Machine Translated by Google

Chemosphere 308 (2022) 136113

Contents lists available at ScienceDirect

Chemosphere

journal homepage: www.elsevier.com/locate/chemosphere

Evaluating disinfection performance of ultraviolet light-emitting diodes

against the microalgae Tetraselmis sp.: Assay methods, inactivation
efficiencies, and action spectrum
Diya Wen a,b, Yuelu Jiang a,b,**, Daoyi Chen a,b,*
to
School of Environment, Tsinghua University, Beijing, 100084, China
b
Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China

HIGHLIGHTS GRAPHICAL ABSTRACT

• Disinfection performance of Tetraselmis

sp. with different UV-LED wavelengths
was evaluated.
• The MPN assay had the most sensitive
endpoint for UV-treated Tetraselmis sp..
• The action spectrum of Tetraselmis sp.
peaked at 265 nm LED.
• The optimal energy efficiency was ob
tained at 285nm LED.
• The UV action spectrum of inactivation
matched the DNA absorbance spectrum.

ARTICLE INFO ABSTRACT

Handling Editor: Xiangru Zhang Ultraviolet light-emitting diodes (UV-LEDs) are among the most compact devices and safest technologies in water
disinfection systems. However, the validation of different assay methods to evaluate the disinfection performance
Keywords: of different wavelengths (265, 280, 285, and 300 nm) of UV-LEDs toward marine microalgae remains poorly
UV light-Emitting diode characterized. In this study, several detection assays, namely the culture-based most probable number (MPN)
Phytoplankton inactivation
Ballast water treatment
assay, membrane integrity-based vital stain (VS) assay, chlorophyll fluorescence assay, and photochemical
efficiency assay, were compared to assess the viability of the marine microalga Tetraselmis sp., with results
Tetraselmis sp.
Inactivation efficiencies indicating the MPN assay to be the most sensitive. In addition, this study compared the inactivation kinetics,
inactivation efficiency, and energy efficiency of Tetraselmis sp. under different UV wavelengths, as assessed by
the VS and MPN assays. The fluence-response curves of Tetraselmis sp. varied with assay and wavelength, with
Geeraerd's model fitting all fluence-response microalgal inactivation curves. The results showed a non-significant
difference in inactivation efficiency among different wavelengths of UV-LEDs (except for 300 nm) when using the
VS assay. On the contrary, significant differences among all wavelengths were observed with respect to inactivity
efficiency when using the MPN assay. The wavelength of 265 nm exhibited maximum inactivation efficiency,
whereas 285 nm achieved optimal energy efficiency. The UV action spectrum of Tetraselmis sp. exhibited the
peak at 265 nm, a finding which matched well with the absorbance spectrum of DNA. The observations from

* Corresponding author. Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China.
**Corresponding author. Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China.
E-mail addresses: jiang.yuelu@sz.tsinghua.edu.cn (Y. Jiang), chen.daoyi@sz.tsinghua.edu.cn (D. Chen).

