On the use of the serial dilution culture method to enumerate viable phytoplankton in natural communities of plankton subjected to ballast water treatment

Abstract

Discharge standards for ballast water treatment (BWT) systems are based on concentrations of living cells, for example, as determined with vital stains. Ultraviolet radiation (UV) stops the reproduction of microorganisms without killing them outright; they are living, but not viable, and ecologically as good as dead. Consequently, UV-treated discharge can be compliant with the intent of regulation while failing a live/dead test. An alternative evaluation of BWT can be proposed based on the assessment of viable, rather than living, cells in discharge water. In principle, the serial dilution culture-most probable number (SDC-MPN) method provides the appropriate measure for phytoplankton. But, the method has been criticized, particularly because it is thought that many phytoplankton species cannot be cultured. A review of the literature shows that although SDC-MPN has been used for more than 50 years—generally to identify and count phytoplankton species that cannot be preserved—its application to enumerate total viable phytoplankton seems to be new, putting past criticisms of the method in a different light. Importantly, viable cells need to grow only enough to be detected, not to be brought into sustained culture, and competition between species in a dilution tube is irrelevant as long as the winner is detectable. Thorough consideration of sources of error leads to recommendations for minimizing and quantifying uncertainties by optimizing growth conditions and conducting systematic comparisons. We conclude that with careful evaluation, SDC-MPN is potentially an effective method for assessing the viability of phytoplankton after BWT.

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

“…False negatives are cells that have been classified as dead that are in fact alive. As discussed by Cullen and MacIntyre () in the context of compliance with ballast water regulations, false negatives are potential propagules that are undetected. These may lead to regulatory acceptance of systems that exceed discharge limits for living cells, rendering the regulations less protective of the environment than intended.…”

“…False positives are nonvital cells that are erroneously counted as live: in effect, they make the regulations more stringent than specified by discharge standards. These errors are conservative with respect to regulation because risk to the environment is reduced (DHS , as discussed by Cullen and MacIntyre ), but manufacturers or ship operators could be unfairly penalized if compliant ballast water management systems failed to meet the standard due to erroneous test results. Both types of errors reduce the accuracy of ballast water testing, but false negatives are of particular concern because systematic errors of this type correspond to increased risk to the environment.…”

“…, Davey , Berges and Choi ). However, recognizing that the distinction between “viable” and “living” can be critically important in the evaluation of ballast water management systems (First and Drake , Cullen and MacIntyre ), we define our terms specifically as they apply to microorganisms such as phytoplankton subjected to binary classification as live versus dead.…”

Evaluation of ultraviolet disinfection of microalgae by growth modeling: application to ballast water treatment

“…According to the BWMC-Guideline 8, the evaluation of the treatment efficacy is based on the determination of living organisms by widely accepted standard methods (Gollasch et al 2012;Cullen and MacIntyre 2016), such as vital fluorescence stains detected using flow cytometry or fluorescence microscopy, ATP content, photosynthetic activity, or motility assessment (Hessen et al 1997;Sato et al 2004;Gavand et al 2007;Gallo-Villanueva et al 2011;Steinberg et al 2011Steinberg et al , 2012Fan et al 2013;Martínez et al 2013;Stehouwer et al 2013;Adams et al 2014;First and Drake 2014;Tilney et al 2014;Feng et al 2015;Gollasch et al 2015;Olsen et al 2015;van Slooten et al 2015;Wright et al 2015). However, the application of UV treatment does not cause immediate changes in the concentration of living organisms, although their growth curves become different over the time, depending on the applied UV dose (Tao et al 2010;Gorokhova et al 2012;Martínez et al 2012).…”

“…Therefore, the enumeration of living organisms after the treatment does not provide direct information about the threats derived from a ballast discharge. In this sense, authors made a distinction between the terms Bvitality,û nderstood as the living or dead status of organisms, and Bviability,^meaning the ability of organisms to reproduce (Peperzak and Brussaard 2011;Cullen and MacIntyre 2016). Threats derived from the ballast water discharge are thus related with the content of viable organisms, as the subset of the living organisms able to reproduce.…”