Ultraviolet radiation as a ballast water treatment strategy: Inactivation of phytoplankton measured with flow cytometry

Abstract

This study investigates different UV doses (mJ/cm(2)) 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 ≥400mJ/cm(2) rendered inactivation after 1day as measured by all analytical methods, and are recommended for ballast water treatment if immediate impairment is required. Irradiation with lower UV doses (100-200mJ/cm(2)) gave considerable differences of inactivation between experiments and analytical methods. Nevertheless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥100mJ/cm(2) and ≤200mJ/cm(2) 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.

Full text

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
abstractarticle info
Article history:
Received 27 August 2015
Received in revised form 8 December 2015
Accepted 10 December 2015
Available online xxxx
This study investigates different UV doses (mJ/cm
2
) and the effect of dark incubation on the survival of the algae
Tetraselmis suecica, to simulate ballast water treatment and subsequent transport.
Samples w ere UV irradiated and analyzed by flow cytometry and standard culturing methods. Doses of
≥ 400 mJ/cm
2
rendered inactivation after 1 day as measured by all analytical methods, and are recommended
for ballast water treatment if immedi ate impairm ent is required. Irradiation with lower UV doses (100–
200 mJ/cm
2
) gave considerable differences of inactivation between experiments and analytical methods. Never-
theless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥ 100 mJ/cm
2
and ≤ 200 mJ/cm
2
can be sufficient if the water is treated at intake and left in dark ballast tanks. The variable re-
sults demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation need-
ed 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/).
Keywords:
Tetraselmis suecica
Ultraviolet irradiation
Esterase substrate
Flow cytometry
Inactivation
Dark incubation
1. Introduction
Ships use water as bal last to ensure stability an d 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, 199 1). Some organisms survive in ballast tanks and are
released into new environments. If nonindigenous species adapt and es-
tablish 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 ecosys-
tems, 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 Con-
vention for the Control and Management of Ship's Ballast Water and
Sediments (International Maritime Organization, 2004). Regulation D-
2 of the Convention sets the standard regarding category and concen-
tration of organisms at discharge. The Convention will enter into force
12 months after being ratified by 30 States representing 35% of the mer-
chant 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 treat-
ment 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 nationa l 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 con-
sumption (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;
Marine Pollution Bulletin xxx (2015) xxx–xxx
⁎ Corresponding author.
E-mail address: ingunn.hoell@hsh.no (I.A. Hoell).
MPB-07359; No of Pages 6
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/).
Contents lists available at ScienceDirect
Marine Pollution Bulletin
journal homepage: www.elsevier.com/locate/marpolbul
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

Zimmer and Slawson, 2002). LP lamps emit UV-C radiation, primarily
at 254 nm, which is most effi ciently 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
photoreactivati on 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; Holz inger 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 inac-
tivation 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 uncer-
tain (Olsen et al., 2015). Damaged cells can be unculturable, though they
can be m etabolic active and may pose a health risk (Oliver, 2010).
Further, cellular DNA repair mechanisms can restore the genetic infor-
mation (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 ter-
minology describing the organisms at discharge can be confusing or un-
clear. 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 “organ-
isms 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 compli-
ance 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. Ex-
amples 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 determinin g the concen-
tration of viable organism in ballast water discharge accord ing to
Regulation D-2. Quantification of live ba cteria traditionally relies on
cultivation me thods, 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 detaile d an alysis
(Inte rnational 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 latt er contain both dying and repairable cells.
The current study uses the FCM protocol to elaborate on different UV
doses and the effec t of dark incubation on inactivation of the alga e
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.
2) Quantify effects of different UV doses on T. suecica.
3) Estimate the time of dark incubation required to permanently inac-
tivate the algae treated with UV doses lower than minimum perma-
nently 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 medi-
um to a density of 10
4
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/cm
2
)of
the UV lamp was fixed and the exp osure times used were 155, 233,
311, 622 and 1244 s for UV doses 100, 150, 200, 400 and 800 mJ/cm
2
,re-
spectively. The irradiated sam ples were tr ansferred to sterile 50 ml
polypropylene tubes (Fisher Scientific), so was the control samples, in-
cluding 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 inactiva-
tion effect of different UV doses and dark incubation, and to test wheth-
er this effect was in terpretable with FCM. This was followed by two
complete experiments, denoted as exp-I and exp-II, and an overview
oftheset-upfortheseexperimentsisgiveninFig. 1.
For FCM analysis, th e samples were stained with 5-ca rboxy fluo-
rescein 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 aft er 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.
2 R.O. Olsen et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
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

defined based on plate count results from non-irradiated cells, and re-
gression 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 dil uted in growth medium (24 PPT ar tificial sea water
added 0.12% Substral) in 10-fold series up to 10
− 4
dilution to a total
volume of 1 ml pr. we ll 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 in to green as
detected by the eye, a nd scored against the MPN table for a three-
replicate 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
out at day 22 for samples treated with 100–200 mJ/cm
2
and at day 2
for samples treated with 400 and 800 mJ/cm
2
, and for exp-II at day 20
for samples treated with 100–200 mJ/cm
2
and at day 3 for samples
treated with 400 and 800 mJ/cm
2
. 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 fl asks, 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/cm
2
inactivated the algae perma-
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).Errorbarsindicate1
standard deviation of 3 replicates.
3R.O. Olsen et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
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