https://doi.org/10.1016/j.chemosphere.2022.136113
Received 3 April 2022; Received in revised form July 5, 2022; Accepted 16 August 2022
Available online 22 August 2022
0045-6535/© 2022 Published by Elsevier Ltd.
Machine Translated by Google
D. Wen et al.
This study provides a theoretical basis and technical support for the application of the emerging UV-LED light
sources in the algicidal treatment of marine water.
1.Introduction
The spread of non-indigenous species (NIS) via ballast water has
become a major threat to the biodiversity and integrity of marine eco
systems worldwide and can lead to industrial and economic damage,
posing a threat to public health. Among various NIS, invasive phyto
plankton species have raised significant concerns regarding their ability
to alter resource availability, decrease biodiversity, and even change the
energy flow and nutrient circulation in ecosystems, possibly leading to
the collapse of ecosystems or posing a threat to public safety as a result
of mass proliferation. Numerous events have occurred due to the pro
liferation and adhesion of phytoplankton, which have had severe
impacts on human activities such as thalassotherapy, aquaculture, and
desalination. For instance, in the Scottish Shetland Islands in 2013,
large blooms of the dinoflagellate Dinophysis impacted fish and shellfish
aquaculture and severely affected the local aquaculture industries
through physical interference, de-oxygenation, and shellfish poisoning
(Berdalet et al., 2015; Davidson et al., 2015; al., 2021). In 2014, a
Phaeocystis globosa outbreak bloomed in the coastal waters of Guangxi
in China, posing a threat to the operational safety of the Fangchenggang
nuclear power plant (Kang et al., 2020).
To prevent the spread of NIS, both the Maritime Environment
Protection Committee and the International Maritime Organization (IMO)
approved the guidelines and procedures for uniform implementation of
the International Convention for the Control and Management of Ships'
Ballast Water and Sediments (BWM Convention) in 2004 (IMO, 2004),
which came into force in 2017. The BWM Convention requests that all
ships use a ballast water management system (BWMS) (IMO, 2008a,b).
In addition, the IMO and the United States Coast Guard have approved
different treatment systems by which to minimize the population size of
microorganisms in ballast water, including an initial mechanical filtration
step plus a physical or chemical treatment, or a combination of both
physical and chemical processes.
Ultraviolet (UV) disinfection is one of the most effective disinfection
technologies used to inactivate various waterborne organisms (Linde
nauer and Darby, 1994; Nebot Sanz et al., 2007). The most commonly
used UV-based BWMSs are medium-pressure (MP) and low-pressure
(LP) mercury UV systems, which comprised 46% of all approved
treatment systems as of December 2018. The MP UV system emits a
broad spectrum of UV wavelengths, ranging from 200 to 400 nm; By
contrast, the LP UV system emits monochromatic UV radiation at 254
nm. The broad wavelength spectrum emitted by MP UV makes it
challenging to identify which wavelength causes the microbial inactivation
and to determine the inactivation mechanism. Recently, the use of UV-
emitting diodes (UV-LEDs) in water disinfection has been considered a
potential alternative to the traditional ultraviolet mercury lamps because
the LEDs can emit specific algicidal wavelengths in selected optimum
narrow-wavebands (Song et al., 2016 ; Chen et al., 2017). Furthermore,
LEDs are more widely applicable because of a number of additional
benefits, such as compact size, no need for a warm-up time, and the
absence of mercury (Kebbi et al., 2020). Therefore, UV-LEDs have
particular applications to maritime situations with fragile ecological
environments and to ships where space is valuable and at a premium.
Appropriate detection assays, generating reliable fluence-response
data, are essential to assess the performance of UV disinfection
systems. Several parameters have been used to study the effect of UV
systems, including the vital stain (VS) assay, which is a method for
obtaining quantitative estimates of the membrane integrity of phyto
plankton cells, and the most probable number (MPN) assay, which is a
method based on the viability of phytoplankton cells (Steinberg et al.,
2011; Lundgreen et al., 2018; MacIntyre et al., 2018). The MPN assay
2
Chemosphere 308 (2022) 136113
requires lengthy incubation periods (ranging from days to weeks),
whereas the time required for a VS assay of each sample includes 10
min of incubation and additional time for subsequent counting. These
two detection assays, approved by the IMO and the USCG (IMO, 2008a;
US EPA, 2010) and widely used in research into the inactivation of
phyto plankton (Breeuwer and Abee, 2000; MacIntyre and Cullen, 2016),
can be used to quantify the densities of viable or living phytoplankton cells.
More rapid assays have been developed to assess the number of viable/
living organisms in samples, such as the chlorophyll fluorescence
(CHLF) assay, the adenosine triphosphate assay, and the photochemical
efficiency (Fv/Fm) assay, which reflect the biomass , the cellular energy,
and the photosynthetic activity of phytoplankton, respectively (First and
Drake, 2013; Rantalankila et al., 2016; Bradie et al., 2018; Castro et al.,
2018; First et al., 2018). For example, Fv/Fm is a relative measure of
electron transport efficiency, which has been shown to reflect rates of
phytoplankton production (Barranguet and Kromkamp, 2000), whereas
CHLF measured in vivo is frequently used as a proximal measurement
of the abundance of phytoplankton in water and sediment. However,
previous studies have failed to compare the sensitivity of various assay
methods under different UV wavelengths and various UV fluences.
Therefore, although a number of promising approaches have been
developed, they require further study to assess their comparability, ac
curacy, and predictability in terms of UV-inactivation of phytoplankton.
The action spectrum (spectral sensitivity) of the target organism is
the basis of process design and operation in UV-LED treatment systems
(Song et al., 2016; Kebbi et al., 2020). It can be used to calculate algid
UV fluence at a specific wavelength. The action spectrum has been
studied in various microorganisms, and results have revealed peak
spectral sensitivities ranging from 220 nm to 270 nm. Previous research
has shown that bacteria, such as Bacillus pumilus and the spores of Ba
cillus subtilis, have a peak sensitivity at around 220 nm, whereas
various co lipages, such as MS2, T1UV, and T7, exhibit a peak sensitivity
at around 260 nm ( Beck et al., 2015). There have been only a few
studies on the spectral sensitivity of phytoplankton, and the results were
inconsistent. Sun and Blatchley (2017) reported that the action spectrum
of Tetraselmis sp. showed essentially flat behavior from 254 to 280 nm.
Park et al. (2014) found that the greatest inactivation of Tetraselmis sp.
was achieved at 265 nm, but the action spectrum between 254 and 280
nm was missing from their study. Understanding the fluence response
of the targeted phytoplankton at multiple UV wavelengths would enable
the prediction of microalgal inactivation as a result of wavelength-specific
UV-LED disinfection technologies. Therefore, a systematic study is
needed to illustrate the fluence-response behavior and the action
spectrum of phytoplankton inactivation under algicidal wavelengths (254–
285 nm), using narrow-waveband UV-LEDs.
In the current study, Tetraselmis sp., a potential NIS and an indicator
organism in BWMS, was used to verify the inactivation kinetics of UV
