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Destruction of microcystins (cyanotoxins) byUV-254 nm-based direct photolysis and advancedoxidation processes (AOPs): Influence of variableamino acids on the degradation kinetics andreaction mechanisms

Xuexiang He a,b, Armah A. de la Cruz c, Anastasia Hiskia d,Triantafyllos Kaloudis e, Kevin O'Shea f, Dionysios D. Dionysiou a,b,*

a Environmental Engineering and Science Program, University of Cincinnati, Cincinnati, OH 45221-0012, United

Statesb Nireas-International Water Research Centre, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprusc Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH 45268, United Statesd Institute of Advanced Materials, Physicochemical Processes, Nanotechnology and Microsystems, National Center

for Scientific Research “Demokritos”, Patriarchou Grigoriou & Neapoleos, 15310 Agia Paraskevi, Athens, Greecee Water Quality Department, Athens Water Supply and Sewerage Company (EYDAP SA), Oropou 156, 11146

Galatsi, Athens, Greecef Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, United States

a r t i c l e i n f o

Article history:

Received 18 September 2014

Received in revised form

4 February 2015

Accepted 6 February 2015

Available online 17 February 2015

Keywords:

Microcystins

UV-254 nm

Hydrogen peroxide

Second-order rate constant

Mechanism

Variable amino acids

* Corresponding author. Environmental EngiStates. Tel.: þ1 513 556 0724; fax: þ1 513 556

E-mail address: dionysios.d.dionysiou@uhttp://dx.doi.org/10.1016/j.watres.2015.02.0110043-1354/© 2015 Elsevier Ltd. All rights rese

a b s t r a c t

Hepatotoxic microcystins (MCs) are the most frequently detected group of cyanobacterial

toxins. This study investigated the degradation of common MC variants in water, MC-LR,

MC-RR, MC-YR and MC-LA, by UV-254 nm-based processes, UV only, UV/H2O2, UV/S2O2�8

and UV/HSO�5 . Limited direct photolysis of MCs was observed, while the addition of an

oxidant significantly improved the degradation efficiency with an order of UV/

S2O2�8 > UV/HSO�

5 > UV/H2O2 at the same initial molar concentration of the oxidant. The

removal of MC-LR by UV/H2O2 appeared to be faster than another cyanotoxin, cylin-

drospermopsin, at either the same initial molar concentration or the same initial organic

carbon concentration of the toxin. It suggested a faster reaction of MC-LR with hydroxyl

radical, which was further supported by the determined second-order rate constant of

MCs with hydroxyl radical. Both isomerization and photohydration byproducts were

observed in UV only process for all four MCs; while in UV/H2O2, hydroxylation and diene-

Adda double bond cleavage byproducts were detected. The presence of a tyrosine in the

structure of MC-YR significantly promoted the formation of monohydroxylation

byproduct m/z 1061; while the presence of a second arginine in MC-RR led to the elim-

ination of a guanidine group and the absence of double bond cleavage byproducts. It was

neering and Science Program, University of Cincinnati, Cincinnati, OH 45221-0012, United2599.

c.edu (D.D. Dionysiou).

rved.

wat e r r e s e a r c h 7 4 ( 2 0 1 5 ) 2 2 7e2 3 8228

therefore demonstrated in this study that the variable amino acids in the structure of

MCs influenced not only the degradation kinetics but also the preferable reaction

mechanisms.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Cyanobacteria are a photosynthetic e prokaryotic group that

