in compliance with the performance standard, even without treatment, while ballast water having the same concentration of larger cysts (>50 microm) could require a multiple-log reduction in abundance before its permissible discharge. Also of concern, it remains uncertain whether ballast-water treatment can remove sufficient organisms, including dinoflagellate cysts, to meet the performance standard.     

Bacteriological examination of ballast water in Singapore Harbour by flow cytometry with FISH

In this study the concentrations of total bacteria, enterobacteria, Vibrio spp., and E. coli have been compared for ballast water samples taken from ships in Singapore Harbour. The cell concentrations were enumerated using FISH and flow cytometry. The data were highly variable, reflecting the many influences upon ballast water as it is utilized in the shipping industry. The concentration of bacterial species was determined as a proportion of the total concentration of cells for the ballast water sampled. For the ballast water sampled these concentrations were 0.67-39.55% for eubacteria, 0-2.46% for enterobacteria, 0.18-35.82% for Vibrio spp., and 0-2.46% for E. coli. Using FISH and flow cytometry, an informative determination of the bacterial hazards of ship ballast water can be made.     

Mesocosm experiments for evaluating the biological efficacy of ozone treatment of marine ballast water

Ballast water is a major pathway for the transfer of non-indigenous species in aquatic environments. The objectives of this study were to determine the ability of ozone to reduce the numbers of a spectrum of marine organisms collected from Puget Sound, Washington in replicated mesocosm (280 l) experiments, and estimate the minimum ozone concentrations as measured by total residual oxidant (TRO) required to reduce organism densities. Ozone treatment was effective in removing bacteria, phytoplankton, and mesozooplankton with initial TRO concentrations of 2–5 mg l⁻¹ as Br₂. Persistence of TRO resulted in an extended period of toxicity and cumulative mortality. TRO decay allowed bacteria populations to multiply when TRO levels fell below 0.5–1.0 mg l⁻¹ as Br₂. Phytoplankton chlorophyll a concentrations were rapidly reduced by ozone treatment and did not increase in any treatments or controls because of lack of light. Overall mesozooplankton viability was rapidly reduced by 90–99% in treatment TRO levels above 1.85 mg l⁻¹ as Br₂. Our study outlines novel protocols that can be used for testing different potential ballast water treatment systems in replicated and controlled mesocosm experiments.     

The effects of freshwater flushing on marine heterotrophic protists--implications for ballast water management.

Survivorship of ballast-entrained marine heterotrophic protists was examined following freshwater flushing. The recovered taxa, including typical marine rhizopods such as Platyamoeba murchelanoi, Labyrinthula spp, Pontifex maximus, Thecamoeba orbis, and the ciliate Condylostoma arenarium, were reared in waters of various salinities. After 2 months, the original salinity subsample retained five protist taxa, the freshwater six, including the amoeba Cochliopodium bilimbosum, the brackish water 22 taxa, and the seawater 19 taxa. Since protists form a major component of marine microbial food webs, their survival may be instrumental in supporting complex ballast-entrained food webs. Our study raises questions as to the reliability of open-ocean exchange (OOE) or freshwater flushing as effective control measures.     

Macroalgal survival in ballast water tanks

Despite a large amount of research into invasive species and their introductions, there have been no studies focused on macroalgal transport in ballast water. To address this, we collected replicate samples of ballast water from 12 ships in two Mediterranean harbours (Naples and Salerno). Filtered samples were kept in culture for a month at Mediterranean mean conditions (18 degrees C, 12:12h LD, 60micromol photons m(-2)s(-1)). Fifteen macroalgal taxa were cultured and differed according to the geographic origin of the ballast water. Most of the cultured algae were widely distributed species (e.g. Ulva spp. and Acinetospora-phase). However, Ulva ohnoi Hiraoka and Shimada, described from Japan, was hitherto unknown in the Mediterranean Sea. We show for the first time that ballast water can be an important vector for the transport of microscopic stages of macroalgae and that this can be a vector for the introduction of alien species.     