LEDs on phytoplankton. Various assays, namely VS, MPN, Fv/Fm, and
CHLF assays, were used to evaluate and compare the inactivation
efficiencies and to obtain a credible fluence-response behavior profile.
In addition, the inactivation rate constants of algicidal UV wavelengths
(254 nm LP UV, 265, 280, 285, and 300 nm LEDs) were studied to
provide a convincing UV action spectrum for Tetraselmis sp., which
would be valuable for future development of algicidal technologies and
systems based on UV-LEDs.
2. Materials and methods
2.1. UV-LED set-up
The experimental device is a UV-LED system (Nikkiso Giken, Japan)
Machine Translated by Google
D. Wen et al.
equipped with peak emission wavelengths of 265 nm, 280 nm, 285 nm, and 300
nm. The emission spectra of UV-LEDs were determined using a micro-spectrometer
(USB6500; JINGYI, China), and the results are shown in Fig. S1. The UV fluence
rate of different UV-LEDs was adjusted as 1.5 ± 0.12 mW/cm2
, the distance between the sample and UV-LEDs was
1 cm. In addition, collimated beam apparatus equipped with LP mercury UV (LP
UV) was also used to investigate the inactivation efficiency at 254 nm. The fluence
rate of LP UV was adjusted to 1.46 ± 0.16 mW/cm2 . UV fluence rates of the UV-
LED and LP UV devices were measured and calibrated using a potassium
ferrioxalate actinometer (Goldstein and Rabani, 2008; Bolton et al., 2011). Detailed
information, including device configuration, system schematics, and the
measurement of optical parameters, had been described in previous studies (Wen
et al., 2021).
2.2. Experimental procedure
The cultures of Tetraselmis sp. (obtained from the Center for Collections of
Marine Algae, Xiamen University, Xiamen, China) were main tained and grown in
f/2-Si medium prepared with sterilized natural seawater from Yan Tian Port,
Shenzhen, China (pH 8.2 and salinity 35 PSU), under a 14-h/10-h light/dark cycle
and 60 ÿmol photons/m2 /s at 20 ± 0.5 ÿC (Wen et al., 2021). The culture
suspensions in the loga rithmic phase were diluted to achieve a cell concentration
of 6–9 × 104 cells/mL. Tetraselmis sp. suspensions in a Peri dish (Ø 50 mm) were
irradiated by UV-LEDs or LP UV devices at particular wavelengths. UV influences
tested in this study ranged from 50 to 1000 mJ/cm2 . The exposure times (s) were
calculated as the target UV fluence over the UV fluence rate (mW/cm2 ). The
sample volume of each experiment was 30 mL. Triplicate subsamples were taken
to test for the various parameters.
The determination of fluence-response curves was carried out according to the
methodology described in previous research (Beck et al., 2017; Rattanakul and
Oguma, 2018).
2.2.1. VS assay
The cell density were counted by a flow cytometer (FACSCCalibur; Becton
Dickinson Co., USA) with CountBright™ Absolute Counting Beads (Invitrogen™,
USA) and nucleic acid stain SYTOX Green (S7020, Invitrogen™, USA), following
the methodology described previously (Wen et al., 2021).
2.2.2. MPN assay
The MPN assay was used to evaluate the viability of Tetraselmis sp. after
exposure to UV-LED radiation following the methodology of Cullen and MacIntyre
(2016). The phytoplankton suspension was subjected to seven 1:10 serial dilutions
with sterile f/2 Si medium with three tripli cate subsamples per dilution. All tubes
were incubated for 4 weeks at 25 ÿC to ensure that all samples had sufficient time
for adequate growth to occur. Tubes were scored as positive or negative using a
96-well plate reader (Spectramax i3, Molecular Devices, USA). The excitation
wave length of 430 nm and the emission wavelength of 680 nm were used to
quantify the fluorescence responses of the cultures (Sun and Blatchley, 2017).
2.2.3. Fv/ Fm and CHLF assays
Following exposure to a saturating light pulse that closed photo system II
(PSII) reaction centers, initial fluorescence (F0) and maximum fluorescence (Fm)
were determined using a pulse-modulated fluorescence fluorescence system
(FL3500; Photon Systems Instruments, Czech Republic) after a 15-min period of
dark adaptation. The maximum quantum yield of PSII (Fv/Fm) was used to reflect
the photosynthetic efficiency, which was defined as Eq. (1) (Baker, 2008).
Fm ÿ F0
(1)
FV / Fm = FM
3
Chemosphere 308 (2022) 136113
The in-vivo fluorescence of the algal samples before and after UV exposure
was measured by the in-vivo module in the chlorophyll fluo rometer (Model 7200;
Turner Designs, USA). The unit is the relative fluorescence unit (RFU).
2.2.4. DNA damage analysis
The Tetraselmis sp. cultures were incubated for 0, 3, 6, 12, 24, or 48 h in batch
mode after being treated with UV-LEDs of different wavelengths at UV fluence of
200 mJ/cm2 . All experiments were conducted at 25 ÿC with
three biological replicates of each sample. After incubation, each culture was
pelleted by centrifugation (3724 g for 40 min at 4 ÿC) and the pellet was quickly
frozen and stored at ÿ 80 ÿC for subsequent testing.
Cyclobutane–pyrimidine dimer (CPD)-DNA, which make up around 60% of the
UV-induced DNA damage products (Sinha and H¨ ader, 2002), were analyzed as
described previously (Hull et al., 2017). A DNeasy Blood & Tissue Kit (QIAGEN,
USA) was used to extract DNA from Tet raselmis sp., the extracted DNA being
detected spectrophotometrically by a NanoDrop 2000 UV–Vis spectrophotometer
(ThermoFisher, USA).
An OxiSelect UV-Induced DNA Damage ELISA Kit (Cell Biolabs, USA) was used
to quantify CPDÿ DNA using a microplate reader (Epoch; BioTek Instruments,
USA).
23. Data analysis and statistics
2.3.1. Comparison of relative UV responses from different assays
The relative responses to UV of assays based on different physio logical
parameters, such as membrane integrity, culturability, photo chemical efficiency,
and chlorophyll fluorescence, were calculated by Eq. (2), where r and r0 are the
measured responses of the UV-irradiated sample and the control sample,
respectively. Variations in UV wave lengths or fluences were fitted with linear or
non-linear functions, the half-maximal effective UV fluence (ED50) was determined
(Table S1).
The ED50 for a particular assay method, based on membrane integrity, culturability,
photochemical efficiency, or chlorophyll fluorescence, was derived from a UV
fluence dose-response curve, in which the ED50 is the UV fluence dosage at
which 50% of the desired maximum response
occurs.
r
Relative response (% of control) = × 100 (2)
r0
2.3.2. Inactivation kinetics
The inactivation kinetics model proposed by Geeraerd et al. (2000) was applied
to the fluence-response curve, which includes a shoulder and/or a tail region.
Geeraerd's model is derived from Eq. (3).
Nt Nres 10ktl Nres
= 10ÿ (3)
N0 kF ( 1 ÿ N0 )( 1 + ( 10k(tlÿ t) ÿ 10ÿ kt ) ) + N0
where N0 and Nt are the initial and residual concentration of cells of Tetraselmis
sp. (cells/mL) at time 0 and t, respectively. The linear part of Geeraerd's model
conforms to first-order kinetics, and the slope of the linear part of the relationship
was used to represent the fluence-based inactivation constant k; t1 is the shoulder
length (mJ/cm2 ), and the calculations of k and tl refer to Rattanakul and Oguma
(2018). Nres implies the existence of a subpopulation and would lead to the tailing
of the inactivation curve. The Geeraerd's model can be reduced to Eq. (4), if only
the shoulders are considered in the inactivation curve.
Nt
log ÿ 10ÿ kt ) ÿ ktl + kF (4)
= log( 1 + 10k(tlÿ t)
N0
2.3.3. Energy efficiency
The electric energy for each 2-log inactivation (EE,2), as determined by Eq.
(5), was used to represent the energy efficiency (Rattanakul and Oguma, 2018),
where A is the irradiation area (cm2 ), F2 is the UV
Machine Translated by Google

