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

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

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  Potential microbial bioinvasions via ships’ ballast water,sediment, and biofilm Lisa A. Drake  a,* , Martina A. Doblin  b , Fred C. Dobbs  c a Department of Science, US Coast Guard Academy, 27 Mohegan Avenue, New London, CT 06320, USA b University of Technology, Sydney, P.O. Box 123, Broadway New South Wales 2007, Australia c Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, 4600 Elkhorn Avenue, Norfolk, VA 23529, USA Abstract 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 mech-anisms 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 theUS East and Gulf coasts. All habitats contained bacteria and viruses. By extrapolating the measured concentration of a microbial metricto the estimated volume of ballast water, biofilm, or residual sediment and water within an average vessel, we calculated the potentialtotal 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 microorganismsmay be transported within ships in a variety of ways. Using temperature tolerance as a measure of survivability and the temperaturedifference between ballast-water samples and the water into which the ballast water was discharged, we estimated 56% of microorganismscould survive in the lower Bay. Extrapolated delivery and survival of microorganisms to the Port of Hampton Roads in lower Chesa-peake Bay shows on the order of 10 20 microorganisms (6.8  ·  10 19 viruses and 3.9  ·  10 18 bacteria cells) are discharged annually to theregion.   2006 Elsevier Ltd. All rights reserved. Keywords:  Aquatic nuisance species; Bacteria; Invasive species; Management; Viruses 1. Why study microorganisms? In the context of invasive species, the reasons for inves-tigating the transfer of aquatic microorganisms, includingviruses, bacteria, protists, and microalgae are threefold:their high densities in natural waters, ability to form restingstages, and potential toxicity or pathogenicity. Aquaticmicroorganisms are orders of magnitude more abundantthan macroorganisms such as copepods and fish: naturallyoccurring bacteria and viruses are found in concentrationson the order of 10 6  –10 11 l  1 (e.g., Ducklow and Shiah,1993; Proctor, 1997; Fuhrman, 1999; Wommack and Col-well, 2000). Given such high densities, microorganisms aretransferred and introduced globally via ships in greaternumbers than any other size class of organisms. Nearly allsuch microorganisms, incidentally, are innocuous tohumans. Instead, the viruses infect naturally occurring bac-terial and phytoplankton hosts, in which they can cause sig-nificantmortality(FuhrmanandNoble, 1996;Suttle,2005).Once released, microorganisms are well poised to beinvasive species. They are small, a size that facilitates theirpassivedispersal. Theyappeartohavesimplerrequirementsfor survival than do metazoans, based upon their ubiquityin the biosphere, including extreme environments (Deming,1997). They predominantly reproduce asexually and grow 0025-326X/$ - see front matter    2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.marpolbul.2006.11.007 * Corresponding author. Tel.: +1 401 789 1461. E-mail address:  lisa.drake1@verizon.net (L.A. Drake). www.elsevier.com/locate/marpolbul Marine Pollution Bulletin 55 (2007) 333–341  rapidly, factors also contributing to their widespread distri-bution. Finally, life cycles of many invertebrate metazoansand unicellular organisms such as bacteria, eukaryotic phy-toplankton (including toxic dinoflagellates), and other pro-tist species, include resting stages (variously called cysts,spores, auxospores, ephippia, or resting eggs according totaxon) capable of surviving prolonged periods of unfavor-able conditions (e.g., Bailey et al., 2003).These resting stages are typically produced at very lowfrequency, if at all, under favorable conditions, and at highfrequency when environmental conditions deteriorate (e.g.,declining nutrient concentrations, shortened photoperiod,or reduced food quality; Blackburn and Parker, 2005). Pro-duction of resting stages ensures long-term viability of thepopulation because they are extremely resistant to adverseconditions, including anoxia, exposure to noxious chemi-cals, freezing, and passage through digestive tracts of fishand waterfowl. Resting eggs of invertebrates