Challenges in global ballast water management

Ballast water management is a complex issue raising the challenge of merging international regulations, ship's specific configurations along with ecological conservation. This complexity is illustrated in this paper by considering ballast water volume, discharge frequency, ship safety and operational issues aligned with regional characteristics to address ecological risk for selected routes. A re-estimation of ballast water volumes gives a global annual level of 3500 Mton. Global ballast water volume discharged into open sea originating from ballast water exchange operations is estimated to approximately 2800 Mton. Risk based decision support systems coupled to databases for different ports and invasive species characteristics and distributions can allow for differentiated treatment levels while maintaining low risk levels. On certain routes, the risk is estimated to be unacceptable and some kind of ballast water treatment or management should be applied.     

The effects of prolonged darkness on temperate and tropical marine phytoplankton, and their implications for ballast water risk management

Phytoplankton assemblages from tropical (Goa) and temperate (UK) locations were exposed to a 28 day dark period, followed by a period of re-exposure to light. During this time phytoplankton survival and changes in nutrient concentrations were mapped. The tropical plankton water samples showed high nutrient levels after the dark period which were utilised by cells during the re-exposure period. UK experiments looked at the effect of three different water types on population recovery after the 28 day dark period, and differences due to seasonal effects.The population growth observed during the re-exposure period in the tropical population was comparable to that of the temperate population. Water type affected recovery and of the three tested media fresh seawater promoted the highest levels of growth. Seasonality had a significant influence on species survival. Understanding the effects of all these factors can aid the development of effective risk assessments in ballast water management.     

Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand

Research to understand and remediate coastal pollution is moving toward a multitiered approach in which traditional enumeration of fecal indicators is accompanied by molecular analysis of a variety of targets. Technology that rapidly detects multiple microbial contaminants would benefit from such an approach. The Luminex 100 system is a suspension array that assays multiple analytes rapidly in a single well of a microtiter plate. The ability of the system to simultaneously detect multiple fecal indicating bacteria in environmental samples was tested. Primer/probe sets were designed to simultaneously detect the following fecal indicators: the Bacteroides fragilis group, Enterococcus spp., Escherichia coli and Shigella spp., Bacteroides distasonis, and Ent. faecalis. Specificity and sensitivity of the Luminex probes was tested against laboratory cultures. In addition, sequencing, culture plate testing, and specificity testing with environmental isolates were steps taken to validate the function of the assay with environmental samples. Luminex response to cultures and to environmental samples was consistent with sequencing results, suggesting that the technology has the potential to simultaneously detect multiple targets for coastal water quality applications, particularly as progress is made to efficiently extract DNA from water and sediment matrices.     

Continuous flushing of contaminants from ballast water tanks

The transport of non-indigenous species (NIS) with ship ballast water is a major environmental problem. The International Maritime Organisation (IMO) have recommended that ballast tanks are flushed through with sea water to remove NIS contaminants. The flushing efficiency is studied using mathematical models and a scaled experimental model of a ballast tank. The density contrast between the ballast water and water used for flushing is important when the Froude number Fr(w)=U(w)/sqr rt|g(')|H < 1 (defined in terms of average horizontal flow U(w), reduced buoyancy g', and H the vertical dimension in the tank). When denser water is used to flush a ballast tank, from below, it efficiently displaces lighter ballast water; but flushing through with light water creates a buoyant gravity current which effectively short circuits part of the tank. When Fr(w)>1, the density contrast between the ballast water and water used for flushing is not important and flushing is controlled by a bulk Péclet number, Pe(w). For Pe(w)<1 perfect mixing occurs, while for Pe(w)>1 displacement flushing occurs. Laboratory experiments of flushing were performed using a model two-dimensional ballast tank employing dye attenuation to measure the whole concentration field and these experiments confirm the essential features of the mathematical models. The results of this study are discussed in the context of current IMO flushing protocols.     