D. Wen et al. Chemosphere 308 (2022) 136113

fluence (mJ/cm2 ) required to obtain 2-log inactivation, V is the volume of the
sample (cm3 ), C is the wall-plug efficiency of different UV-LEDs,
WF is the water factor, ÿ is the absorbance for a 1-cm path length ), and l is the
(cmÿ 1vertical path length of the water in the Petri dish
(cm) (Eq. (6)).
A× F2
EE,2 = 3.6 × 103 × V × C × WF
1 ÿ 10ÿ ÿl
WF =
ÿlln(10)
2.3.4. Data statistical analysis
Analysis of covariance was used to indicate a difference in the inactivation
rate in the linear part of fluence-response curves, with p values equal to or less
than 0.05 indicating a significant difference. In addition, goodness-of-fit testing of
the inactivation kinetic models for Tetraselmis sp. was conducted Microsoft Excel
(Microsoft Excel 2013; Microsoft, USA), and root mean square error (RMSE)
was determined to assess the goodness-of-fit with the observed data. A lower
value for RMSE indicated a predicted value (from the model) which fitted more
closely to the observed data.
3. Results and discussion
3.1. Measurements of UV sensitivity from different assays
The relative response of Tetraselmis sp. to exposure to UV-LED of different
wavelengths was assessed using different assays (Fig. 1 and Table S2). The
VS assay, using SYTOX Green as the stain, reflected the membrane integrity of
Tetraselmis sp. after exposure to UV radiation,
whereas the MPN assay, based on the viability of Tetraselmis sp., reflected the
ability of the microalga to reproduce or complete its life cycle. The membrane
integrity of Tetraselmis sp. assessed by the VS assay decreased progressively
in response to 50–300 mJ/cm2 , and then
decreased significantly to 400 mJ/cm2 at all wavelengths tested except for 300
nm, indicating that the cell membrane of the microalga was more sensitive to UV
(5) radiation at 265–285 nm than at 300 nm. The viability of Tetraselmis sp
determined by the MPN assay decreased significantly in response to increasing
UV fluence at all UV wavelengths.
(6)
On the other hand, photosynthetic activity based on Fv/Fm or CHLF was
insensitive to increasing UV fluence. In the CHLF assays, the decrease in CHLF
of Tetraselmis sp. in response to increasing UV fluence was only detected over
the range 0–100 mJ/cm2 , with more than 60% of CHLF at
100 mJ/cm2 being retained at all UV wavelengths tested. This result is in line
with findings from a previous study, where the residual CHLF of Microcystis
aeruginosa and Phaeocystis globosa was up to 90% after
exposure.
The ED50 of either assay is the median effective UV fluence dose that
produces 50% of the maximal response (Table S1). The ED50 values assessed
by the VS assay showed no significant differences among treatments by 265–
285 nm UV-LEDs (p > 0.05), although the values were 3- to 6-fold higher than
the corresponding values determined by the MPN assay. The lower the value of
ED50, the greater the UV sensitivity of the assay. The ED50 values obtained
by the Fv/Fm and CHLF assays could not be calculated using the four-parameter
log logistic model, indicating that photosynthesis-related performance was not
sensitive to UV treatments over the fluence range 0–600 mJ/cm2 . According to
a previous study, the ED50 values of Chlorella autotrophica, Chaetoceros
calcitrans, and Phaeocystis globosa determined by the Fv/Fm assay were close
to or higher than 1000 mJ/cm2 (Romero-Martínez et al., 2016).