and cysts of dinoflagellates are usually negatively buoyant and sinkwhen released or formed. Resting stages may remain viablein sediments in a virtual suspended metabolic state for dec-ades or even centuries (Hairston et al., 1995) and can germi-nate under a combination of favorable light, temperature,and other environmental conditions (e.g., Kremp andAnderson, 2000; Itakura and Yamaguchi, 2001; Figueroaet al., 2006).Pathogenic or toxic aquatic bacteria, viruses, protists,and microalgae can have devastating effects on ecosystemsand economic resources. There are well-studied pathogen– host systems among many aquatic phyla; for example,virusesterminatingalgalblooms(e.g.,MilliganandCosper,1994; Nagasaki et al., 1994; Van Etten et al., 1991; Shortand Suttle, 2002), viruses infecting seals (e.g., Osterhauset al., 1985; Grachev et al., 1989), and protists decimatingseagrass beds (e.g., Muehlstein, 1992; Ralph and Short,2002). Furthermore, the apparent increasing frequencyand distribution of toxic microalgal blooms has receivedmuch attention in the past two decades (see reviews by Hal-legraeff, 1993; CENR, 2000). Given marine pathogens canspreadlocallymuchmorequicklythanterrestrialpathogens(even when instances of obvious human intervention areexcluded, McCallum et al., 2003), and considering the rela-tively fast transport by ships, the threat of global dispersalof aquatic pathogens appears more immediate than thethreat of invasion by other groups of organisms.Thus, by virtue of their abundance, life-history charac-teristics, and potential pathogenicity or toxicity, microor-ganisms possess a great capacity to invade and causedetrimental effects in new environments. This paper willexplore the extent and potential consequences of bacteriaand virus transport within ships. 2. Types of habitats within ships Microorganisms can be found in several locations withina ship—ballast water, residual sediment and water, andbiofilms formed on interior tanks surfaces—each of whichwill be considered separately. Transfer among these habi-tats has not been fully explored (although see Meyeret al., 2000). We do not consider microorganisms withinhull fouling communities in this review.  2.1. Ballast water Ships’ ballast waters are the best investigated of thesehabitats. Although water has been used regularly as ballastsince the 1880s (Carlton, 1985), the transfer of organismsby ballast-water discharge was investigated only sporadi-cally until the late 1980s. The interest in ballast water atthat time stemmed largely from the dramatic ecologicaland economic impacts of introduced species, such as comb jellies ( Mnemiopsis leidyi  ) in the Black Sea and zebra mus-sels ( Dreissena polymorpha ) in the North American GreatLakes (International Maritime Organization, 1999). Muchof the work on ballast invaders has been dedicated tostudying metazoans (Fofonoff et al., 2003), despite the highdensities of naturally occurring microorganisms in aquaticenvironments. To test the hypothesis that vast quantities of bacteria and viruses are carried in ships’ ballast tanks, Ruizet al. (2000) quantified their abundance in ballast water of vessels arriving to Chesapeake Bay from foreign ports.Indeed, the numbers were high: mean abundances of 8.3  ·  10 8 bacteria l  1 and 7.4  ·  10 9 virus-like particles(VLPs) l  1 were documented.  2.2. Sediment and water residuals We now know ships declaring no (pumpable) ballast onboard may also serve as vectors. Concerns about no ballaston board (NOBOB) invasions have risen from a position of relative obscurity a few years ago to one of the chief envi-ronmental concerns in the Great Lakes basin today (e.g.,Grigorovich et al., 2003). The potential for NOBOB-med-iated invasions lies within tanks’ muddy puddles; residualsof sediment and water can contain an assortment of meta-zoans and microorganisms, including resting stages (e.g.,Hallegraeff and Bolch, 1992; Galil and Hu¨lsmann, 1997;Gollasch et al., 1998; Hamer et al., 2000). When NOBOBtanks are later filled with ballast water, the accumulatedsediment (and associated biota) may be resuspended anddischarged immediately or at subsequent ports of call.Sediment accumulation can be appreciable, dependingon elapsed time since the ship was last dry-docked. Forexample, double-bottom ballast tanks of a cargo vesselcontained up to 30 cm of sediment after only two yearsof