Ultraviolet radiation as a ballast water treatment strategy

MPB-07359; No of Pages 6 Marine Pollution Bulletin xxx (2015) xxx–xxx

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Ultraviolet radiation as a ballast water treatment strategy: Inactivation of phytoplankton measured with flow cytometry Ranveig Ottoey Olsen a, Friederike Hoffmann b,c, Ole-Kristian Hess-Erga d, Aud Larsen c, Gunnar Thuestad a, Ingunn Alne Hoell a,⁎ a

Stord/Haugesund University College, Klingenbergvegen 8, 5414 Stord, Norway University of Bergen, P.O. Box 7800, 5020 Bergen, Norway Uni Research Environment, Thormoehlensgt. 49b, 5006 Bergen, Norway d Norwegian Institute for Water Research, Thormoehlensgt. 53 D, 5006 Bergen, Norway b c

a r t i c l e

i n f o

Article history: Received 27 August 2015 Received in revised form 8 December 2015 Accepted 10 December 2015 Available online xxxx Keywords: Tetraselmis suecica Ultraviolet irradiation Esterase substrate Flow cytometry Inactivation Dark incubation

a b s t r a c t This study investigates different UV doses (mJ/cm2) and the effect of dark incubation on the survival of the algae Tetraselmis suecica, to simulate ballast water treatment and subsequent transport. Samples were UV irradiated and analyzed by flow cytometry and standard culturing methods. Doses of ≥ 400 mJ/cm2 rendered inactivation after 1 day as measured by all analytical methods, and are recommended for ballast water treatment if immediate impairment is required. Irradiation with lower UV doses (100– 200 mJ/cm2) gave considerable differences of inactivation between experiments and analytical methods. Nevertheless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥100 mJ/cm2 and ≤200 mJ/cm2 can be sufficient if the water is treated at intake and left in dark ballast tanks. The variable results demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation needed for inactivation when algae are treated with low UV doses. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction Ships use water as ballast to ensure stability and trim during the voyage, and ambient water is pumped into ballast tanks in the hull of the ships. It is traditionally discharged without any treatment and represents a global vector for aquatic invasion. A multitude of organisms like virus, bacteria, algae and zooplankton are carried around the world in ship's ballast tanks (David et al., 2007; Drake et al., 2007; Hallegraeff and Bolch, 1991). Some organisms survive in ballast tanks and are released into new environments. If nonindigenous species adapt and establish in a new environment, they might have an impact on the native species and cause ecological change in the ocean (Gollasch et al., 2015; Ruiz et al., 1997). It is of importance to minimize and prevent dispersal of species by ballast water discharge to hinder potential harm to ecosystems, the economy, or human health (Ruiz et al., 2000). In 2004 the International Maritime Organization (IMO) established standards for ballast water treatment through the International Convention for the Control and Management of Ship's Ballast Water and Sediments (International Maritime Organization, 2004). Regulation D2 of the Convention sets the standard regarding category and concentration of organisms at discharge. The Convention will enter into force ⁎ Corresponding author. E-mail address: [email protected] (I.A. Hoell).

12 months after being ratified by 30 States representing 35% of the merchant shipping tonnage. In August 2015 44 States, representing 32.86% of the world tonnage, have ratified the Convention. The upcoming IMO regulations have led to development of various ballast water treatment systems (BWTS) that facilitate disinfection of ballast water (David and Gollasch, 2015; Delacroix et al., 2013; Lloyd's Register Marine's, 2015a, 2015b; Stehouwer et al., 2015; Werschkun et al., 2012, 2014). All BWTS have to be approved by national authorities according to IMO regulations and/or the regulations of other national bodies (e.g. U.S. Coast Guard (USCG)). When selecting and installing a BWTS, the shipping companies have to consider different technical and operational aspects (Lloyd's Register Marine's, 2015a, 2015b). The BWTS use a range of different treatment technologies, from processing the water with solid–liquid separation to chemical- (active substances) and/or physical disinfection (e.g. UV). The main operational cost for UV based BWTS is related to power consumption (Werschkun et al., 2014). Ship owners can reduce such costs by lowering the UV intensity, providing that the ship's discharged ballast water still complies to Regulation D-2 (International Maritime Organization, 2008a). It is therefore of interest to determine the lowest lethal UV dose and to estimate the time required for inactivation when stored in ballast tanks after irradiation. UV irradiation is performed either by low pressure (LP) or medium pressure (MP) UV lamps (Oguma et al., 2002; Werschkun et al., 2012;

http://dx.doi.org/10.1016/j.marpolbul.2015.12.008 0025-326X/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article as: Olsen, R.O., et al., Ultraviolet radiation as a ballast water treatment strategy: Inactivation of phytoplankton measured with flow cytometry, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.12.008

