The Biological Basis for Ballast Water Performance Standards: âViable

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Policy Analysis

The Biological Basis for Ballast Water Performance Standards: “Viable/Non-Viable” or “Live/Dead”? Ernest R. Blatchley, John Cullen, Brian Petri, Keith Bircher, and Nicholas Welschmeyer Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00341 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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Environmental Science & Technology

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The Biological Basis for Ballast Water Performance Standards:

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“Viable/Non-Viable” or “Live/Dead”?

3 Ernest R. Blatchley III1,*, John J. Cullen2, Brian Petri3, Keith Bircher4, Nicholas Welschmeyer5

4 5 1

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Lyles School of Civil Engineering and Division of Environmental & Ecological Engineering, Purdue

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University, 550 Stadium Mall Drive, West Lafayette, IN 47907 USA 2

Department of Oceanography, Dalhousie University, P.O. Box 15000, Halifax, Nova Scotia B3H 4R2,

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Canada 3

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11 12

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Trojan Technologies, 3020 Gore Road, London, Ontario, N5V 4T7, Canada

Calgon Carbon Corporation, 3000 GSK Drive, Moon Township, PA 15108 USA

Moss Landing Marine Laboratories, 8272 Moss Landing Rd., Moss Landing CA 95039 USA

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*Corresponding Author

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Email: [email protected]; Phone: 1-765-494-0316; Fax: 1-765-494-0395

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ABSTRACT

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The shipping industry is critical to international commerce; however, contemporary shipping

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practices involve uptake and discharge of ballast water, which introduces the potential for transfer of non-

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indigenous, invasive species among geographically distinct habitats.

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regulations for ballast water management have been implemented by the International Maritime

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Organization (IMO) and by regulatory agencies such as the United States Coast Guard (USCG). IMO

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and USCG discharge standards are numerically identical, but involve different definitions of treatment

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endpoints, which are based on fundamentally-different biological assays for quantification of ballast

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water treatment effectiveness. Available assays for quantification of the responses of organisms in the

ACS Paragon Plus Environment

To counteract this hazard,


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10-50 µm size range include vital stains based on fluorescein diacetate (FDA), sometimes used in

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combination with 5-chloromethylﬂuorescein diacetate (CMFDA), observations of motility, and the most

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probable number dilution culture method (MPN). The mechanisms and implications of these assays are

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discussed relative to the Type Approval process, which quantitatively evaluates compliance with ballast

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water discharge standards (BWDSs) under controlled shipboard and land-based tests. For antimicrobial

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processes that accomplish treatment by preventing subsequent replication of the target species, the

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FDA/CMFDA and MPN methods can yield dramatically different results. An important example of a

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treatment process that is affected by the choice of assay is ultraviolet (UV) irradiation. Results of

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laboratory and field experiments have demonstrated UV-based technologies to be effective for

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accomplishing the objectives of ballast water treatment (inactivation of cellular reproduction), when the

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MPN assay is used as the basis for evaluation. The FDA, CMFDA, motility, and MPN methods are

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subject to well recognized sources of error; however, the MPN method is based on a response that is

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consistent with the objectives of ballast water management as well as the mechanism of action of UV-

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based inactivation. Complementary assays are available for use in compliance testing; however, the

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development of relevant indicative tests remains as a research priority. Historical lessons learned from

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applications of vital stains (and other indirect methods) for quantification of microbial responses to UV

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irradiation in other settings also support the use of assays that provide a direct measure of growth and

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reproduction, such as MPN. Collectively, these observations point to the use of MPN assays as the

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standard for Type Testing, especially when UV-based treatment is employed.

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KEY WORDS

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Ballast Water, FDA/CMFDA, MPN, Phytoplankton, Vital Stain, UV.

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INTRODUCTION

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Roughly 90% of global commerce depends on the international shipping industry.1, 2 As such, the

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adoption of safe shipping practices is critical to commercial activity and necessary for protection of the


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health and well-being of those involved in shipping, as well as the environment. Since the adoption of

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steel-hulled ships roughly 120 years ago, uptake and discharge of liquid ballast (i.e., ballast water) has

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evolved as a standard practice.3 Ballast water is taken on-board to counteract the off-loading of cargo.

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Deballasting is then conducted during the subsequent loading operation where cargo is transferred onto

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the ship.

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Ballasting and deballasting practices are necessary to maintain proper balance and trim attributes

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for ships, regardless of the amount of cargo onboard. However, these practices are also known to

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represent a mechanism for transfer of species that are non-indigenous to ports in which deballasting takes

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place.