Shipboard trials of an ozone-based ballast water treatment system

Legislation introduced by the United Nations International Maritime Organization (IMO) has focused primarily on standards defining successful treatments designed to remove invasive species entrained in ballast water. An earlier shipboard study found that ozone introduced into salt water ballast resulted in the formation of bromine compounds, measured as total residual oxidants (TRO) that were toxic to both bacteria and plankton. However, the diffuser system employed to deliver ozone to the ballast water tanks resulted in patchiness in TRO distribution and toxicity to entrained organisms. In this follow-up study, the shipboard diffuser system was replaced by a single Venturi-type injection system designed to deliver a more homogeneous biocide distribution. Within-tank variability in TRO levels and associated toxicity to zooplankton, phytoplankton and marine bacteria was measured via a matrix of tubes deployed to sample different locations in treated and untreated (control) tanks. Three trials were conducted aboard the oil tanker S/T Prince William Sound in the Strait of Juan de Fuca off Port Angeles, Washington State, USA, between June and December 2007. Mortalities of plankton and bacteria and oxidant concentrations were recorded for treated and untreated ballast water up to 3 days following treatment, and residual toxicity beyond this period was measured by bioassay of standard test organisms. Results indicated uniform compliance with current IMO standards, but only partial compliance with other existing and pending ballast water legislation.     

Ship board testing of a deoxygenation ballast water treatment

A ship board trial of a deoxygenation method for treating ballast water was carried out during a voyage from Southampton (United Kingdom) to Manzanillo (Panama). A nutrient solution added to two ballast tanks encouraged bacterial growth, resulting in a gradual change to an anoxic environment. Samples were taken from two treated tanks and two untreated tanks to assess changes in the abundance and viability of zooplankton, phytoplankton and bacteria. The work was carried out before the International Maritime Organization (IMO) standard was agreed so only a broad indication of whether the results achieved the standard was given. For the zooplankton, the standard would have been achieved within 5 or 7 days but the phytoplankton results were inconclusive. The biological efficacy was the result of the combination of several factors, including the treatment, pump damage and an increase in the water temperature during the voyage.     

EU shipping in the dawn of managing the ballast water issue

After almost two decades of intensified research, regulatory and political activities focussed on the prevention of harmful organisms and pathogen transfers around the world in 2004 the International Convention on the Management of Ships’ Ballast Water and Sediments (BWM Convention) was adopted to provide a common and globally uniform ballast water management (BWM) approach. Nevertheless, regionally different BWM approaches are developing. By now, many countries around the world seem to be aware of the ballast water issue and its management limitations. In the EU, different approaches have been identified at regional and national scales. The first voluntary BWM requirements at the regional level have been introduced by the HELCOM and OSPAR countries, Adriatic countries have prepared a common approach considering a new legal framework for implementation, and some national level requirements have also been identified. However, a common EU wide BWM approach has not yet clearly emerged. In this paper the authors review the BWM approaches developing in Europe, and describe the EU response on BWM. The authors further provide recommendations which may be considered when developing BWM measures in the EU. This contribution focuses on the BWM issue in European seas in light of the EU Maritime Policy and EU Marine Strategy. The Caspian Sea was also considered.     

The dilution and dispersion of ballast water discharged into Goderich Harbor

Observations are presented on dilution and dispersion rates of ballast water discharged under normal operational conditions at the semi-enclosed port of Goderich, Ontario. The ballast water was tagged with Rhodamine-WT dye and microscopic magnetically-attractive tracer particles. Maximum concentrations of dye immediately after discharge were diluted to 1-5% of initial ballast tank concentrations, and within 3 days had decreased to less than 0.1% of initial concentrations. Inside the harbor, there was 10-20% of the ballast water still present after 2 days, consistent with a flushing rate of 0.8-1.15 day⁻¹. Magnetic particles were collected up to 7.5 km outside the harbor after one day, consistent with a dilution factor of order 10⁵ outside the harbor. The results of this study are discussed in the context of ballast water discharge standards proposed by the International Maritime Organization to minimize the introduction of aquatic nonindigenous species through ships’ ballast water and sediments.     