use (Hamer et al., 2000). While circumstances vary fromship to ship, the unpumpable water that remains in mostvessels, together with any residual sediment, potentiallyharbors nonindigenous organisms. A metastudy of 13European studies recorded 990 species in a combinationof ballast water and sediment samples (Gollasch et al.,2002). Furthermore, Kelly (1993) reported Japanese ships visiting the USA carried viable cysts and spores of nonin-digenous species after 11–15 days’ voyage. Finally, Mac- 334  L.A. Drake et al. / Marine Pollution Bulletin 55 (2007) 333–341  Isaac et al. (2002) modeled NOBOB vessel-mediated inoc-ulation of the Great Lakes using available plankton densi-ties in coastal European waters, ship transit times, species’survival curves, and residual ballast volumes; individualNOBOB vessels potentially discharge >10 7 individuals of rotifers, cladocerans, and copepods, and 10 11 bacteria,depending on conditions inside ballast tanks.In this regard, tank sediments serve as a repository forparticles, living or otherwise, that settle from water withinthe tank. With respect to issues of invasions, therefore, sed-iment and water residuals in a NOBOB tank will contain atemporally integrated assortment of organisms found inthe water columns that overlay it days and weeks earlier,and possibly months and years earlier, in the case of restingstages of organisms.  2.3. Biofilms on ballast-water tank surfaces Aquatic surfaces are colonized to some degree withbiofilms, organic matrices that can contain bacteria, micro-algae, and associated protists, sometimes including patho-genic forms (e.g., Decho, 1990, 2000). Microorganisms inmature biofilms are notoriously resistant to chemical disin-fectants for reasons that have yet to be satisfactorilyexplained (Costerton et al., 1999). The production of exo-polymer secretions, ‘‘slimes’’, by surface-bound bacteria isa well-recognized—but insufficient—mechanism to explainsuchresistancetochemicaltreatment.Confocalmicroscopyand other tools have demonstrated the complicated, heter-ogeneous architecture of thick biofilms (many as thick ashundreds of micrometers, Baier, 1984; Cook et al., 2000).In addition to providing a chemical barrier to potentiallylethalagents,biofilmsmightalsoproviderefugeforbacteriafrom predatory protists (Hu¨lsmann et al., 2000) or promoteinteractions among pathogenic bacteria and protist grazerssuch that evolutionarily successful pathogens survive diges-tion byprotistsorbecome their endosymbionts (Barker andBrown, 1994).For these reasons, it is worthwhile to consider ships’ bal-last-water biofilms, but there are only two such reports.First, biofilm communities formed on multiple types ofarti-ficial surfaces deployed in a ballast tank during a trans-oceanic voyage (Meyer et al., 2000). When the substrataand associated biofilms were removed and submerged inartificial seawater, they seeded secondary biofilms, whichsurvived for years (Meyer et al., 2000). Second, bacteriaand VLPs have been enumerated in biofilms collected fromships arriving to Chesapeake Bay and the Great Lakes.Microbial concentrations were up to ten times greaterthan those in ballast water, and some samples containedpotentially harmful bacteria and microalgae (Drake et al.,2005). 3. Ship-borne microbial transport: case studies Here we consider examples of ship-borne microbialtransport, discharge, and subsequent deleterious effect.First in most such litanies is the introduction of toxic dino-flagellates to Australia (Hallegraeff et al., 1988; Hallegraeff,1998). Upon commencement of woodchip export fromsouthern Tasmania to Japan and South Korea, the previ-ously undetected toxic dinoflagellate  Gymnodinium catena-tum  was likely introduced to Tasmania (McMinn et al.,1997). It is impossible to say ships’ ballast water wasresponsible for the translocation, but the evidence to thateffect is compelling. First, the large, chain-forming dinofla-gellate and associated human poisonings were previouslyunknown in this area prior to the opening of the woodchipmill. Second, radioisotope dating of sediment cores showsthe appearance of the dinoflagellate’s cysts coincident withthe beginning of woodchip export (McMinn et al., 1997).This species is now well established in the Derwent andHuon estuaries, and high concentrations of paralytic shell-fish toxins in shellfish have caused regular closure of