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consequences. Prominent examples of invasions that are believed to be associated with ballast water

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management practices include the introduction of zebra mussels (Dreissena polymorpha) and Eurasian

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ruffe (Gymnocephalus cernuus) throughout the Laurentian Great Lakes and their tributary rivers.4-8

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Invasions of these organisms were motivating factors behind the development of United States policy

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regarding ballast water management.9 Recent reviews have provided detailed summaries of invasions that

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have occurred globally that are linked to ballast water.9-12 Direct economic impacts due to known

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invasions have been estimated to exceed US$100 billion per year.13, 14

Invasions by non-indigenous species can have devastating environmental and economic

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To address this issue, the International Maritime Organization (IMO) adopted regulations to limit

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the discharge of organisms in ballast water beginning with an interim D-1 standard requiring ships to

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exchange their ballast water in the open seas and eventually requiring all ships to conform to the D-2

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Performance Standard.15 Table 1 provides a summary of the numerical limits that were established by the

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IMO for this purpose. The IMO used the word “viable” to describe numerical limits for two organism

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groups that were defined by minimum dimension (dmin), defining viable organisms as those that have the

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ability to successfully generate new individuals.16 This is consistent with the metrics for microbial

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(bacterial) indicators — quantified in colony forming units, cfu — that are based on conventional

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measures of viability, such as plating, growth after membrane filtration, or most probable number (MPN)

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assays (i.e., cultivation-based assays).



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Table 1. Ballast water discharge Performance Standard, as defined by IMO Convention D-2.15 The IMO

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defines viable organisms as those that have the ability to successfully generate new individuals. Organism Class

Acceptable Concentration

dmin ≥ 50 µm

< 10 viable organisms/m3

10 µm ≤ dmin < 50 µm

< 10 viable organisms/mL Indicator Microbes1

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Vibrio cholerae (serotypes O1 and O139)

< 1 cfu/100 mL

Escherichia coli

< 250 cfu/100 mL

Intestinal Enterococci

< 100 cfu/100 mL

1

cfu = colony forming unit.

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The size class 10 µm ≤ dmin < 50 µm (10-50 µm) comprises predominantly phytoplankton,

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whereas the size class dmin ≥ 50 µm corresponds predominantly to zooplankton. Numerical limits for

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microbes with dmin < 10 µm were restricted to specific indicator microbes to limit distribution of

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waterborne, microbial pathogens that could be attributed to ballast water discharges to coastal waters and

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to reduce the complexity of accurately enumerating total natural bacteria which are typically present at

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concentrations exceeding 105 cells/mL.

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Research and development of ballast water management systems (BWMSs) is driven by the

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stringent Type Approval process of IMO and USCG which demands demonstrated compliance with

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BWDSs (Table 1) under lengthy, controlled shipboard and land-based test scenarios. Successful Type

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Approval of a BWMS for use on all global ship routes includes 1) five consecutive successful

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“shipboard” tests over a mandatory six-month observation period under routine ship operations executed

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at geographically distinct ports and 2) five consecutive successful “land-based” tests in each of three

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salinity types, representing marine, brackish and freshwater salinity types. The land-based test scenario


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includes requirements for high concentrations of “challenge” organisms, particle loads, and dissolved

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organic substances, all of which may challenge the efficacy of a given BWMS.

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completion of the Type Approval process for both shipboard and land-based testing will easily exceed

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one year and the test assays chosen for laboratory analysis should represent the best available technology,

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regardless of assay time-scale and complexity, to properly evaluate results relative to BWDSs.

The successful

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Ballast water treatment systems designed to date generally involve combinations of one or more

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forms of physical separation (e.g., filtration) with one or more active antimicrobial processes. Common

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examples of antimicrobial processes used in ballast water treatment include chlorination, ozonation, metal

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ions, quaternary ammonium compounds, and ultraviolet (UV) irradiation;13, 17, 18 however, chlorine and

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UV-based processes are by far the most commonly-applied processes, being collectively responsible for

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nearly 100% of the market.19, 20 Physical separation processes can efficiently remove zooplankton from

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ballast water, and properly-designed antimicrobial processes can be highly effective in limiting releases of

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viable indicator bacteria, as indicated by decades of successful management of wastewater and drinking

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water. On the other hand, control of organisms in the 10-50 µm size range is less reliable by these

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methods. Therefore, control of phytoplankton is often a limiting factor in the design of ballast water

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treatment systems. As such, the development of systems to meet the Performance Standard for the 10-50

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µm size range can not only govern the overall design of a ballast water treatment system but also suffer

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from test protocols that do not adequately report on the inactivation efficiency for the 10-50 µm group.