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R.O. Olsen et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Zimmer and Slawson, 2002). LP lamps emit UV-C radiation, primarily at 254 nm, which is most efficiently absorbed by nucleic acids and causes DNA damages (Sinha and Häder, 2002). UV induced DNA damages can be reversed by DNA repair mechanisms, referred to as photoreactivation and dark repair (Sancar and Sancar, 1988; Sinha and Häder, 2002). MP UV lamps emit radiation spanning the UV-A, -B and -C bands causing additional damage to proteins and enzymes. For instance, UV-B radiation can affect key components in photosynthesis (Fiscus and Booker, 1995; Holzinger and Lütz, 2006; Kottuparambil et al., 2012), causing energy deprivation in phytoplankton cells. Thus, it has been argued that MP UV lamps can cause a higher degree of inactivation compared to LP UV lamps (Kalisvaart, 2001; Oguma et al., 2002). UV irradiation can leave cells in different conditions (live, dead or damaged), whereof the viability of damaged cells at discharge is uncertain (Olsen et al., 2015). Damaged cells can be unculturable, though they can be metabolic active and may pose a health risk (Oliver, 2010). Further, cellular DNA repair mechanisms can restore the genetic information (Sancar and Sancar, 1988; Sinha and Häder, 2002; Zimmer and Slawson, 2002) causing the cell to grow and replicate after discharge (Liebich et al., 2012; Martínez et al., 2012, 2013). Additionally, the terminology describing the organisms at discharge can be confusing or unclear. The IMO Convention refers to “viable” organisms (International Maritime Organization, 2004), and the Guidelines for approval of ballast water management systems (G8) define “viable organisms” as “organisms and any life stages thereof that are living” (International Maritime Organization, 2008a). USCG also uses the term “living” (United States Coast Guard, 2012). Determining the condition of UV irradiated cells is a complex task. On the other hand, cheap, fast and reliable methods to analyze ballast water are necessary for approval of BWTS technologies and for compliance testing of ballast water discharge (International Maritime Organization, 2013). Testing for compliance can be performed in two steps; an indicative and a detailed analyses. An indicative analysis is a relatively simple and quick measurement that gives a rough estimate of the number of viable organisms in the ballast water at discharge. Examples of indicative analysis methods are e.g. BallastCAM and various fluorescence or ATP detections (Drake et al., 2014; First and Drake, 2013, 2014; Gollasch and David, 2012, 2015; van Slooten et al., 2015). If an indicative analysis shows compliance to Regulation D-2, there is no need for a detailed analysis. Should the indicative analyses be non-compliant, however, a detailed analysis must be undertaken to give robust and direct measurements determining the concentration of viable organism in ballast water discharge according to Regulation D-2. Quantification of live bacteria traditionally relies on cultivation methods, which is time-consuming and may give false negatives as several species are uncultivable although viable (Roszak and Colwell, 1987; Staley and Konopka, 1985). Flow cytometry (FCM) has been suggested as a promising method for detailed analysis (International Maritime Organization, 2013; Peperzak and Gollasch, 2013). FCM facilitates rapid detection, enumeration and characterization of organisms in combination with fluorescent dyes, and enables to study populations and communities indirectly (Peperzak and Brussaard, 2011; Shapiro, 2000). Previously a FCM protocol was developed to distinguish between live and dead Tetraselmis suecica cells (Olsen et al., 2015). For UV irradiated samples the FCM protocol could not distinguish between live and damaged cells, as the latter contain both dying and repairable cells. The current study uses the FCM protocol to elaborate on different UV doses and the effect of dark incubation on inactivation of the algae T. suecica, to simulate a ballast water treatment and subsequent transport. Our specific objectives were to: 1) Determine the minimum UV dose that permanently inactivates the algae. 2) Quantify effects of different UV doses on T. suecica.

