Marine Pollution Bulletin 103 (2016) 270–275

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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
b
University of Bergen, P.O. Box 7800, 5020 Bergen, Norway
c
Uni Research Environment, Thormoehlensgt. 49b, 5006 Bergen, Norway
d
Norwegian Institute for Water Research, Thormoehlensgt. 53 D, 5006 Bergen, Norway

a r t i c l e i n f o a b s t r a c t

Article history: This study investigates different UV doses (mJ/cm2) and the effect of dark incubation on the survival of the algae
Received 27 August 2015 Tetraselmis suecica, to simulate ballast water treatment and subsequent transport.
Received in revised form 8 December 2015 Samples were UV irradiated and analyzed by flow cytometry and standard culturing methods. Doses of
Accepted 10 December 2015
≥ 400 mJ/cm2 rendered inactivation after 1 day as measured by all analytical methods, and are recommended
Available online 21 December 2015
for ballast water treatment if immediate impairment is required. Irradiation with lower UV doses (100–
Keywords:
200 mJ/cm2) gave considerable differences of inactivation between experiments and analytical methods. Never-
Tetraselmis suecica theless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥100 mJ/cm2
Ultraviolet irradiation and ≤200 mJ/cm2 can be sufficient if the water is treated at intake and left in dark ballast tanks. The variable re-
Esterase substrate sults demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation need-
Flow cytometry ed for inactivation when algae are treated with low UV doses.
Inactivation © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
Dark incubation (http://creativecommons.org/licenses/by/4.0/).

1. Introduction 12 months after being ratified by 30 States representing 35% of the mer-
chant shipping tonnage. In August 2015 44 States, representing 32.86%
Ships use water as ballast to ensure stability and trim during the of the world tonnage, have ratified the Convention. The upcoming
voyage, and ambient water is pumped into ballast tanks in the hull of IMO regulations have led to development of various ballast water treat-
the ships. It is traditionally discharged without any treatment and ment systems (BWTS) that facilitate disinfection of ballast water (David
represents a global vector for aquatic invasion. A multitude of organisms and Gollasch, 2015; Delacroix et al., 2013; Lloyd's Register Marine's,
like virus, bacteria, algae and zooplankton are carried around the world 2015a, 2015b; Stehouwer et al., 2015; Werschkun et al., 2012, 2014).
in ship's ballast tanks (David et al., 2007; Drake et al., 2007; Hallegraeff All BWTS have to be approved by national authorities according to
and Bolch, 1991). Some organisms survive in ballast tanks and are IMO regulations and/or the regulations of other national bodies (e.g.
released into new environments. If nonindigenous species adapt and es- U.S. Coast Guard (USCG)).
tablish in a new environment, they might have an impact on the native When selecting and installing a BWTS, the shipping companies have
species and cause ecological change in the ocean (Gollasch et al., 2015; to consider different technical and operational aspects (Lloyd's Register
Ruiz et al., 1997). It is of importance to minimize and prevent dispersal Marine's, 2015a, 2015b). The BWTS use a range of different treatment
of species by ballast water discharge to hinder potential harm to ecosys- technologies, from processing the water with solid–liquid separation
tems, the economy, or human health (Ruiz et al., 2000). to chemical- (active substances) and/or physical disinfection (e.g. UV).
In 2004 the International Maritime Organization (IMO) established The main operational cost for UV based BWTS is related to power con-
standards for ballast water treatment through the International Con- sumption (Werschkun et al., 2014). Ship owners can reduce such
vention for the Control and Management of Ship's Ballast Water and costs by lowering the UV intensity, providing that the ship's discharged
Sediments (International Maritime Organization, 2004). Regulation D- ballast water still complies to Regulation D-2 (International Maritime
2 of the Convention sets the standard regarding category and concen- Organization, 2008a). It is therefore of interest to determine the lowest
tration of organisms at discharge. The Convention will enter into force lethal UV dose and to estimate the time required for inactivation when
stored in ballast tanks after irradiation.
⁎ Corresponding author. UV irradiation is performed either by low pressure (LP) or medium
E-mail address: ingunn.hoell@hsh.no (I.A. Hoell). 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/).
 R.O. Olsen et al. / Marine Pollution Bulletin 103 (2016) 270–275 271

