J Appl Phycol DOI 10.1007/s10811-016-0798-3
Comparison of three techniques to evaluate the number of viable phytoplankton cells in ballast water after ultraviolet irradiation treatment Oscar Casas-Monroy 1 & Po-Shun Chan 2 & R. Dallas Linley 1 & Julie Vanden Byllaardt 1 & Jocelyn Kydd 1 & Sarah A. Bailey 1
Received: 19 August 2015 / Revised and accepted: 17 January 2016 # Her Majesty the Queen in Right of Canada as represented by: the Minister of Fisheries and Oceans 2016
Abstract The unintentional release of aquatic nonindigenous species (NIS) via ballast water has long been recognized as a primary vector of biological invasions. To reduce the risk of ballast-mediated invasions, the International Maritime Organization (IMO) will direct ships to meet standards regarding the maximum number of viable organisms discharged in ballast water, with most ships expected to install ballast water management systems (BWMSs). Currently, filtration + ultraviolet (UV) irradiation is utilized as a common BWMS. There are issues, however, with enumerating viable phytoplankton after treatment at the low UV doses used onboard ships Electronic supplementary material The online version of this article (doi:10.1007/s10811-016-0798-3) contains supplementary material, which is available to authorized users. * Oscar Casas-Monroy
[email protected] Po-Shun Chan
[email protected] R. Dallas Linley
[email protected]
because the physiological effect occurs at the DNA level— organisms are reproductively sterilized but may remain alive for hours or days after treatment. The objective of this study is to examine three techniques to measure the number of viable phytoplankton cells following filtration + UV treatment: pulse amplitude modulation (PAM) fluorometry, epifluorescence microscopy using fluorescein diacetate (FDA) stain, and the serial dilution culture most probable number (MPN) method. PAM and staining techniques demonstrated similar patterns of phytoplankton reduction after UV irradiation. After 14 days, the MPN method confirmed viability of treated samples in enriched culture medium incubations and may be used to indicate potential recovery of damaged cells (i.e., “re-growth”). All cells that survived treatment and showed detectable growth after 14 days of incubation were smaller than 10 µm, as determined by microscopy. Combinations of direct and/or indirect measurements and culture-based methods might be the best solution to improve detection strategies and eliminate nonindigenous species. Keywords Pulse amplitude modulation (PAM) fluorometry . Epifluorescence microscopy . Fluorescein diacetate (FDA) . Serial dilution culture . Most probable number (MPN)
Julie Vanden Byllaardt
[email protected] Jocelyn Kydd
[email protected]
Introduction
Sarah A. Bailey
[email protected]
Discharge of ballast water is considered one of the main anthropogenic vectors in the transfer of nonindigenous species (NIS) to marine, estuarine, and freshwater ecosystems (Ruiz et al. 2000; Drake et al. 2007; Chapman et al. 2012). Nonindigenous species have the potential to establish populations in new environments resulting in negative impacts on human health, economy, biodiversity, and ecosystem function (Sala et al. 2000; Kolar and Lodge 2001). Currently, the most
1
Great Lakes Laboratory for Fisheries and Aquatic Sciences, Fisheries and Oceans Canada, 867 Lakeshore Road, Burlington, ON L7S 1A1, Canada
2
Trojan Technologies, 3020 Gore Road, London, ON N5V 4T7, Canada
J Appl Phycol
widely used strategy to reduce the risk of ballast-mediated NIS transfer is mid-ocean ballast water exchange (BWE). Properly executed, BWE can reduce concentrations of freshwater organisms by 99% (Gray et al. 2007). In contrast, the efficacy of BWE appears lower and more variable for coastal marine organisms (Wonham et al. 2001); thus, BWE is not considered comprehensively protective (see Miller et al. 2011, National Research Council 2011; United States Coast Guard 2012). Based on the International Convention for the Control and Management of Ships’ Ballast Water and Sediments, established by the International Maritime Organization (IMO), governments will soon require ships to comply with performance standards (Regulation D-2) for viable organisms in ballast water. The standards specify a maximum allowable number of viable individuals for three groups of aquatic organisms in ballast water discharge: organisms ≥ 50 μm in minimum dimension (less than 10 individuals m −3 ), organisms ≥ 10 μm, and < 50 μm in minimum dimension (less than 10 individuals mL−1) and indicator bacteria (less than one colony forming unit (cfu) of Vibrio cholerae per 100 mL−1; less than 250 cfu of Escherichia coli per 100 mL−1 and less than 100 cfu of intestinal Enterococci per 100 mL−1). In general, ships are expected to comply with the IMO discharge standards by installing ballast water management systems (BWMS), although options such as shore-based ballast water treatment or potable water ballast are possible. There are