3) Estimate the time of dark incubation required to permanently inactivate the algae treated with UV doses lower than minimum permanently inactivation dose. 4) Provide recommendations for ballast water management. 2. Material and methods The phytoplankter specie T. suecica (Strain K-0297, Scandinavian Culture Collection of Alga and Protozoa, University of Copenhagen, Denmark) was selected as a test organism. It was cultured in 24 PPT artificial sea water (Marine SeaSalt, Tetra, Melle, Germany) added 0.12% Substral (The Scotts Company (Nordic) A/S, Naverland, Glostrup, Denmark), at 15 °C, 100 rpm, 14:10 light:dark cycle and 90 lx light intensity (Flora-Glo, T8, 20 W). The culture was diluted in growth medium to a density of 104 live cells ml−1 prior to irradiation, monitored by FCM. Irradiation was performed using a collimated beam MP UV lamp (800 W) (BestUV, Hazerswoude, The Netherlands) (Olsen et al., 2015). For each experiment three samples of 15 ml diluted T. suecica culture were irradiated with the same UV dose in a petri dish (inner diameter 6 cm, culture depth 7 mm) while mixed with a 1 × 0.4 cm magnetic stir bar at 200 rpm in room temperature (RT). The intensity (mW/cm2) of the UV lamp was fixed and the exposure times used were 155, 233, 311, 622 and 1244 s for UV doses 100, 150, 200, 400 and 800 mJ/cm2, respectively. The irradiated samples were transferred to sterile 50 ml polypropylene tubes (Fisher Scientific), so was the control samples, including 2 × 15 ml non-irradiated cells and 10 ml dead cells. The dead cells were killed by fixation with formaldehyde at 5% final concentration (36.5–38% formaldehyde, Sigma-Aldrich). All tubes were wrapped in aluminum foil and incubated in the dark with loosened lids at 15 °C. First, a pre-study over 5 days was performed to observe the inactivation effect of different UV doses and dark incubation, and to test whether this effect was interpretable with FCM. This was followed by two complete experiments, denoted as exp-I and exp-II, and an overview of the set-up for these experiments is given in Fig. 1. For FCM analysis, the samples were stained with 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and analyzed with an Attune Acoustic Focusing Cytometer (Olsen et al., 2015). The samples in the pre-study were analyzed at days 1, 3 and 5 after treatment. In exp-I samples were analyzed at days 1, 3, 6, 9, 13 and 22, and in exp-II samples were analyzed at days 1, 3, 6, 10, 15 and 22 after treatment (Fig. 1). The samples in exp-I and -II were analyzed at different intervals due to logistics. A previously defined gate (i.e. a collection of single cell FCM-signals) in the FCM dot plots was used for analysis. The gate was

Fig. 1. Experimental set-up showed by a flow diagram. This set-up was followed in the pre-study, exp-I and exp-II.



defined based on plate count results from non-irradiated cells, and regression analysis was used for validation of the gate (Olsen et al., 2015). To determine the number of culturable cells, plate count analysis was performed 1 day after UV treatment (Olsen et al., 2015). Also at day 1, a most probable number (MPN) analysis was performed. The samples were diluted in growth medium (24 PPT artificial sea water added 0.12% Substral) in 10-fold series up to 10−4 dilution to a total volume of 1 ml pr. well in 48 well plates (Greiner Bio-One, Austria) and incubated at 15 °C, 14:10 light:dark cycle and 90 lx light intensity. Positive growth was determined by a change in color into green as detected by the eye, and scored against the MPN table for a threereplicate design from FDA's Bacterial Analytical Manual (U. S. Food and Drug Administration (FDA), 2010), which gives rough results in intervals. When the numbers of live/damaged cell signals in FCM dot plots were approximately 10% of the total number of cells, a regrowth check was performed for verification. For exp-I this procedure was carried

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out at day 22 for samples treated with 100–200 mJ/cm2 and at day 2 for samples treated with 400 and 800 mJ/cm2, and for exp-II at day 20 for samples treated with 100–200 mJ/cm2 and at day 3 for samples treated with 400 and 800 mJ/cm2. 1 ml of the sample was added to 9 ml growth medium in 50 ml Erlenmeyer flasks. No visible change to green color indicated that there were no reproductive cells in the sample. The flasks, trays and plates were incubated at 15 °C in the dark for 3 weeks. 3. Results FCM analysis in the pre-study showed that inactivation increased with higher UV doses and during the dark incubation period (data not shown). Based on these results, two complete experiments were carried out (exp-I and -II), and these results are presented below. UV irradiation with doses ≥400 mJ/cm2 inactivated the algae permanently as demonstrated by all analysis methods. FCM analyses (Fig. 2b, d)

Fig. 2. Line graphs showing % gated signals (=live and damaged cells) of the total number of cells (=live, damaged and dead cells) from exp-I (a, b) and exp-II (c, d). Error bars indicate 1 standard deviation of 3 replicates.