aqua-culture farms there.A second example of probable microbial transport byships is the oft-cited discovery of the bacterium  Vibrio chol-erae , agent of human cholera, in ships arriving to ports inthe Gulf of Mexico (McCarthy and Khambaty, 1994). Dur-ing routine monitoring of shellfish and fish in the Gulf, anepidemic-causing strain of   V. cholerae , serotype O139, pre-viously unreported in the Gulf, was detected (DePaolaet al., 1992). At the time, an epidemic caused by  V. cholerae O139 was underway in South America. When ships withlast ports of call in South America were sampled in MobileBay, Alabama, their ballast water contained the epidemicstrain of cholera. Subsequent testing showed it to be indis-tinguishable from the strain found in Gulf fish and shellfish(McCarthy et al., 1992). Although no illnesses werereported in the US from this strain, the incidents illustratethe potential for ships to transport—and, importantly, deli-ver—viable, toxic bacteria.Lastly, there are a growing number of data sets illustrat-ing the  transport  of potentially toxic microorganisms byships. Organized by broad taxonomic groups, they include:bacteria, such as  V. cholerae  (e.g., Knight et al., 1999;Zo et al., 1999; Ruiz et al., 2000) and  Vibrio  spp. (Mimuraet al., 2005); autotrophic protists (e.g., Hallegraeff andBolch, 1992; McCollin et al., 2000; Doblin et al., inpress); and heterotrophic protists (e.g., Galil and Hu¨ls-mann, 1997). 4. Can free-living microorganisms be invaders?Biogeographical considerations Except for spectacular examples provided by highlyinfectious pathogens (see review in McCallum et al.,2004), the invasion biology of aquatic microorganisms isnot as well understood as those of vertebrates, macroinver-tebrates, and macroalgae. In general, many members of thelatter groups have biogeography, i.e., their distribution isspecific to certain geographical areas. If we assume, how-ever, free-living microorganisms have no biogeographyand are global in their distribution, as some researchers L.A. Drake et al. / Marine Pollution Bulletin 55 (2007) 333–341  335  contend (e.g., Finlay, 2002; Finlay and Fenchel, 2004), thenthey cannot be considered to ‘‘invade’’ environments. Thistopic is explored with respect to ship-borne microorgan-isms by Dobbs and Rogerson (2005). We will not reiteratetheir arguments here, but in short, they contend resolutionof the microbial-ubiquity hypothesis is highly relevant toconsiderations of ballast management. 5. Methods to determine numbers of microorganisms,estimate their survival, and calculate the total number of microorganisms delivered 5.1. Relative propagule pressure among habitats Propagule pressure (i.e., the number of individualsintroduced into a given environment) is understood as aprime factor explaining the success of an invasion (seeOcchipinti–Ambrogi in this volume). Furthermore, theconcentration of microorganisms in ballast waters has beenconsidered a proxy for propagule pressure in most studiesand for devising microbiological standards for ballastwater treatment. We know, however, there are microorgan-isms in ballast-tank sediments and biofilms. How mightthese other microbiological repositories figure into treat-ment strategies and regulatory policy? To address thequestion, we calculated the number of microorganismscontained within each ballast-tank habitat (ballast water,biofilm, and residual sediment and water) to create anumerical hierarchy based on potential delivery of micro-organisms. We extrapolated the average concentrationsof microorganisms within ships sampled to their estimatedconcentrations within a so-called ‘‘average’’ vessel. Weconsidered our average vessel to be a bulk carrier, becausemost ballast-tank microbiology has been gleaned fromsuch ships, they contain the largest volume of ballast watercompared to other ship types, and they are highly abun-dant in the world fleet and therefore representative.The dynamics of biofilm detachment from ballast-tankwalls remain uninvestigated, as do the dynamics of residu-als’ resuspension and entrainment into ballast water.Therefore, the calculations below relate to the number of microorganisms within each ballast-tank habitat prior toballast discharge at a receiving port.Between 1996 and 2003, we sampled bulk carriers andcolliers (coal-carrying ships) arriving to the lower andupper Chesapeake Bay from