Fig. 1. Effect of UV-LEDs irradiation on different detection assays and UV wavelengths of Tetraselmis sp.. a)-d) vital stain, e)-h) most probable number, i)-l) Fv/Fm,
and m )-p) chlorophyll fluorescence.

4
Machine Translated by Google

D. Wen et al. Chemosphere 308 (2022) 136113

3.2. Inactivation kinetics of Tetraselmis sp. assessed by VS and MPN Table

assays 1 Fitting parameter of the fluence-response curves of Tetraselmis sp. based on
different UV wavelength and detection assays.
The inactivation kinetics of Tetraselmis sp. over the fluence-response curves ÿ VS assays MPN assays
were evaluated using the VS and MPN assays. As shown in Fig. 2, there was an (nm)
tl D4 RMSE tl (mJ/ D4 RMSE
exponential increase in response with increasing fluence, with a shoulder in its (mJ/ D1 (mJ/ cm2 ) D1 (mJ/
initial stage of response to increasing UV fluence. cm2 ) (mJ/cm2 ) cm2 ) (mJ/cm2 ) cm2 )
Geeraerd's model closely fitted all fluence-response curves from either assay: the 265 410 581 1093 0.205 64.3 107 235 0.315
goodness-of-curve-fitting at all wavelengths was based on RMSE (Table 1). ± ± ± ± ± ±
Exponential UV-response curves had also been observed in previous studies on 51.5 24.1 56.8 7.89 5.44 1.92
280 409 610 1212 0.144 86.0 139 297 0.310
bacteria and viruses, including Bacillus subtilis spores, Pseudomonas aeruginosa,
± ± ± ± ± ±
and adenoviruses (Beck et al., 2017; Rattanakul and Oguma, 2018), with the
41.3 16.1 59.6 9.46 6.06 4.14
shoulder of the response curve being one of the common features. In exponential 285 356 596 1315 0.121 112 241 628 0.383
curves, the shoulder indicates some factors that affect the overall inactivation effi ± ± ± ± ± ±
37.7 14.3 56.0 14.3 6.12 18.5
300 – > > 0.086 125 460 1538 0.337
science, such as the microorganism being clumped, or the rate of repair of essential
2000 2000
components being higher than the rate of destruction (Mossel et al., 1995), or the ± ± ±121
22.9 13.1
damage being cumulative rather than immediately fatal when a large number of the
same target organism is inactivated (Geeraerd et al., 2000). VS: vital stain, MPN: most probable number. Data are shown as mean ± standard
deviation.
The length of the shoulder (tl) (Table 1) in the response curve represents the
difficulty or ease of the inactivation in its initial stage. In the present investigation,
the tl on the fluence-response curves varied ac cording to the UV wavelength and
the assay method, with values in the range 356–410 mJ/cm2 for VS assays and
64.3–125 mJ/cm2 for MPN assays. The tl in fluence-response curves based on
MPN assays increased significantly in response to increasing UV wavelengths (p <
0.05), with viability being largely suppressed at 265 nm LED. To the contrary, with
the VS assay, the t1 value at 265 nm was slightly longer than for other wavelengths,
but not significantly (p > 0.05). Compared with the UV fluence required for 1-log10
(D1) and 4-log10 (D4) from previously published studies (Table 1), Tetraselmis sp.
showed higher D1 but lower D4 values than those of the cyanobacterium Microcystis
aeruginosa or the green alga Chlorella ellipsoidea (Tao et al., 2010; Romero-
Martínez et al., 2016), suggesting that more energy might need to be consumed to
inactivate Tetraselmis sp. than for the other algae in the initial stage of UV radiation.

3.3. Inactivation efficiency

Inactivation rate constants (k) were calculated from the linear part of the fluence- Fig. 3. The inactivation rate constants of Tetraselmis sp. under different UV
wavelengths/light sources. Living assays: vital stain, Viability assays: super script
response curves (Fig. 2), using Eq. (4) to compare the UV inactivation efficiencies
a, b and c represent plate cultivation, most probable number and growth
at different wavelengths. The kVS is the inactivation rate constant based on the
monitoring, respectively.
VS assay, whereas the kMPN is the inactivation rate constant based on the MPN
assay (Fig. 3). The k values

Fig. 2. Inactivation behavior and model fittings for Tetraselmis sp. under exposure to 265, 280, 285, and 300 nm UV-LEDs. The log inactivation was assessed by a) vital
stain (VS) assay, and b) most probable number (MPN) assay.