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of samples irradiated with 400 and 800 mJ/cm2 displayed b 4% and ≤0.1% live/damaged cell signals after 1 day, respectively. The numbers of live and damaged cells remained at this level or were further reduced during the incubation period. Plate count and MPN did not show any growth at UV doses ≥400 mJ/cm2 and neither did regrowth check performed at days 2 and 3 for exp-I and exp-II, respectively. As observed in the pre-study, FCM analysis of UV irradiated samples showed a relationship between inactivation and UV doses. This was examined at day 1, when an immediate effect of UV treatment was observed, and again the number of live/damaged cells decreased with higher UV doses. Table 1 shows a comparison of the results from FCM, MPN and plate count from exp-I and -II. The control samples were all in the same range, but MPN results showed N11,000 cells ml−1, indicating that the samples should have been diluted further. The results for the UV irradiated samples, varied when using different analysis methods. In exp-I there were good agreements between live/damaged cell numbers obtained by FCM and MPN analysis, but the plate count analysis resulted in considerably lower numbers at day 1. For exp-II, comparable results were obtained using plate count and MPN analysis whereas the results using FCM gave considerable higher live/damaged cell numbers. For samples treated with UV doses 100, 150 and 200 mJ/cm2 inactivation of cells was dependent on time of dark incubation as demonstrated by FCM (Fig. 2a, c). Generally, the numbers of live/damaged cells decreased during incubation, and were fewer in UV irradiated samples than in the stained controls. In exp-I the percentage of live/damaged cells in the UV irradiated samples (Fig. 2a) decreased throughout the incubation period and amounted to ≤3% at day 22. The samples treated with 100 mJ/cm2 behaved similar to the stained controls during incubation, but at day 22 the percentage of live/damaged cells was lower than the stained controls also for samples treated with this UV dose. In exp-II (Fig. 2c) inactivation increased during incubation, and b3% live/ damaged cells were observed at days 22, 10 and 3 in the samples treated with 100, 150 and 200 mJ/cm2, respectively. Regrowth checks performed at days 22 and 20 for exp-I and exp-II, respectively, were negative for all UV irradiated samples and positive for the control samples (data not shown). Considerable variations were observed between the results from the two experiments when looking at a detailed level. Firstly, the percentage of live/damaged cells in the stained controls varied at day 1 (Fig. 2a, c), being 92% and 54% in exp-I and exp-II, respectively. Secondly, for the samples UV irradiated with ≤200 mJ/cm2, the inactivation rate varied between the experiments (Fig. 2a, c) and the number of FCM live/ damaged cells fluctuated (Table 1). Thirdly, some replicates showed

large standard deviations; most evident in exp-I (Fig. 2a). Fourthly, at the first days after UV irradiation, it was observed more cells in the treated samples than in the controls (Table 1). 4. Discussion The aim of this study was to evaluate inactivation by different UV doses and dark incubation on the algae T. suecica, to give recommendations for ballast water management regarding treatment and transport. UV doses 400 and 800 mJ/cm2 rendered T. suecica cells unculturable and without esterase activity 1 day after irradiation whereas doses ≤ 200 mJ/cm2 did not necessarily inactivate the cells. This indicates that the minimum UV dose that permanently inactivates this algal specie is somewhere between 200 and 400 mJ/cm2 which is similar to the dose Ou et al. (2012) found to be lethal after UV-C radiating the cyanobacteria Microcystis aeruginosa (Ou et al., 2012). The samples irradiated with UV doses 100, 150 and 200 mJ/cm2 contained culturable and esterase active T. suecica cells 1 day after irradiation, but the inactivation increased with higher UV doses. Our results are in line with previous studies of freshwater green algae Chlorella ellipsoidea, Chlorella vulgaris, and Scenedesmus quadricanda, and the cyanobacteria M. aeruginosa which showed limited sensitivity to UV-C irradiation with doses ≤200 mJ/cm2 (Ou et al., 2012; Tao et al., 2010). Comparing the different analysis methods at day 1 for the samples UV irradiated with doses ≤ 200 mJ/cm2 revealed that the numbers of FCM gated cells were higher than the numbers of cfu detected by plate count. Such discrepancy between plate count and FCM results has also been observed in other studies of UV irradiated bacteria and alga (Kramer and Muranyi, 2014; Olsen et al., 2015; Schenk et al., 2011). UV induced DNA damage can block transcription and replication, inhibiting growth and reproduction (Oguma et al., 2002; Sinha and Häder, 2002). DNA damaged cells are not detected as live by growth assays, though they can express activity (Davey, 2011; Hammes et al., 2011; Villarino et al., 2003); explaining the contradicting results from FCM and plate count analysis. Plate count and MPN analysis are based on reproductive capacity and one could expect these growth assays to give comparable results. However, the results obtained by the MPN were similar to the plate count results in exp-II and to FCM in exp-I. This illustrates the challenge of getting reproducible results when analyzing UV irradiated organisms with methods that analyze different cellular characteristics. Further, growth assays may introduce errors as a majority of the microbes are uncultivable (Roszak and Colwell, 1987; Staley and Konopka, 1985). In addition, the MPN positive growth was determined by the eye, though