Changes in zooplankton abundance and diversity after ballast water exchange in regional seas

A study of the efficiency of ballast water exchange (BWE) in regional seas was carried out to assess this management method in reducing the risk of introducing non-native species. Zooplankton samples were taken before and after exchange on ten voyages where BWE took place (North Sea, English Channel, Irish Sea and Bay of Biscay). Zooplankton abundance was always reduced after exchange, but diversity increased on eight occasions. The greatest changes occurred when the source port water was of low salinity and when exchange took place in deeper waters further from land. However, it was clear that BWE did not remove all the source port taxa and this method is unlikely to provide a consistent and effective method of managing ballast water in regional seas.     

Marine testing board for certification of ballast water treatment technologies

The proposed MTB is a process to expedite the implementation of international standards and regulations, and the subsequent testing, certification, and regulatory approval of new ballast water treatment ("control") technologies. This would expedite their acceptance in the global marketplace and reduce risks of shipowners following international regulation. The cost to test and evaluate and certify new ballast water treatment technologies for the global marketplace has been estimated to be less than US $1 per day per ship. It is time for the shipping industry, national regulatory bodies, and IMO to endorse the concept of the MTB and for the shipping industry to support a proactive cost-saving solution for sustainable shipping and protecting the environment from unwanted invasions of aquatic species with their potential negative impacts.     

Biological controls on bacterial populations in ballast water during ocean transit

Bacteria (and viruses) numerically dominate ballast water communities, but what controls their population dynamics during transit is largely unexplored. Here, bacterial abundance, net and intrinsic growth rates, and grazing mortality were determined during a trans-Atlantic voyage. The effects of grazing pressure by microzooplankton on heterotrophic bacteria during transit were determined for source port, mid-ocean exchange (MOE), and six-day-old source port ballast water. When the grazer component was removed, bacterial abundances significantly increased. Additionally, we determined that the grazer-mediated mortality for ballast water originating from ports was greater than MOE water and that mortality decreased over time for the source port ballast water. This study shows that bacterial populations in transit are controlled by microzooplankton grazing. If these findings are representative of ballast water environments, they suggest that if the grazing component is selectively removed by various treatment methods, bacterial populations may increase; this could have environmental and human health consequences.     

Management and environmental risk study of the physicochemical parameters of ballast water

Shipping is a vital industry for the global economy. Stability of ships, provided by ballast water, is a crucial factor for cargo loading and unloading processes. Ballast water treatment has practical significance in terms of environmental issues, ecosystem, and human health, because ships discharge this water into the environment before loading their cargos. This study reviews the common methods for ballast water management – exchange, heating, filtration, ultrasonic treatment, ultraviolet irradiation, chemicals, and gas supersaturation – to select the best one. This study compares water temperature, salinity, dissolved oxygen, polycyclic aromatic hydrocarbons (PAHs), and heavy metals (Co, Cr, Ni, Pb) for ballast tanks of selected ships with the recipient port environment in the Persian Gulf as a case study. The exchange of ballast water in the ocean and/or its treatment on board to prevent inadvertent effects on the environment's physicochemical conditions is related to vessel characteristics, legislation, and the environmental condition. Ecological risk study showed that the salt content in ballast water is close to that of seawater, but the values of Cr (2.1 mg/l) and Ni (0.029 mg/l) in ballast water are higher than those in seawater (1 and 0.004 mg/l, respectively).     

Real-time PCR and NASBA for rapid and sensitive detection of Vibrio cholerae in ballast water

Transport of ballast water is one major factor in the transmission of aquatic organisms, including pathogenic bacteria. The IMO-guidelines of the Convention for the Control and Management of Ships' Ballast Water and Sediments, states that ships are to discharge <1 CFU per 100 ml ballast water of toxigenic Vibrio cholerae, emphasizing the need to establish test methods. To our knowledge, there are no methods sensitive and rapid enough available for cholera surveillance of ballast water. In this study real-time PCR and NASBA methods have been evaluated to specifically detect 1 CFU/100ml of V. cholerae in ballast water. Ballast water samples spiked with V. cholerae cells were filtered and enriched in alkaline peptone water before PCR or NASBA detection. The entire method, including sample preparation and analysis was performed within 7 h, and has the potential to be used for analysis of ballast water for inspection and enforcement control.