5
Machine Translated by Google
D. Wen et al.
varied between different assays and with different UV-LED wavelengths.
For the VS assay, there was no significant differences in kVS values
among the 265, 280, and 285 nm wavelengths (n = 12, p > 0.05), although,
when the MPN assay was used, significant differences in the kMPN
constants were found among the 265, 280, 285, and 300 nm wavelength
LEDs (n = 10, p < 0.05). Regardless of the assay method, the maximum k
value within the wavelength range of 265–300 nm in the present study
was obtained at 265 nm.
The value of k quantifies the UV tolerance of an organism: a high k
indicates high UV sensitivity and, therefore, low UV tolerance (Rivas-
Zaballos et al., 2021). According to Fig. 2, comparable damage to cell
membranes occurred when the microalga was exposed to UV irradiation
in the range 265–285 nm, indicating that the cell membrane of Tetra
selmis sp. has similar tolerance to the different UV wavelengths. How
ever, when the photo-repair or regrowth of microorganisms is taken into
account in the analysis of the k (ie, kMPN) value, the viability of the
microalgal cells was more sensitive to specific UV wavelengths than the
integrity of their cell membranes. As noted in previous studies, the
inactivation effect of UV irradiation is related to inhibiting the organ ism's
viability rather than to killing the cells directly (Blatchley III et al., 2018).
Thus, the MPN assays provide a more comprehensive assessment that
depends on the immediate UV tolerance and photo-repair ability (or
regrowth ability) of the microalgae tested.
Fig. 3 presents the inactivation rate constants from the literature of
Tetraselmis sp. measured by different assays under various wavelengths/
light sources. Reported values from using LP UV and viability-focused
assays, such as MPN, plate culture, and liquid culture growth monitoring,
were between 0.020 and 0.034 cm2 / mJ, which is in line with or slightly
higher than the values of 0.017 cm2 / mJ obtained in the current study.
The findings in the present study were consistent with the conclusion of
other authors, with slightly higher UV tolerance of Tet raselmis sp. than of
other phytoplankton species, such as Chlorella vul garis with 0.032 cm2 /
mJ, Tisochrysis lutea with 0.044 cm2 /mJ, Phaeodactylum tricornutum
with 0.054 cm2 /mJ, and Anabaena sp. with 0.042 cm2 /mJ (Tao et al.,
2010; Romero-Martinez et al., 2020; Rivas-Zaballos et al., 2021),
indicating that Tetraselmis sp. was an appropriate indicator organism for
such studies, with considerable UV tolerance in BWMS development (Sun
and Blatchley, 2017; Lundgreen et al., 2019).
The k values for the inactivation of Tetraselmis sp. in the present and
previous studies using living-focused assays, such as combined
fluorescein microscopy (vital stain), or flow cytometry, ranged from 0.0008
to 0.006 cm2 /mJ (Olsen et al., 2016; Lundgreen et al., 2019) , which were
one or two orders of magnitude lower than those of viability-focused
assays. It showed that the assay method greatly influenced the evaluation
of inactivation rate of microalgae irradiated by UV.
Different results may still be obtained using the same assay method
on different experimental set-ups, such as UV fluence rate, initial algal cell
density, and UV wavelength. In general, the higher the UV fluence rate,
the higher the inactivation rate (Nyangaresi et al., 2018; Qiao et al., 2018).
For instance, the UV fluence rate used by Romero-Martinez et al. (2020)
(30 mW/cm2 ) was 20 times that obtained in the current study, and the k
value was 1.5 times the corresponding value determined in the current
study. However, the lower the initial algal concentration, the higher the UV
transmittance (UVT), which can compensate for the effect of a low UV
fluence rate on the inactivation efficiency. The UV fluence rate (0.25 mW/
cm2 ) and the initial algal cell density used by Lundgreen et al. (2019)
were 5.82 times and 10–20 times lower than those in the current study,
respectively; Thus, the k value is still higher than this study. In addition,
UV sources are another reason for the difference in k value under the
same UV fluence rate, assay method and initial algal cell density. The
kMPN values obtained by UV-LEDs at four wavelengths in the present
study were all lower than those in the studies of Sun and Blatchley (2017),
which used MP UV (200–400 nm) sources; the narrow-band wavelengths,
which were obtained by the use of optical filters, introduced variations in
the estimates of a rate constant based on regression analysis. In general,
published studies into the
6
Chemosphere 308 (2022) 136113
UV-inactivation of microalgae involve different assay methods,
experimental set-ups, and conditions, so the systematic comparison of k
is needed to obtain a reasonable conclusion and comparison.
3.4. Action spectrum and DNA absorption spectrum
Fig. 4a illustrates the inactivation constants and DNA absorption
characteristics as a function of wavelength, known as the action spectrum
and the DNA absorption spectrum of the test organism, respectively. Both
DNA absorbance and inactivation constants were normalized to their
corresponding values at 254 nm. In addition, in order to achieve the
comparability of the action spectrum with the previous study, the
inactivation efficiency of 254 nm (LP UV) was also tested, and the fluence-
response profile and inactivation constant are shown in Fig. S2 and Fig .
4a , respectively.
Knowledge of the action spectrum of model microorganisms is vital for
improving UV fluence monitoring and for developing practical UV LED
disinfection technology guidelines. The most effective wave lengths for
the inactivation of Tetraselmis sp. is 265 nm. The kMPN values at 265 nm
UV-LEDs are 1.48, 1.18, 3.14, and 9.20 times higher than those of 254
nm, 280, 285, and 300 nm UV-LEDs, respectively (Fig. 4a ) . In addition,
the action spectrum mirrored the peak wavelength of DNA absorption.
Such consistency suggested that the mechanism of Tetra selmis sp.
inactivation may be due to photochemical damage to DNA rather than to
other biomolecules. This result might provide a possible explanation as to
why higher UV fluence rates were used in previous studies, but that
damage to non-DNA biomolecules, such as chlorophyll a and proteins,
was limited. Furthermore, this finding is consistent with our results
regarding the variation in CHLF and Fv/Fm following UV irradiation.
Previous studies had also investigated the inactivation behavior of
Tetraselmis sp. at wavelengths of 254 nm–300 nm, using MP UV with
optical filters (Sun and Blatchley, 2017), which may result in low wavelength
accuracy and inconsistent average UV fluence rates. Thus, the algicidal
effect in the 254–280 nm wavelength range could not be clearly
distinguished, and the action spectrum had a low resolution within the
range 254–285 nm. In the present study, more detail is shown for the
action spectrum in the range 254–285 nm, with the maximum response
peak being obtained at 265 nm (Fig. 4a).
3.5. DNA damage products of Tetraselmis sp.
It has been confirmed that UV radiation induces two of the most
abundant mutagenic and cytotoxic products of DNA damage, namely
CPDs and 6–4 photoproducts, which make up around 60% and 20–30%
of the UV-induced DNA damage products, respectively ( Sinha and H¨
ader, 2002). The DNA-damaging effects of the UV wavelengths 265, 285,
and 300 nm, as well as those of the different UV-fluence rates, on the
formation of CPDs in the cells of Tetraselmis sp. were evaluated. The
fluence-dependent CPD concentration at different wavelengths is shown
in Fig. 4b. In this study, irradiation with 265 nm LEDs was found to cause
CPD formation very efficiently. The UV fluence and the wave length are
the main reasons for any discrepancies between CPD forma tion. The
mean ± standard deviation CPD concentration in Tetraselmis
sp. after 265 nm irradiation at UV influences of 200 mJ/cm2 and 400 mJ/
cm2 was 8.16 ± 4.94 and 13.7 ± 9.77 ng/mL, respectively, being
comparable with the values reported from other studies (Hull et al., 2017).
Song et al. (2016) concluded that wavelengths between 255 nm and 265
nm, corresponding with the absorbance spectrum of DNA, can induce
higher levels of DNA damage. A similar trend was found under 285 nm
LED irradiation, but the formation of CPDs was significantly lower at that
wavelength than in samples irradiated at 265 nm LED (p <
0.05). The wavelengths between 280 nm and 285 nm were widely
separated from the absorption peak of DNA. The inactivation mechanism
appears to be due mainly to the destruction of algal biomolecules such as
proteins and lipids (Kalisvaart, 2004). UV-C irradiation can cause more
significant DNA damage than UV-B (Sinha and Hader, ¨
Machine Translated by Google
D. Wen et al.