of samples irradiated with 400 and 800 mJ/cm
2
displaye d 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/cm
2
and neither did regrowth check per-
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
showed a re lationship between in activation and UV doses. Th is 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 co ntrol samples were
all in the same range, but MPN results showed N 11,000 cells ml
−1
,indi-
cating 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/cm
2
inacti-
vation of cells was dependent on time of dark incubation as demonstrat-
ed 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/cm
2
behaved similar to the stained controls during incuba-
tion, 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 b 3% live/
damaged cells were observed at days 22, 10 and 3 in the samples treated
with 100, 150 and 200 mJ/cm
2
, respectively. Regrow th check s per-
formed at days 22 and 20 for exp-I and exp-II, respectively, were nega-
tive for all UV irradiated samples and positive for the control samples
(data not shown).
Considerable variations were observed betwee n the results from
the two experiments when looking at a detailed level. Firstly, the per-
centage 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. Second-
ly, for the samples UV irradiated with ≤ 200 mJ/cm
2
, the inactivation rate
varied between the experiments (Fig. 2a, c) and the number of FCM live/
damaged cells fluctuated (Table 1). T hirdly, some rep licates showed
large standard deviations; most evident in exp-I (Fig. 2a). Fourthly, at
the first days after UV irra diati on, 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 recommenda-
tions for ballast water management regarding treatment and transport.
UV doses 400 and 800 mJ/cm
2
rendered T. suecica cells unculturable
and without esterase activi ty 1 day after irradiation whereas doses
≤ 200 mJ/cm
2
did not necessarily inactivate the cells. This indicates
that the minim um UV dose that permanently inactivates this algal
specie is somewhere between 200 and 400 mJ/cm
2
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/cm
2
contained
culturable and esterase active T. suecica cells 1 day a fter 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,andScenedesmus quadricanda, and the cyanobacteria
M. aeruginosa which showed li mited sensitivity to UV-C irradiation
with doses ≤ 200 mJ/cm
2
(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/cm
2
revealed that the numbers of
FCM gated cells were higher than the numbers of cfu detecte d 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 as-
says, though they can express activity (Davey, 201 1; 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 obta ined by the MPN were similar to th e 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. Furt her,
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 “b 10” as the results are obtained
by 100 µl being spread on the agar plates. The MPN values show “b 3” 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/cm
2
150 m J/cm
2
200 mJ/cm
2
400 m J/cm
2
800 m J/cm
2
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 N 11,000
(4200–40,000)
11,000
(1800–41,000)
11,000
(1800–41,000)
11,000
(1800–41,000)
b 3(0–9.5) b 3(0–9.5)
Rate count 1 8667 (577) 7467 (503) 8400 (1249) 5040 (170) b 10 b 10
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
(900–20,000)
930
(180–4200)
b 3(0–9.5) b 3(0–9.5) b 3(0–9.5)
Rate count 1 10,187 (2510) 4107 (508) 950 (416) 48 (50) b 10 b 10
4 R.O. Olsen et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx
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

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 incuba-
tion 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 ap-
propriate indicator organism for ballast water monitoring.
Some of the variation on results between exp-I and -II, such as vari-
ation in the stained controls day 1, difference in inactivation rate when
irradiated with doses ≤ 200 mJ/cm
2
, 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 var-
iability and contains a diverse community of algae species in different
cellular phases varying with time and space and may have different tol-
erance 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 re-
quired 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/cm
2
are inactivated
when left in dark ballast tanks over a period of time. Our results also
demonstrate the challenge of giving recommendations regarding dura-
tion of dark incubation needed for inactivation when using lower UV
doses. It is, however, important to keep in mind that controllable exper-
iments in a laboratory differ from a flow-through chamber in a commer-
cial BWTS and that a B WTS 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 pre-
vent inactivation.
UV irradiation as a treatment technology has been criticized due to
the uncertainty regarding inactivation at discharge but is more environ-
mental friendly than chemical disinfection and creates no harmful by-
products (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 stud-
ies 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 ou r FCM protocol is applic able 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 re-
peate d 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 environmen-
tal conditions. The level of metabolic activity can vary between various
algae species, and the response to environmental changes and inactiva-
tion treatments can differ (Jochem, 1999, 2000; Olsen et al., 2015). Al-
though there are in dications 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/cm
2
in order to
permanently inactivate the organisms. The variable results for UV
doses 100–200 mJ/cm
2
demonstrate the challenge of giving unambigu-
ous recommendations on duration of dark incubation needed for
inactivation.
Acknowledgments
This research was founded by the Norwegian Research Council
(project BallastFlow, project no. 208 653) 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 discus-
sions. Specia l thanks to Sa ndra Schöttner fo r assistance with the UV
lamp and experimental set-up.
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