Table 1 FCM, plate count and MPN results from exp-I and exp-II. Results are all in cells ml−1. When 0 cfu was detected by the plate count method, the values show “b10” as the results are obtained by 100 µl being spread on the agar plates. The MPN values show “b3” when no visible green color was observed, according to the MPN table (U. S. Food and Drug Administration (FDA), 2010). n.d. = no data. 1 standard deviation (±) for FCM and plate counts, as well as 95% confidence intervals for MPN, are in brackets. Experiment

Analysis

Time (days)

Control

100 m J/cm2

150 m J/cm2

200 mJ/cm2

400 m J/cm2

800 m J/cm2

Exp-I

FCM

1 3 6 9 13 22 1

3 (1) 2 (1) 2 (2) 1 (1) 2 (2) 5 (4) b3 (0–9.5)

b10 169 (37) 126 (23) 100 (40) 73 (11) 105 (22) 111 (50) b3 (0–9.5)

b10 21 (12) 13 (2) 11 (6) 7 (1) 9 (3) 11 (3) b3 (0–9.5)

10,187 (2510)

10,003 (318) 8896 (2926) 9453 (343) 10,195 (1183) 5533 (2666) 512 (627) 11,000 (1800–41,000) 8400 (1249) 3946 (1464) 2893 (1203) 1587 (607) 597 (275) 427 (74) 406 (223) 930 (180–4200) 950 (416)

8531 (1437) 6402 (3494) 5468 (1959) 4812 (1386) 2239 (958) 304 (321) 11,000 (1800–41,000) 5040 (170) 973 (682) 617 (382) 344 (197) 180 (45) 172 (53) 214 (57) b3 (0–9.5)

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13,902 (572) 12,202 (1012) 12,221 (1601) 12,218 (1549) 9949 (2110) 1354 (970) 11,000 (1800–41,000) 7467 (503) 9016 (332) 8429 (1518) 5320 (966) 2663 (363) 1190 (167) 642 (141) 4600 (900–20,000) 4107 (508)

488 (348) 55 (45) 16 (6) 12 (6) 5 (2) 8 (7) b3 (0–9.5)

1 1 3 6 10 15 22 1

8231 (1222) 5248 (183) 4373 (1185) 8485 (2377) 8258 (714) 4160 (968) N11,000 (4200–40,000) 8667 (577) 10,419 (883) 9915 (198) 10,688 (954) 6366 (577) 4327 (476) 2441 (481) n.d.

48 (50)