Fig. 4. UV inactivation action spectrum and DNA absorption spectrum for Tetraselmis sp (a), UV-induced cyclobutane–pyrimidine dimers (CPD) concentration and
fitted curves at different UV-LEDs (b).
2002), which is consistent with the log inactivation results from the
current study. No significant levels of CPDs were detected following 300
nm LED irradiation, and the goodness-of-fit of the equation was weak
(r2 = 0.83). A possible reason for this may be because UV-A radiation is
poorly absorbed by DNA and is thus less efficient at inducing damage
to DNA. However, the primary mechanism of UV-A inactivation involves
an indirect effect, by the generation of reactive intermediates and
oxidative damage to DNA and other cellular components (Jiang et al.,
2009; Xiao et al., 2018).
3.6. Energy efficiency
In addition to considering the inactivation rate, the power
consumption caused by the different wall-plug efficiencies (WPE) of the
various UV-LEDs should be considered in order to meet the current
applicability of each system. The electrical energy per 2-log10 inactivity
(EE,2) was used to compare the energy efficiencies of different UV
LEDs (Table S3). According to the initial algal cell density concentration
in the current study (an average of 7.5 × 104 cells/mL), the logarithmic
inactivation required to comply with the IMO D-2 standard (10 cells/ mL)
is 3.87 log. Extrapolation of k showed that the UV fluences of 228, 285,
and 611 mJ/cm2 for 265, 280, and 285 nm LEDs, respectively, based
on MPN assays, would be required to reach the IMO D2 level; this cor
responded to a minimum value at 265 nm LED. However, given careful
consideration of the algicidal efficiency and energy efficiency of different
UV-LEDs, 285 nm is the optimal wavelength for inactivating Tetraselmis
sp., which was 0.20 kWh/m3 /2-log, based on MPN assays.
Ultraviolet LEDs have advantages in large-scale application systems,
such as ballast water treatment systems for ocean transportation, due
to their long lifespan, absence of pollution, and low maintenance costs.
At present, the application of short-wavelength UV LEDs (265 nm) is
mostly affected by the efficiency of WPE. LED boards with high
arrangement-densities need to be used to meet the required UV power.
Thus, higher costs are caused in practical applications. It makes the 285
nm LED, a wavelength with both strong algicidal and high WPE prop
erties, advantageous in practical applications. Currently, the WPE of
commercial-grade and research-grade LEDs have been greatly
enhanced, reaching 5% and 20%, respectively, at a wavelength of 285
nm (Sun et al., 2019). If the price of UV LED chips is reduced, the impact
of wall-plug efficiency on cost will be compensated. From 2008 to 2018,
the power of UV LEDs increased twenty-fold and the cost decreased
ten-fold. By 2026, UV-LEDs are expected to be as cheap as $0.10/mW
(Lawal et al., 2018). Combined with the above, UV-LEDs have broad
prospects for practical applications in the field of marine engineering in
the future.
7
Chemosphere 308 (2022) 136113
4. Conclusion
UV-LED irradiation has proven to be an effective way of achieving
inactivation of Tetraselmis sp. in seawater. Choosing the right assay
from the options available can be a challenging task and will influence
decisions related to UV and UV-based treatments, such as reactor
design, inactivation fluence setting, etc. In the present study, the most
sensitive response of Tetraselmis sp. was the culturability of the
microalga, quantified by the MPN assay. The highest inactivation rate
was 0.0236 cm2 /mJ obtained at 265 nm LED. The best energy
efficiency was 0.20 kWh/m3 /2-log obtained at 285 nm LED. It is
challenging to use the Fv/ Fm or CHLF assays to reflect the viability of
phytoplankton immediately after ultraviolet radiation, because
photosystem II and chloroplasts are not the biological targets of UV inactivation.
The emerging UV-LED light sources can construct the UV action
spectrum of Tetraselmis sp. more accurately than mercury lamps, a
finding which is important for the validation of UV-LED disinfection
systems. Tetraselmis sp. exhibited a maximum peak of inactivation at
265 nm, which matched well with the relative absorbance spectrum of
DNA. The yield of DNA damage products (in terms of CPDs) confirmed
that the inhibition of culturability caused by DNA damage is the primary
inactivation mechanism of Tetraselmis sp. by UV. These results can be
used in the development of algicidal seawater disinfection systems,
based on UV-LEDs.
CREdiT authorship contribution statement
DW = Conceptualization, methodology, formal analysis, investigation,
data curation, writing (original draft, review and editing), visualization.
YJ = Conceptualization, methodology, formal analysis and validation,
writing (review and editing), supervision, funding acquisition. DC =
Methodology, validation, project administration, funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Machine Translated by Google
D. Wen et al.
Acknowledgments
This work was supported by the Sustainable Development Project of
Shenzhen (Grant no. KCXFZ20201221173401005), the Stable Support
Funding from Technology and Innovation Commission of Shenzhen
(Grant no. WDZC20200818183253001) and Research Grant from
Shenzhen International Graduate School, Tsinghua University (Grant
no. JC2021002) to Yuelu Jiang.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/
j.chemosphere.2022.136113.
References
Baker, N.R., 2008. Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu.
Rev Plant Biol. 59 (1), 89–113. https://doi.org/10.1146/annurev.
arplant.59.032607.092759.
Barranguet, C., Kromkamp, J., 2000. Estimating primary production rates from
Photosynthetic electron transport in estuarine microphytobenthos. Mar. Ecol. Prog.
Ser. 204, 39–52. https://doi.org/10.3354/meps204039.
Beck, SE, Wright, HB, Hargy, TM, Larason, TC, Linden, KG, 2015. Action spectra for
validation of pathogen disinfection in medium-pressure ultraviolet (UV) systems.
Water Res. 70, 27–37. https://doi.org/10.1016/j.watres.2014.11.028.
Beck, S.E., Ryu, H., Boczek, L.A., Cashdollar, J.L., Jeanis, K.M., Rosenblum, J.S.,
Lawal, OR, Linden, KG, 2017. Evaluating UV-C LED disinfection performance and
investigating potential dual-wavelength synergy. Water Res. 109, 207–216. https:// doi.org/
10.1016/j.watres.2016.11.024.
Berdalet, E., Fleming, LE, Gowen, R., Davidson, K., Hess, P., Backer, LC, Moore, SK,
Hoagland, P., Enevoldsen, H., 2015. Marine harmful algal blooms, human Health and
wellbeing: challenges and opportunities in the 21st century. J. Mar. Biol. Assoc. UK 2015
(1), 1–31. https://doi.org/10.1017/S0025315415001733.
Blatchley III, E.R., Cullen, J.J., Petri, B., Bircher, K., Welschmeyer, N., 2018. The
biological basis for ballast water performance standards: "Viable/Non-Viable" or "Live/
Dead. Environ. Sci. Technol. 52 (15), 8075–8086. https://doi.org/10.1021/acs.est .
8b00341.
Bolton, JR, Stefan, MI, Shaw, PS, Lykke, KR, 2011. Determination of the quantum yields of
the potassium ferrioxalate and potassium iodide–iodate actinometers and a method for
the calibration of radiometer detectors. J. Photochem. Photobiol., A: Chemistry 222
(1), 166–169. https://doi.org/10.1016/j.jphotochem.2011.05.017.
Bradie, J., Broeg, K., Gianoli, C., He, J., Heitmüller, S., Curto, A.L., Nakata, A., Rolke, M.,
Schillak, L., Stehouwer, P., Vanden Byllaardt, J., Veldhuis, M., Welschmeyer, N.,
Younan, L., Zaake, A., Bailey, S., 2018. A shipboard comparison of analytical methods
for ballast water compliance monitoring. J. Sea Res. 133, 11–19. https://doi.org/ 10.1016/
j.seares.2017.01.006.
Breeuwer, P., Abee, T., 2000. Assessment of viability of microorganisms employing
fluorescence techniques. Int. J. Food Microbiol. 55 (1), 193–200. https://doi.org/ 10.1016/
S0168-1605(00)00163-X.
Castro, MCT, Veldhuis, MJW, Fileman, TW, Hall-Spencer, JM, 2018. Different
Approaches and limitations for testing phytoplankton viability in natural assemblies and
treated ballast water. Mar. Pollut. Bull. 137, 172–179. https://doi.org/10.1016/
j.marpolbul.2018.10.013.
Chen, J., Loeb, S., Kim, J.-H., 2017. LED revolution: fundamentals and prospects for UV
disinfection applications. Environ. Sci.: Water Res. Technol. 3 (2), 188–202. https://
doi.org/10.1039/c6ew00241b.
Cullen, JJ, MacIntyre, HL, 2016. On the use of the serial dilution culture method to enumerate
viable phytoplankton in natural communities of plankton subjected to ballast water
treatment. J. Appl. Phycol. 28 (1), 279–298. https://doi.org/10.1007/
s10811-015-0601-x.
Davidson, K., Whyte, C., Aleynik, D., Dale, A., Gontarek, S., Kurekin, AA, McNeill, S., Miller,
P.I., Porter, M., Saxon, R., Swan, S., 2021. HAB reports: online early warning of harmful
algal and biotoxin risk for the Scottish shellfish and finfish aquaculture industries. Front.
Mar. Sci. 8, 631732 https://doi.org/10.3389/fmars.2021.631732.