foreign and domestic ports,largely from Western Europe, the Mediterranean region,and the US East and Gulf coasts. Data sources for esti-mates of microbial concentrations are described in Table1. Despite the large number of ballast-water samples col-lected for microbial enumeration ( n  = 31 for VLPs, 53 forbacteria), there was little if any replication for a givenport/season combination, so data were not analyzed bycombination. Likewise, the number of vessels sampled for Table 1Data used to estimate the total number of microorganisms contained within ballast-tank habitats in an ‘‘average’’ bulk carrierParameter Value (SD) Sample size and data source Ballast water Volume 18,484 MT a (11,988)  n  = 10; category 1Bacteria concentration 0.803  ·  10 9 l  1 (1.88)  n  = 53, 2 subsamples; category 2VLP concentration 13.9  ·  10 9 l  1 (15.7)  n  = 31, 2 subsamples; category 2 Residuals: sediment pore water Volume 25 MT  n  = 8; category 3Bacteria concentration 26.3  ·  10 9 l  1 (36.2)  n  = 12, 0–2 subsamples; category 4VLP concentration 1170  ·  10 9 l  1 (1124)  n  = 12, 0 subsamples; category 4 Residuals: overlying water Volume 17 MT  n  = 8; category 3Bacteria concentration 0.439  ·  10 9 l  1 (0.342)  n  = 13, 0–2 subsamples; category 4VLP concentration 62.4  ·  10 9 l  1 (48.7)  n  = 13, 0–2 subsamples; category 4 Biofilm Area covered by biofilm b 4,459 m 2 Pers. com c Thickness of biofilm 250  l m Baier (1984); Meyer et al. (2000)Volume 1.1 MT Calculated using above two variablesBacteria concentration 6.62  ·  10 9 l  1 (8.83)  n  = 3, 2–3 subsamples; category 5VLP concentration 633  ·  10 9 l  1 (869)  n  = 5, 2–3 subsamples; category 6HR—Hampton Roads, which encompasses ship terminals in the lower Chesapeake Bay cities of Norfolk, Newport News, Portsmouth, and Chesapeake,Virginia, USA; category 1—colliers and bulk carriers arriving to HR from foreign and domestic ports in 2001–2003; category 2—colliers arriving fromforeign ports to upper and lower Chesapeake Bay in 1996–2001; category 3—bulk carriers arriving to HR from foreign and domestic ports in 2003–2004;category 4—bulk carriers arriving from foreign and domestic ports to HR in 2003; category 5—colliers and bulk carriers arriving from foreign anddomestic ports to HR and the Great Lakes in 2002 (Drake et al., 2005); category 6—colliers and bulk carriers arriving from foreign and domestic ports toHR and the Great Lakes in 2002–2003 (Drake et al., 2005). VLP—virus-like particle. a 1 MT = 1000 kg = 1000 l of freshwater. b Assumes 10% of the total tank surface area covered by biofilm. c J. Kelly, International Paint Inc.336  L.A. Drake et al. / Marine Pollution Bulletin 55 (2007) 333–341  residuals or biofilms precluded analysis by port/seasoncombination.For a complete description of field and laboratory meth-ods, see Drake et al. (2001, 2005). Briefly, field sampleswere collected as follows: ballast-water samples were col-lected by hand or using a bleached Niskin bottle and dis-pensed into sterile containers. Because residual samplesconsisted of sediment and a layer of overlaying water, bothfractions were collected independently and aseptically intosterile containers. Finally, biofilm samples on ballast tanksurfaces were scraped from an area of known dimensionswith a sterile polystyrene scraper, and the scraper withattached material was placed in a sterile container. In allcases, samples were transported from vessel to laboratoryin a cooler.In the laboratory, bacteria were enumerated using thenucleic acid stain DAPI (4 0 ,6-diamidino-2-phenylindole;Sigma Chemical Company, St. Louis, Missouri; Porterand Feig, 1980) or by flow cytometery using PicoGreen  (Molecular Probes, Inc., Eugene, Oregon; Veldhuis et al.,1997). VLPs were visualized and enumerated with thenucleic acid stains YO-PRO TM -1 (Hennes and Suttle,1995) or SYBR  Green I (Molecular Probes, Inc., Eugene,Oregon; Noble and Fuhrman, 1998). When different meth-ods were used, an intercalibration was conducted to ensuredata from both methods could be pooled for analysis. Sta-tistical analyses were conducted using SPSS for WindowsRelease 11.0.1.Most of the vessels sampled for ballast water had under-gone open ocean exchange prior to sampling (70% of ves-sels sampled for bacteria analysis, 77% of vessels sampledfor VLP analysis). Because there were no significant differ-ences between mean bacteria