b10

b10

MPN

Exp-II

Rate count FCM

MPN Rate count



fluorometry determination would have been a more objective analysis method and could potentially have given a slightly different outcome. However, the MPN method was validated by another researcher and by microscopy. The FCM results showed that inactivation increased with the time of dark incubation. Phytoplankton are photosynthetic organisms requiring light as an energy source (McMinn and Martin, 2013) and dark incubation will naturally affect their viability over time (Gollasch and David, 2010), especially if the cells continue with unchanged activity causing them to run out of energy (Jochem, 1999). Additionally, incubation in darkness will limit photoreactivation; the repair mechanism which removes UV induced DNA lesions and reverses damages by using the energy of light (Sinha and Häder, 2002), leaving the cells unrepaired and dying. Other studies have reported limited or no photoreactivation in UV irradiated Escherichia coli during incubation at darkness (Oguma et al., 2001; Yin et al., 2015; Zimmer and Slawson, 2002). In agreement with other studies the control samples contained reproducible cells even after 3 weeks of dark incubation (Gollasch and David, 2010; Olsen et al., 2015). T. suecica's ability to survive in darkness over time implies it does not overrate effects of UV treatments and makes it an appropriate indicator organism for ballast water monitoring. Some of the variation on results between exp-I and -II, such as variation in the stained controls day 1, difference in inactivation rate when irradiated with doses ≤200 mJ/cm2, large standard deviations and more cells in treated samples than in untreated ones, indicates that the algal culture used in exp-I contained a greater portion of fresh and healthy cells than the culture used in exp-II. Natural sea water shows great variability and contains a diverse community of algae species in different cellular phases varying with time and space and may have different tolerance and response to the same UV doses applied (Rastogi et al., 2010; Sinha et al., 1998; Xiong et al., 1997). Although not intentionally, our study indeed demonstrated that the UV treatment/dark incubation required for inactivation is dependent on the status of the ballast water inhabiting organisms. Therefore, our results imply that organisms in ballast water treated with UV doses 100–200 mJ/cm2 are inactivated when left in dark ballast tanks over a period of time. Our results also demonstrate the challenge of giving recommendations regarding duration of dark incubation needed for inactivation when using lower UV doses. It is, however, important to keep in mind that controllable experiments in a laboratory differ from a flow-through chamber in a commercial BWTS and that a BWTS comprise two or more treatment stages, enhancing the inactivation efficiency. Other factors can, however, also influence the inactivation efficiency in a BWTS, like biotic and abiotic particles in the sea water protecting the microbes during UV irradiation (Hess-Erga et al., 2008; Tang et al., 2011) and a BWTS has to be optimal under the prevailing circumstances whatever factors exist that may prevent inactivation. UV irradiation as a treatment technology has been criticized due to the uncertainty regarding inactivation at discharge but is more environmental friendly than chemical disinfection and creates no harmful byproducts (Jung et al., 2012). In addition, UV irradiation represents little risk for the operators and less training is required to run the systems (International Maritime Organization, 2008b). This study was performed in a laboratory with cultures of T. suecica and with the MP UV lamp as the sole treatment source. For future studies it would be of interest to do experiments with T. suecica in real BWTS with MP UV technology. Such studies would improve our ability to give recommendations regarding UV doses and duration of dark incubation of T. suecica. To evaluate whether our FCM protocol is applicable to other microbes, laboratory studies should be performed with natural sea water irradiated with different UV doses. If analysis of natural sea water is feasible with our FCM protocol, the experiment needs to be repeated in a BWTS to evaluate inactivation in a system where it is intended to be used. The microbial community in ballast water is very diverse and organisms will vary with season, location and environmental conditions. The level of metabolic activity can vary between various

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algae species, and the response to environmental changes and inactivation treatments can differ (Jochem, 1999, 2000; Olsen et al., 2015). Although there are indications that the majority of phytoplankton species can be detected by the esterase substrates fluorescein diacetate (FDA) and 5-chloromethylfluoorescein diacetate (CMFDA) (Peperzak and Brussaard, 2011), we are therefore aware that fluorescing signal from esterase substrates can vary over a large range of intensities (Dorsey et al., 1989). Our recommendation at this point is thus to treat the ballast water with UV doses close to 400 mJ/cm2 in order to permanently inactivate the organisms. The variable results for UV doses 100–200 mJ/cm2 demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation needed for inactivation.

Acknowledgments This research was founded by the Norwegian Research Council (project BallastFlow, project no. 208653) and Knutsen OAS Shipping AS, and supported by Solstad Shipping, Stord/Haugesund University College, VRI Rogaland, UH-nett Vest and TeknoVest. We thank Sandra Schöttner (UiB, Bergen, Norway), Stephanie Delacroix, August Tobiesen (Norwegian Institute for Water Research, Oslo, Norway) and Per Lothe (Knutsen OAS Shipping AS, Haugesund, Norway) for helpful discussions. Special thanks to Sandra Schöttner for assistance with the UV lamp and experimental set-up.

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