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The United States has not acceded to the IMO convention, but were instrumental in securing a

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provision in its regulations to allow member states to unilaterally implement standards that are more

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stringent than those defined by the IMO. Responsibility for regulation of ballast water management

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within the United States is shared between the United States Environmental Protection Agency (EPA) and

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the United States Coast Guard (USCG). The USCG has adopted ballast water discharge standards that

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are numerically identical to IMO D-2; however, in their current form, they regulate the discharge of

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“living” organisms in the size ranges of 10 µm and larger.21 USCG limits for indicator bacteria are

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identical to those of IMO (i.e., based on their ability to reproduce).



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Classification of plankton as “viable” or “living” involves binary decisions about the condition of

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individual organisms within a size class. The biological endpoint(s) and corresponding assay(s) used to

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make these distinctions have important implications with respect to protection of coastal waters against

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potential invaders, and for technologies that may be incorporated as part of ballast water treatment

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systems. Ideally, the assay(s) and endpoint(s) used to make these distinctions should be consistent with

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the mechanism(s) of action for a given antimicrobial process, as well as the intended goal of treatment.

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Differentiation between live/dead and viable/non-viable may be viewed as the distinction

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between “vitality” and “viability”. The term “vitality” refers to evidence of life, identified as intact

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membranes, nucleotide functionality, and metabolic competence.22-24 Motility25 may also be used as a

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positive indicator; however, the distinction between “live” and “dead” in microbes is anything but clear.24,

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indicators of invasive potential.23, 66 In contrast, “viability” refers to the ability of a microbe to increase

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cellular biomass and to reproduce27-29 The detection of reproduction by single cells in a cultivation-based

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assay proves that they are both alive and viable. Confirmed by their demonstrated utility, cultivation-

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based assays thus represent the “gold standard” for a variety of applications today.24

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discharge of “living” organisms can exceed that of “viable” organisms, but not vice-versa. As the USCG

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continued to stand by its live/dead discharge standard,30,

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document to the IMO suggesting that the vitality of organisms in the 10-50 µm size range could be

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effectively determined using two fluorescent probes and observations of motility16,

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consistent with the USCG Required Method based on the ETV Protocol.32

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description of the method, it is noteworthy that the United States explicitly adopted the IMO definition of

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“viable organisms” as those that are able to reproduce, then subsequently acknowledged that the

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methodology they proposed, based on the USCG final rule, “would be appropriate for ballast water

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treatment technologies designed to remove or kill organisms, rather than render living organisms non-

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reproductive.”28, p. 3 This assessment was subsequently confirmed by the IMO.33 It follows that until or

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unless a regulatory administration accepts a method for enumerating reproductive organisms, it will have

Organisms can exhibit one or more signs of life but never reproduce, so vital signs are inconclusive

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Importantly, the

the United States recently submitted a


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— an approach

Deferring for now a

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no suitable test for ballast water treatment technologies, such as UV irradiation, the most efficient of

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which are designed and function to render organisms non-reproductive rather than to kill them outright.

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The primary goal of this paper is to compare the methods that are used to quantify “viable” and

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“living” organisms in ballast water samples, and to examine the implications of these methods for

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protection of the environment and for selection of ballast water treatment technologies. Additionally, this

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review is intended to address the scientific arguments for and against the use of these methods, as well as

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some of the scientific, technical, environmental, and economic implications of these choices.

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findings of this review are then presented in the context of UV-based ballast water treatment processes,

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which are profoundly affected by the endpoint used to define treatment— living vs. viable. Examination

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in the context of UV-based processes is also helpful in evaluating the general implications of test method

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selection for other treatment technologies.

The

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Testing the Efficacy of Ballast Water Management Systems

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Regulations are enforced by requiring the installation of BWMS that have been granted a Type Approval

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Certificate by the responsible Administration. Type approval conforms to highly detailed guidance29, 30

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and requires extensive documentation of the system’s characteristics as well as its efficacy in meeting the

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Performance Standard. Land-based and shipboard tests, conducted by independent laboratories, can

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extend over many months. Costs for these tests are typically on the order of several million USD for

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land-based tests and an additional several million USD for ship-based tests.34 Type approval testing

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requires binary classification of individuals: if an organism is determined to be viable (IMO) or living

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(USCG), it is counted against the discharge standard; otherwise, it is not.