Zimmer and Slawson, 2002). LP lamps emit UV-C radiation, primarily 3) Estimate the time of dark incubation required to permanently inac-
at 254 nm, which is most efficiently absorbed by nucleic acids tivate the algae treated with UV doses lower than minimum perma-
and causes DNA damages (Sinha and Häder, 2002). UV induced DNA nently inactivation dose.
damages can be reversed by DNA repair mechanisms, referred to as 4) Provide recommendations for ballast water management.
photoreactivation and dark repair (Sancar and Sancar, 1988; Sinha
and Häder, 2002). MP UV lamps emit radiation spanning the UV-A, -B 2. Material and methods
and -C bands causing additional damage to proteins and enzymes. For
instance, UV-B radiation can affect key components in photosynthesis The phytoplankter specie T. suecica (Strain K-0297, Scandinavian
(Fiscus and Booker, 1995; Holzinger and Lütz, 2006; Kottuparambil Culture Collection of Alga and Protozoa, University of Copenhagen,
et al., 2012), causing energy deprivation in phytoplankton cells. Thus, Denmark) was selected as a test organism. It was cultured in 24 PPT
it has been argued that MP UV lamps can cause a higher degree of inac- artificial sea water (Marine SeaSalt, Tetra, Melle, Germany) added
tivation compared to LP UV lamps (Kalisvaart, 2001; Oguma et al., 0.12% Substral (The Scotts Company (Nordic) A/S, Naverland, Glostrup,
2002). Denmark), at 15 °C, 100 rpm, 14:10 light:dark cycle and 90 lx light
UV irradiation can leave cells in different conditions (live, dead or intensity (Flora-Glo, T8, 20 W). The culture was diluted in growth medi-
damaged), whereof the viability of damaged cells at discharge is uncer- um to a density of 104 live cells ml−1 prior to irradiation, monitored by
tain (Olsen et al., 2015). Damaged cells can be unculturable, though they FCM.
can be metabolic active and may pose a health risk (Oliver, 2010). Irradiation was performed using a collimated beam MP UV lamp
Further, cellular DNA repair mechanisms can restore the genetic infor- (800 W) (BestUV, Hazerswoude, The Netherlands) (Olsen et al., 2015).
mation (Sancar and Sancar, 1988; Sinha and Häder, 2002; Zimmer and For each experiment three samples of 15 ml diluted T. suecica culture
Slawson, 2002) causing the cell to grow and replicate after discharge were irradiated with the same UV dose in a petri dish (inner diameter
(Liebich et al., 2012; Martínez et al., 2012, 2013). Additionally, the ter- 6 cm, culture depth 7 mm) while mixed with a 1 × 0.4 cm magnetic stir
minology describing the organisms at discharge can be confusing or un- bar at 200 rpm in room temperature (RT). The intensity (mW/cm2) of
clear. The IMO Convention refers to “viable” organisms (International the UV lamp was fixed and the exposure times used were 155, 233,
Maritime Organization, 2004), and the Guidelines for approval of ballast 311, 622 and 1244 s for UV doses 100, 150, 200, 400 and 800 mJ/cm2, re-
water management systems (G8) define “viable organisms” as “organ- spectively. The irradiated samples were transferred to sterile 50 ml
isms and any life stages thereof that are living” (International Maritime polypropylene tubes (Fisher Scientific), so was the control samples, in-
Organization, 2008a). USCG also uses the term “living” (United States cluding 2 × 15 ml non-irradiated cells and 10 ml dead cells. The dead
Coast Guard, 2012). cells were killed by fixation with formaldehyde at 5% final concentration
Determining the condition of UV irradiated cells is a complex task. (36.5–38% formaldehyde, Sigma-Aldrich). All tubes were wrapped in
On the other hand, cheap, fast and reliable methods to analyze ballast aluminum foil and incubated in the dark with loosened lids at 15 °C.
water are necessary for approval of BWTS technologies and for compli- First, a pre-study over 5 days was performed to observe the inactiva-
ance testing of ballast water discharge (International Maritime tion effect of different UV doses and dark incubation, and to test wheth-
Organization, 2013). Testing for compliance can be performed in two er this effect was interpretable with FCM. This was followed by two
steps; an indicative and a detailed analyses. An indicative analysis is a complete experiments, denoted as exp-I and exp-II, and an overview
relatively simple and quick measurement that gives a rough estimate of the set-up for these experiments is given in Fig. 1.
of the number of viable organisms in the ballast water at discharge. Ex- For FCM analysis, the samples were stained with 5-carboxyfluo-
amples of indicative analysis methods are e.g. BallastCAM and various rescein diacetate acetoxymethyl ester (CFDA-AM) and analyzed with
fluorescence or ATP detections (Drake et al., 2014; First and Drake, an Attune Acoustic Focusing Cytometer (Olsen et al., 2015). The samples
2013, 2014; Gollasch and David, 2012, 2015; van Slooten et al., 2015). in the pre-study were analyzed at days 1, 3 and 5 after treatment. In
If an indicative analysis shows compliance to Regulation D-2, there exp-I samples were analyzed at days 1, 3, 6, 9, 13 and 22, and in exp-II
is no need for a detailed analysis. Should the indicative analyses samples were analyzed at days 1, 3, 6, 10, 15 and 22 after treatment
be non-compliant, however, a detailed analysis must be undertaken (Fig. 1). The samples in exp-I and -II were analyzed at different intervals
to give robust and direct measurements determining the concen- due to logistics. A previously defined gate (i.e. a collection of single cell
tration of viable organism in ballast water discharge according to FCM-signals) in the FCM dot plots was used for analysis. The gate was
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 characteriza-
tion 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 irradi-
ated 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 trans-
port. Our specific objectives were to:

1) Determine the minimum UV dose that permanently inactivates the

algae. Fig. 1. Experimental set-up showed by a flow diagram. This set-up was followed in the
2) Quantify effects of different UV doses on T. suecica. pre-study, exp-I and exp-II.
272 R.O. Olsen et al. / Marine Pollution Bulletin 103 (2016) 270–275

defined based on plate count results from non-irradiated cells, and re- out at day 22 for samples treated with 100–200 mJ/cm2 and at day 2
gression analysis was used for validation of the gate (Olsen et al., 2015). for samples treated with 400 and 800 mJ/cm2, and for exp-II at day 20
To determine the number of culturable cells, plate count analysis for samples treated with 100–200 mJ/cm2 and at day 3 for samples
was performed 1 day after UV treatment (Olsen et al., 2015). Also at treated with 400 and 800 mJ/cm2. 1 ml of the sample was added
day 1, a most probable number (MPN) analysis was performed. The to 9 ml growth medium in 50 ml Erlenmeyer flasks. No visible change
samples were diluted in growth medium (24 PPT artificial sea water to green color indicated that there were no reproductive cells in the
added 0.12% Substral) in 10-fold series up to 10−4 dilution to a total sample. The flasks, trays and plates were incubated at 15 °C in the
volume of 1 ml pr. well in 48 well plates (Greiner Bio-One, Austria) dark for 3 weeks.
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 3. Results
detected by the eye, and scored against the MPN table for a three-
replicate design from FDA's Bacterial Analytical Manual (U. S. Food FCM analysis in the pre-study showed that inactivation increased
and Drug Administration (FDA), 2010), which gives rough results in with higher UV doses and during the dark incubation period (data not
intervals. shown). Based on these results, two complete experiments were carried
When the numbers of live/damaged cell signals in FCM dot plots out (exp-I and -II), and these results are presented below.
were approximately 10% of the total number of cells, a regrowth check UV irradiation with doses ≥400 mJ/cm2 inactivated the algae perma-
was performed for verification. For exp-I this procedure was carried nently 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.
 R.O. Olsen et al. / Marine Pollution Bulletin 103 (2016) 270–275 273