or VLP concentrations inexchanged vs. unexchanged tanks (Mann–Whitney  U  -test,  p  = 0.69 for bacteria and 0.345 for VLPs), samples fromboth types of vessels were pooled to calculate averagemicrobial concentrations. Biofilm and residual samplesinherently represent an amalgamation of previous ballast-ing operations, so their prior condition as exchanged orunexchanged was not considered in these analyses.To determine the average quantity of ballast water andresidualvolumepresentinabulkcarrier,vesselofficerswereinterviewed. In the case of residual volume, vessel captainsdid not partition unpumpable ballast between sedimentand overlying water. Based on personal observations, themajority of residual volume was sediment, so residualvolume was apportioned as 80% sediment and 20% overly-ing water. The microbial abundance of each fraction wascalculated, and the two were added to determine the totalmicrobial abundance inthe residualvolume.The finalnum-ber underestimates the total residual abundance becausemicroorganisms attached to sediment grains or meiofaunawere not removed and counted. Instead, the microbial com-ponent of sediment residuals was measured by enumeratingmicroorganisms in sediment pore water. Pore water wasanalyzed after it was expressed from sediment samples bycentrifugation at 1000  g   at 4   C for 10 min in a Marathon2100R centrifuge (Fisher Scientific, Hampton, New Hamp-shire). The volume of pore water contained in an averagesediment residual volume was 38% (SD = 14%,  n  = 10 withup to four subsamples per sample).The amount of biofilm on ballast tank walls was deter-mined by multiplying the average surface area of the tanksand the thickness of aquatic biofilms (Table 1). Field sam-pling showed only a small portion of the tanks’ surfaceswere covered by biofilm, so the calculations include a tankarea coverage of 10%. 5.2. Survival success of discharged microorganisms Once organisms are delivered to a new location, theirinvasion success is a function of their ability to surviveand reproduce (Carlton, 1985). With respect to microor-ganisms, little is known in this regard. One proxy of sur-vival used for invertebrates determines the hydrographicmatch between the ballast water and receiving (pier side)waters (e.g., Smith et al., 1999). We used this model andestimated the percentage of vessels arriving to lower Ches-apeake Bay that encounter optimum temperature condi-tions for microorganisms upon ballast-water discharge.Using empirical data from ships arriving to the lowerBay, we calculated temperature differences between ballastwater and receiving waters and then applied assumptionsabout bacteria temperature tolerances. Our discussion islimited to ballast water because the dynamics of sedimentand biofilm discharge are more complex; that is, theamount of sediment and biofilm discharged by a vessel willdepend on vessel type, operations, tank history, and char-acteristics of the tank habitats themselves.Thirty-two commercial ships were boarded in the Portof Hampton Roads, Virginia, which encompasses ship ter-minals in the lower Chesapeake Bay cities of Norfolk,Newport News, Portsmouth, and Chesapeake. The vesselsarrived from foreign and domestic ports, largely from Wes-tern Europe, the Mediterranean region, and the US Eastand Gulf coasts between 1999 and 2003. Most vessels(72%, 23 of 32) had undergone open ocean exchange. Tem-perature was measured in surface water samples of ballasttanks and surface water collected adjacent to the vesselpier.First, we considered the  tolerance  of microorganisms.Assuming bacteria have a temperature tolerance range of 30   C (Madigan et al., 2003) and they inhabit ballast waterat the midpoint of that range, then they can tolerate dis-charge into water ±15   C that of the ballast water. Second,we considered the  optimum  temperature conditions. If bac-teria have a 10   C temperature range for optimum growth(Madigan et al., 2003), and if they inhabit ballast water atthe midpoint of their optimal range, then they will growbest when discharged into receiving water ±5   C that of the ballast water. We have simply and conservativelyassumed virus tolerances and optima reflect those of bacte-ria. Furthermore, our use of the term ‘‘surviving viruses’’ is L.A. Drake et al. / Marine Pollution Bulletin 55 (2007) 333–341  337
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