First, MR, Drake, LA, 2013. Life after treatment: detecting living microorganisms following
exposure to UV light and chlorine dioxide. J. Appl. Phycol. 26 (1), 227–235. https://
doi.org/10.1007/s10811-013-0049-9.
First, MR, Robbins-Wamsley, SH, Riley, SC, Drake, LA, 2018. Assessment of variable
fluorescence fluorometry as an approach for rapidly detecting living
photoautotrophs in ballast water. J. Sea Res. 133, 53–59. https://doi.org/10.1016/j.
seares.2017.02.012.
Geeraerd, AH, Herremans, CH, Van Impe, JF, 2000. Structural model requirements to
describe microbial inactivation during a mild heat treatment. Int. J. Food Microbiol. 59 (3),
185–209. https://doi.org/10.1016/S0168-1605(00)00362-7.
Goldstein, S., Rabani, J., 2008. The ferrioxalate and iodide–iodate actinometers in the UV
region. J. Photochem. Photobiol., A: Chemistry 193 (1), 50–55. https://doi.org/ 10.1016/
j.jphotochem.2007.06.006.
Hull, N.M., Isola, M.R., Petri, B., Chan, P.-S., Linden, K.G., 2017. Algal DNA repair
kinetics support culture-based enumeration for validation of ultraviolet disinfection ballast
water treatment systems. Environ. Sci. Technol. Lett. 4 (5), 192–196. https:// doi.org/
10.1021/acs.estlett.7b00076.
8
Chemosphere 308 (2022) 136113
IMO, 2008a. Guidelines for approval of ballast water management systems (G8).
Resolution 174 (58).
IMO, 2008b. Procedure for approval of ballast water management systems that make use
of active substances (G9). Resolution 169 (57).
IMO, 2004. International Convention for the Control and Management of Ships' Ballast Water
and Sediments. BWM/CONF/36.
Jiang, Y., Rabbi, M., Kim, M., Ke, C., Lee, W., Clark, R.L., Mieczkowski, P.A.,
Marszalek, PE, 2009. UVA generates pyrimidine dimers in DNA directly. Biophys. J. 96
(3), 1151–1158. https://doi.org/10.1016/j.bpj.2008.10.030.
Kalisvaart, B., 2004. Re-use of wastewater: preventing the recovery of pathogens by
using medium-pressure UV lamp technology. Water Sci. Technol. 50 (6), 337–344.
https://doi.org/10.2166/wst.2004.0393.
Kang, Z., Yang, B., Lai, J., Ning, Y., Zhong, Q., Lu, D., Liao, R., Wang, P., Dan, S.F.,
She, Z., Jia, Z., Lao, Y., Li, N., 2020. Phaeocystis globosa bloom monitoring: based on
P. globosa induced seawater viscosity modification adjacent to a nuclear power plant in
Qinzhou Bay, China. J. Ocean Univ. China 19 (5), 1207–1220. https://doi.org/ 10.1007/
s11802-020-4481-6.
Kebbi, Y., Muhammad, AI, Sant'Ana, AS, do Prado-Silva, L., Liu, D., Ding, T., 2020.
Recent advances on the application of UV-LED technology for microbial inactivation:
progress and mechanism. Compr. Rev. Food Sci. Food Saf. 19 (6), 3501–3527.
https://doi.org/10.1111/1541-4337.12645.
Lawal, O., Cosman, J., Pagan, J., 2018. UV-C LED devices and systems: current and
future state. IUVA News 20 (1), 22–28. In: https://iuva.org/resources/20
18_IUVA_Americas_Conference/Technical-Proceedings.
Lindenauer, KG, Darby, JL, 1994. Ultraviolet disinfection of wastewater: effect of dose on
subsequent photoreactivation. Water Res. 28 (4), 805–817. https://doi.org/
10.1016/0043-1354(94)90087-6.
Lundgreen, K., Holbech, H., Pedersen, KL, Petersen, GI, Andreasen, RR, George, C., Drillet,
G., Andersen, M., 2018. UV fluences required for compliance with ballast water
discharge standards using two approved methods for algal viability assessment.
Mar. Pollut. Bull. 135, 1090–1100. https://doi.org/10.1016/j. marpolbul.2018.08.043.
Lundgreen, K., Holbech, H., Pedersen, KL, Petersen, GI, Andreasen, RR, George, C., Drillet,
G., Andersen, M., 2019. Use of standard test organisms for sound validation of UV-
based ballast water treatment systems. Mar. Pollut. Bull. 144, 253–264. https://
doi.org/10.1016/j.marpolbul.2019.04.072.
MacIntyre, HL, Cullen, JJ, 2016. Classification of phytoplankton cells as live or dead using the
vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate.
J. Phycol. 52 (4), 572–589. https://doi.org/10.1111/jpy.12415.
MacIntyre, H.L., Cullen, J.J., Whitsitt, T.J., Petri, B., 2018. Enumerating viable
phytoplankton using a culture-based Most Probable Number assay following
ultraviolet-C treatment. J. Appl. Phycol. 30 (2), 1073–1094. https://doi.org/ 10.1007/
s10811-017-1254-8.
Mossel, DAA, Corry, JE, Struijk, CB, Baird, RM, 1995. Essentials of the
microbiology of foods: A textbook for advanced studies. John Wiley & Sons.
Nebot Sanz, E., Salcedo Davila, I., Andrade Balao, JA, Quiroga Alonso, JM, 2007.
Modeling of reactivation after UV disinfection: effect of UV-C dose on subsequent
photoreactivation and dark repair. Water Res. 41 (14), 3141–3151. https://doi.org/ 10.1016/
j.watres.2007.04.008.
Nyangaresi, P.O., Qin, Y., Chen, G., Zhang, B., Lu, Y., Shen, L., 2018. Effects of single and
combined UV-LEDs on inactivation and subsequent reactivation of E. coli in water
disinfection. Water Res. 147, 331–341. https://doi.org/10.1016/j.
watres.2018.10.014.
Olsen, RO, Hess-Erga, OK, Larsen, A., Hoffmann, F., Thuestad, G., Hoell, IA, 2016.
Dual staining with CFDA-AM and SYTOX Blue in flow cytometry analysis of UV
irradiated Tetraselmis suecica to evaluate vitality. Aquat. Biol. 25, 39–52. https://doi. org/
10.3354/ab00662.
Park, D.-W., Choi, Y.-K., Kil, G.-S., Chang, J.-H., Choi, C.-Y., 2014. Disinfection of
Tetraselmis sp. with UV LED application. J. Int. Counc. Electr. Eng. 1 (1), 1–6.
https://doi.org/10.5370/jicee.2011.1.1.001.
Qiao, Y., Chen, D., Wen, D., 2018. Use of coupled wavelength ultraviolet light-emitting diodes
for inactivation of bacteria in subsea oil-field injection water. Sci. Total Environ. 640–
641, 757–763. https://doi.org/10.1016/j.scitotenv.2018.05.283.
Rantalankila, M., Koivistoinen, H., Sarvasidze, L., Sillanpa¨a, ¨ MET, 2016. Inactivation of
Asterionellopsis glacialis in seawater using combinations of deep ultraviolet light
emitting diodes. Separate Purif. Technol. 169, 247–252. https://doi.org/10.1016/j.
seppur.2016.05.045.
Rattanakul, S., Oguma, K., 2018. Inactivation kinetics and efficiencies of UV-LEDs
against Pseudomonas aeruginosa, Legionella pneumophila, and surrogate
microorganisms. Water Res. 130, 31–37. https://doi.org/10.1016/j.
watres.2017.11.047.
Rivas-Zaballos, I., Romero-Martinez, L., Moreno-Garrido, I., Acevedo-Merino, A.,
Nebot, E., 2021. Evaluation of three photosynthetic species smaller than ten microns as
possible standard test organisms of ultraviolet-based ballast water treatment. Sea.
Pollut. Bull. 170, 112643 https://doi.org/10.1016/j.marpolbul.2021.112643.
Romero-Martínez, L., Moreno-Andr´es, J., Acevedo-Merino, A., Nebot, E., 2016.
Evaluation of ultraviolet disinfection of microalgae by growth modeling: application to
ballast water treatment. J. Appl. Phycol. 28 (5), 2831–2842. https://doi.org/ 10.1007/
s10811-016-0838-z.
Romero-Martinez, L., Rivas-Zaballos, I., Moreno-Andres, J., Moreno-Garrido, I., Acevedo
Merino, A., Nebot, E., 2020. Effect of the length of dark storage following ultraviolet
irradiation of Tetraselmis suecica and its implications for ballast water management.
Sci. Total Environ. 711, 134611 https://doi.org/10.1016/j.scitotenv.2019.134611.
Sinha, R.P., H¨ ader, D.-P., 2002. UV-induced DNA damage and repair: a review.
Photochem. Photobiol. Sci. 1 (4), 225–236. https://doi.org/10.1039/b201230h.
Machine Translated by Google
D. Wen et al.
Song, K., Mohseni, M., Taghipour, F., 2016. Application of ultraviolet light-emitting
diodes (UV-LEDs) for water disinfection: a review. Water Res. 94, 341–349. https://
doi.org/10.1016/j.watres.2016.03.003.
Steinberg, MK, Lemieux, EJ, Drake, LA, 2011. Determining the viability of marine protists
using a combination of vital, fluorescent stains. Mar. Biol. 158 (6), 1431–
1437. https://doi.org/10.1007/s00227-011-1640-8.
Sun, H., Mitra, S., Subedi, R.C., Zhang, Y., Guo, W., Ye, J., Shakfa, M.K., Ng, T.K., Ooi, B.
S., Roqan, IS, 2019. Unambiguously enhanced ultraviolet luminescence of AlGaN
wavy quantum well structures grown on large misoriented sapphire substrate. Adv.
Funct. Mater. 29 (48), 1905445 https://doi.org/10.1002/adfm.201905445.
Sun, Z., Blatchley III, E.R., 2017. Tetraselmis as a challenge organism for validation of
ballast water UV systems. Water Res. 121, 311–319. https://doi.org/10.1016/j.
watres.2017.05.052.
Chemosphere 308 (2022) 136113
Tao, Y., Zhang, X., Au, D.W., Mao,
Chemosphere 78 (5), 541–547. https://doi.org/10.1016/j.
chemosphere.2009.11.016.
US EPA, 2010. Generic Protocol for the Verification of BallastWater Treatment
Technology. Report Number EPA/600/R-10/146.
Wen, D., Chen, D., Jiang, Y., 2021. Synergistic algicidal effects of combined UV-LED/
chlorine treatments on Tetraselmis sp.: optimization and mode-of-action. Chem. Eng.
J. 422, 130043 https://doi.org/10.1016/j.cej.2021.130043.
Xiao, Y., Chu, XN, He, M., Liu, XC, Hu, JY, 2018. Impact of UVA pre-radiation on UVC
disinfection performance: inactivation, repair and mechanism study. Water Res. 141,
279–288. https://doi.org/10.1016/j.watres.2018.05.021.