three fundamental water treatment technologies that may be applied to ballast water, individually or in combination: mechanical separation (using filters or hydrocyclones), physical disinfection (such as ultraviolet or ultrasonic radiation, deoxygenation, or heat treatment), and chemical disinfection (using any biocide such as chlorine, peracetic acid, or ozone). While separation technologies remove organisms from ballast water, disinfection technologies typically inactivate organisms or render them non-viable. Ultraviolet (UV) radiation is commonly used for disinfection of drinking water and waste water and is recently applied also to treat ballast water (Gregg et al. 2009). UV light irradiation, especially UVC (100–290 nm), can be lethal for living microorganisms and can damage cell membranes. The most common effect of UV irradiation is the formation of dimeric photoproducts between adjacent pyrimidine bases within DNA, which interfere with normal base pairing during DNA transcription and replication (Vincent and Roy 1993; Martinez et al. 2013). As a result, genome replication is blocked, and organisms are unable to reproduce (Buma et al. 1996). In many instances, living, but non-reproductive, organisms will persist for a single generation that are, from an invasion viewpoint, as good as dead (Reavie et al. 2010; Cullen and MacIntyre 2015). UV treatment efficacy depends upon the size and morphology of organisms, water turbidity, and recovery processes known as photo-reactivation and dark repair (Gregg et al. 2009). Although it is technically possible to apply UV radiation at
dosages causing immediate cell death, it is most cost- and energy- efficient (i.e., feasible) to apply lower doses rendering organisms non-reproductive. UV irradiation is considered more environmentally friendly than application of chemical biocides like chlorine, but physiological effects on aquatic organisms following low doses are more difficult to measure. Several methods have been proposed to distinguish and enumerate living aquatic organisms (here, we differentiate viable organisms as those having the ability to reproduce while living organisms are alive but may be unable to reproduce) (US EPA 2010). The use of vital stains, such as SYTOX green, fluorescein diacetate (FDA), and FDA combined with 5-chloromethyl fluorescein diacetate (CMFDA), to detect intact cell membranes and cellular activity of phytoplankton has been well established for marine and coastal environments (Veldhuis et al. 2001; Brookes et al. 2000; Villac and Kaczmarska 2011) and has recently been evaluated for multiple freshwater taxa (Adams et al. 2014). As an alternative to staining, photosynthetic efficiency or “health” of phytoplankton can be measured using pulse amplitude modulation (PAM) fluorometry. PAM devices deliver a series of pulses of light while measuring the fluorescence of phytoplankton cells in order to gain information about their photosystem capabilities (Buma et al. 1996). Dimensionless measurements such as the maximum fluorescence (Fm) and minimum (initial) fluorescence (Fo) are used to calculate a dimensionless term Fv/Fm, indicating photosystem health, where Fv is the difference between Fm and Fo (Buma et al. 1996; Martinez et al. 2013). In healthy cells, these values are reduced in the presence of light due to the partial closure of reaction centers and dissipation of light energy and, in turn, are an indication of cells’ photochemical efficiency. Alternatively, the serial dilution culture-based most probable number (SDC-MPN or MPN) method has been used to evaluate viable rather than living cells (Cullen and MacIntyre 2015). This technique measures photosynthetic efficiency (fluorescence) of cells kept in culture over a period of weeks, and uses replicated serial dilutions to estimate cell concentration in the original sample based on the proportion of highly diluted samples containing at least one viable cell (Throndsen 1978; Cullen and MacIntyre 2015). With regards to ballast water treatment, the MPN method can be used to detect regrowth of phytoplankton (photoautotrophs only) after treatment, resulting from cell survival (incomplete treatment) or recovery of damaged cells (photo-reactivation and dark repair) (First and Drake 2014; Liebich et al. 2012). All three techniques above have been used to examine the efficacy of various BWMS; however, previous studies have identified issues enumerating viable vs. living cells after UV-C irradiation (e.g., First and Drake 2014). Only viable cells pose a risk of species establishment, and reducing introductions of harmful aquatic organisms is the intent behind the ballast water
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convention. The three techniques also differ in the length of time required to complete analysis as well as the skills required to perform the measurement. As a result, the present study aims to compare the three laboratory techniques for detection of aquatic organisms sized ≥10 μm to