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After type-approved BWMS are installed, they are to be tested — e.g., in port-state inspection

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programs — using relatively quick direct or indirect measurements (referred to as indicative analysis35, 36)

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typically providing an indication of potential non-compliance (gross exceedance) with Regulation D-2.35,

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Type Approval.37To date, the Marine Environment Protection Committee (MEPC) of the IMO has

By instruction, these analyses should result in no more stringent requirements than what is required for



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recognized two methodologies that may be used to enumerate viable organisms in the 10–50 µm size

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range for Type Approval of BWMS.38 One is based on vital stains, the other on the MPN dilution culture

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method; each is complemented with observations of motility.39 These methods yield counts of viable or

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living organisms by detecting one or more signs of life, such as those listed by IMO: structural integrity,

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metabolism, reproduction, motility, or response to stimuli.40

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Vital Stains

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The term “vital stain” refers to a broad range of chemical treatments that have been used to detect

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signs of vitality in organisms or cells using microscopy or automated methods such as flow cytometry.

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Because some vital stains bind to no substrate, the terms “optically detected chemical probe” or

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“fluorescent probe” may be more accurate than “stain,” but to keep this discussion rooted in established

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terminology,25, 27, 41, 42 common use of the term “stain” will be retained.

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A number of staining methods have been proposed to distinguish between living and dead cells,

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including the use of so-called mortal stains to detect nonviable microbes,43 but most of the interest in vital

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stain assays for protists in ballast water has focused on the fluorescent probe fluorescein diacetate

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(FDA),44, 45 sometimes used with 5-chloromethylﬂuorescein diacetate (CMFDA).25 FDA is a fluorescein

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molecule to which two acetate groups are attached. The hydrophobic, lipophilic, and nonfluorescent FDA

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molecule should readily penetrate the cell plasma membrane.45 Once inside the cell, nonspecific esterases

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cleave the acetate groups, leaving the green-fluorescing, hydrophilic fluorescein molecule that will not

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leak, or leak slowly through a healthy membrane. Green fluorescence accumulates and is detectible using

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epifluorescence microscopy32 or flow cytometry.46

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enzymatic activity, or as the basis for classifying cells as live or dead.25, 27, 41 As reported by Steinberg et

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al.,25, 41 the related fluorescent probe, CMFDA, is mildly thiol reactive and has better cellular retention

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than FDA. Encouraged by preliminary results from their laboratory, Steinberg et al., developed the

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combined FDA+CMFDA method on which the USCG-required ETV Protocol is based.32

The FDA assay is thus used as a measure of


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The assumptions of a live/dead assay with vital stains such as FDA or FDA+CMFDA are

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extensions of the principles underlying their mode of action: i) the non-fluorescent chemical probes will

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penetrate all live cells effectively, ii) inside live cells, they will be converted to the fluorescent product,

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and iii) unable to quickly penetrate the membrane of a living cell, the product will accumulate and be

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retained long enough to be detected as a fluorescent or “stained” cell. Stained cells are thus assumed to

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be living because they possess at least two properties of life, as defined by the IMO and others:22,

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structural integrity and metabolic activity.

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assumption that dead cells do not accumulate the fluorescent product, presumably because the stain is not

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taken up or converted, or the fluorescent product quickly leaks out of dead cells. More to the point, vital

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stain assays for ballast water testing assume that all living organisms will be classified as fluorescent and

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all dead organisms will not.

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This categorical live/dead classification demands the

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Underlying principles notwithstanding, since the earliest use of FDA to examine viability in

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phytoplankton, there has been clear evidence of variable or unreliable results, including high inter- and

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intraspecific variability in staining44-49 and the variation of staining intensity with the cell’s growth

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conditions.48, 50, 51 Results for plankton communities from four locations in the United States,25 two from

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Canada and six ballast water samples52 indicated that errors in the Stain-Motility method were

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promisingly low. However, because there is no assurance that all protists in natural samples are living,53,

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comparing the staining of uniformly living vs. demonstrably dead organisms, e.g., from observations on

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actively growing and heat-killed cultures.16 Consistent with recommendations for validating the

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FDA/CMFDA + motility method,16, 55 but employing flow cytometry to make quantitative measurements

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of stain fluorescence, rather than the microscopist’s judgment of stain fluorescence in the ETV Protocol,

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MacIntyre et al.27 found that FDA+CMFDA stains worked as assumed for some species but not for

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others. Applying a criterion of

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