of samples irradiated with 400 and 800 mJ/cm2 displayed b 4% and large standard deviations; most evident in exp-I (Fig. 2a). Fourthly, at
≤0.1% live/damaged cell signals after 1 day, respectively. The numbers the first days after UV irradiation, it was observed more cells in the
of live and damaged cells remained at this level or were further reduced treated samples than in the controls (Table 1).
during the incubation period. Plate count and MPN did not show any
growth at UV doses ≥400 mJ/cm2 and neither did regrowth check per- 4. Discussion
formed at days 2 and 3 for exp-I and exp-II, respectively.
As observed in the pre-study, FCM analysis of UV irradiated samples The aim of this study was to evaluate inactivation by different UV
showed a relationship between inactivation and UV doses. This was doses and dark incubation on the algae T. suecica, to give recommenda-
examined at day 1, when an immediate effect of UV treatment was tions for ballast water management regarding treatment and transport.
observed, and again the number of live/damaged cells decreased with UV doses 400 and 800 mJ/cm2 rendered T. suecica cells unculturable
higher UV doses. Table 1 shows a comparison of the results from FCM, and without esterase activity 1 day after irradiation whereas doses
MPN and plate count from exp-I and -II. The control samples were ≤ 200 mJ/cm2 did not necessarily inactivate the cells. This indicates
all in the same range, but MPN results showed N11,000 cells ml−1, indi- that the minimum UV dose that permanently inactivates this algal
cating that the samples should have been diluted further. The results specie is somewhere between 200 and 400 mJ/cm2 which is similar to
for the UV irradiated samples, varied when using different analysis the dose Ou et al. (2012) found to be lethal after UV-C radiating the
methods. In exp-I there were good agreements between live/damaged cyanobacteria Microcystis aeruginosa (Ou et al., 2012). The samples
cell numbers obtained by FCM and MPN analysis, but the plate count irradiated with UV doses 100, 150 and 200 mJ/cm2 contained
analysis resulted in considerably lower numbers at day 1. For exp-II, culturable and esterase active T. suecica cells 1 day after irradiation,
comparable results were obtained using plate count and MPN analysis but the inactivation increased with higher UV doses. Our results are in
whereas the results using FCM gave considerable higher live/damaged line with previous studies of freshwater green algae Chlorella ellipsoidea,
cell numbers. Chlorella vulgaris, and Scenedesmus quadricanda, and the cyanobacteria
For samples treated with UV doses 100, 150 and 200 mJ/cm2 inacti- M. aeruginosa which showed limited sensitivity to UV-C irradiation
vation of cells was dependent on time of dark incubation as demonstrat- with doses ≤200 mJ/cm2 (Ou et al., 2012; Tao et al., 2010).
ed by FCM (Fig. 2a, c). Generally, the numbers of live/damaged cells Comparing the different analysis methods at day 1 for the samples
decreased during incubation, and were fewer in UV irradiated samples UV irradiated with doses ≤ 200 mJ/cm2 revealed that the numbers of
than in the stained controls. In exp-I the percentage of live/damaged FCM gated cells were higher than the numbers of cfu detected by
cells in the UV irradiated samples (Fig. 2a) decreased throughout the plate count. Such discrepancy between plate count and FCM results
incubation period and amounted to ≤3% at day 22. The samples treated has also been observed in other studies of UV irradiated bacteria and
with 100 mJ/cm2 behaved similar to the stained controls during incuba- alga (Kramer and Muranyi, 2014; Olsen et al., 2015; Schenk et al.,
tion, but at day 22 the percentage of live/damaged cells was lower than 2011). UV induced DNA damage can block transcription and replication,
the stained controls also for samples treated with this UV dose. In exp-II inhibiting growth and reproduction (Oguma et al., 2002; Sinha and
(Fig. 2c) inactivation increased during incubation, and b3% live/ Häder, 2002). DNA damaged cells are not detected as live by growth as-
damaged cells were observed at days 22, 10 and 3 in the samples treated says, though they can express activity (Davey, 2011; Hammes et al.,
with 100, 150 and 200 mJ/cm2, respectively. Regrowth checks per- 2011; Villarino et al., 2003); explaining the contradicting results from
formed at days 22 and 20 for exp-I and exp-II, respectively, were nega- FCM and plate count analysis.
tive for all UV irradiated samples and positive for the control samples Plate count and MPN analysis are based on reproductive capacity
(data not shown). and one could expect these growth assays to give comparable results.
Considerable variations were observed between the results from However, the results obtained by the MPN were similar to the plate
the two experiments when looking at a detailed level. Firstly, the per- count results in exp-II and to FCM in exp-I. This illustrates the challenge
centage of live/damaged cells in the stained controls varied at day 1 of getting reproducible results when analyzing UV irradiated organisms
(Fig. 2a, c), being 92% and 54% in exp-I and exp-II, respectively. Second- with methods that analyze different cellular characteristics. Further,
ly, for the samples UV irradiated with ≤200 mJ/cm2, the inactivation rate growth assays may introduce errors as a majority of the microbes are
varied between the experiments (Fig. 2a, c) and the number of FCM live/ uncultivable (Roszak and Colwell, 1987; Staley and Konopka, 1985). In
damaged cells fluctuated (Table 1). Thirdly, some replicates showed 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 8231 (1222) 13,902 (572) 10,003 (318) 8531 (1437) 488 (348) 3 (1)
3 5248 (183) 12,202 (1012) 8896 (2926) 6402 (3494) 55 (45) 2 (1)
6 4373 (1185) 12,221 (1601) 9453 (343) 5468 (1959) 16 (6) 2 (2)
9 8485 (2377) 12,218 (1549) 10,195 (1183) 4812 (1386) 12 (6) 1 (1)
13 8258 (714) 9949 (2110) 5533 (2666) 2239 (958) 5 (2) 2 (2)
22 4160 (968) 1354 (970) 512 (627) 304 (321) 8 (7) 5 (4)
MPN 1 N11,000 11,000 11,000 11,000 b3 (0–9.5) b3 (0–9.5)
(4200–40,000) (1800–41,000) (1800–41,000) (1800–41,000)
Rate count 1 8667 (577) 7467 (503) 8400 (1249) 5040 (170) b10 b10
Exp-II FCM 1 10,419 (883) 9016 (332) 3946 (1464) 973 (682) 169 (37) 21 (12)
3 9915 (198) 8429 (1518) 2893 (1203) 617 (382) 126 (23) 13 (2)
6 10,688 (954) 5320 (966) 1587 (607) 344 (197) 100 (40) 11 (6)
10 6366 (577) 2663 (363) 597 (275) 180 (45) 73 (11) 7 (1)
15 4327 (476) 1190 (167) 427 (74) 172 (53) 105 (22) 9 (3)
22 2441 (481) 642 (141) 406 (223) 214 (57) 111 (50) 11 (3)
MPN 1 n.d. 4600 930 b3 (0–9.5) b3 (0–9.5) b3 (0–9.5)
(900–20,000) (180–4200)
Rate count 1 10,187 (2510) 4107 (508) 950 (416) 48 (50) b10 b10
274 R.O. Olsen et al. / Marine Pollution Bulletin 103 (2016) 270–275

fluorometry determination would have been a more objective analysis algae species, and the response to environmental changes and inactiva-
method and could potentially have given a slightly different outcome. tion treatments can differ (Jochem, 1999, 2000; Olsen et al., 2015). Al-
However, the MPN method was validated by another researcher and though there are indications that the majority of phytoplankton
by microscopy. species can be detected by the esterase substrates fluorescein diacetate
The FCM results showed that inactivation increased with the time of (FDA) and 5-chloromethylfluoorescein diacetate (CMFDA) (Peperzak
dark incubation. Phytoplankton are photosynthetic organisms requiring and Brussaard, 2011), we are therefore aware that fluorescing signal
light as an energy source (McMinn and Martin, 2013) and dark incuba- from esterase substrates can vary over a large range of intensities
tion will naturally affect their viability over time (Gollasch and David, (Dorsey et al., 1989). Our recommendation at this point is thus to
2010), especially if the cells continue with unchanged activity causing treat the ballast water with UV doses close to 400 mJ/cm2 in order to
them to run out of energy (Jochem, 1999). Additionally, incubation in permanently inactivate the organisms. The variable results for UV
darkness will limit photoreactivation; the repair mechanism which doses 100–200 mJ/cm2 demonstrate the challenge of giving unambigu-
removes UV induced DNA lesions and reverses damages by using the ous recommendations on duration of dark incubation needed for
energy of light (Sinha and Häder, 2002), leaving the cells unrepaired inactivation.
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 Acknowledgments
with other studies the control samples contained reproducible cells
even after 3 weeks of dark incubation (Gollasch and David, 2010; This research was founded by the Norwegian Research Council
Olsen et al., 2015). T. suecica's ability to survive in darkness over time (project BallastFlow, project no. 208653) and Knutsen OAS Shipping
implies it does not overrate effects of UV treatments and makes it an ap- AS, and supported by Solstad Shipping, Stord/Haugesund University
propriate indicator organism for ballast water monitoring. College, VRI Rogaland, UH-nett Vest and TeknoVest. We thank Sandra
Some of the variation on results between exp-I and -II, such as vari- Schöttner (UiB, Bergen, Norway), Stephanie Delacroix, August Tobiesen
ation in the stained controls day 1, difference in inactivation rate when (Norwegian Institute for Water Research, Oslo, Norway) and Per Lothe
irradiated with doses ≤200 mJ/cm2, large standard deviations and more (Knutsen OAS Shipping AS, Haugesund, Norway) for helpful discus-
cells in treated samples than in untreated ones, indicates that the algal sions. Special thanks to Sandra Schöttner for assistance with the UV
culture used in exp-I contained a greater portion of fresh and healthy lamp and experimental set-up.
cells than the culture used in exp-II. Natural sea water shows great var-
iability and contains a diverse community of algae species in different
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