is among the most ancient organisms on earth (Schopf and

Packer, 1987; Summons et al., 1999; Schopf, 2006). They are

highly adaptive to environmental conditions and their pres-

ence has been found in a wide variety of habitats, such as

ocean, freshwater, brackish water, soil, hot springs and even

Antarctic rocks (Codd, 1995; Ward et al., 1998; Jungblut et al.,

2005). They can release various metabolites such as taste

and odor compounds, anti-microbials, and also problematic

toxins known as cyanobacterial toxins or cyanotoxins

(Izaguirre et al., 1982; Carmichael, 1992; Jaki et al., 1999). The

cyclic hepatotoxic peptides microcystins (MCs) are among the

most important and by far themost studied cyanotoxins. This

group of toxins can be produced by a number of cyanobacteria

genera such as Microcystis, Anabaena, Plankothrix and Nostoc

(Pearson et al., 2010; Moreira et al., 2013). Though MCs mainly

inhibit the serine/threonine phosphatases (PP1 and PP2A),

they may also promote tumor formation, induce apoptosis,

and present long-term chronic toxic effects on wildlife, do-

mestic animals and humans (Nishiwaki-Matsushima et al.,

1992; Honkanan et al., 1994; McDermott et al., 1998; Chen

et al., 2009).

MCs are extremely stable in natural aquatic environments,

being resistant to various natural elimination processes

including chemical oxidation by naturally generated reactive

oxygen species (ROS) and biological transformation by other

microorganisms (Tsuji et al., 1994; de la Cruz et al., 2011;

Sharma et al., 2012; Corbel et al., 2014). Consequently, the

presence of MCs in source water presents a significant threat

to the ecosystem integrity and human health (Catherine et al.,

2013; Gehringer and Wannicke, 2014). Meanwhile, human

activities such as the agricultural and industrial development,

which contribute to eutrophication, water pollution and

climate change, have led to an increasing occurrence of pro-

longed and intense harmful blooms of cyanobacteria around

the world (Gehringer and Wannicke, 2014). Frequent harmful

cyanobacterial blooms especially during summer and early

fall seasons in sources of drinking water brought about chal-

lenges in providing clean and safe drinking water (de la Cruz

et al., 2011; Sharma et al., 2012; Catherine et al., 2013;

Gehringer and Wannicke, 2014). Although MCs mainly exist

as intracellular toxins, cell lysis resulting from death or

external physical force can lead to the release of MCs into the

extracellular water environment (Jones and Orr, 1994; Park

et al., 1998). Conventional water treatment processes may

not effectively remove MCs. For example, a total MC level

higher than 1 mg L�1 was once detected in the treated water in

the Carroll Township, Ohio, 2013, whenwestern Lake Erie was

experiencing a toxic cyanobacterial outbreak (Ohio

Environmental Protection Agency, 2013). A similar case

occurred in August, 2014, when hundreds and thousands of

residents in City of Toledo, Ohio and its surrounding areas

were asked not to drink or boil the water (City of Toledo, 2014).

It is therefore urgent and necessary to develop effective

treatment technologies to deal with such cases. Advanced

oxidation processes (AOPs) through the generation of reactive

radical species have been shown to be a promising alternative

to conventional water treatment processes in degrading cya-

notoxins (Lawton and Robertson, 1999; de la Cruz et al., 2011;

Sharma et al., 2012; Hiskia et al., 2013). Considering the wide

application of germicidal UV-254 nm in water treatment

plants for disinfection purposes, the coupling of UV-254 nm

with H2O2 to achieve both disinfection and pollutant (e.g.,

cyanotoxins) removal has been attracting attention in its po-

tential application in water treatment facilities (Qiao et al.,

2005; Kruithof et al., 2007; Al Momani et al., 2008; Liu et al.,

2010; Metz et al., 2011; He et al., 2012; Zong et al., 2013). Be-

side UV/H2O2 process, UV activated persulfate and perox-

ymonosulfate are capable of generating another radical

species, i.e., sulfate radical, which is also highly reactive but

more selective than hydroxyl radical in degrading organic

contaminants (He et al., 2013; Khan et al., 2013).

Common MC variants contain D-alanine (Ala), X, D-eryth-

romethylaspartic acid (MeAsp), Z, 3-amino-9-methoxy-2,6,8-

trimethyl-10-phenyl-4(E),6(E)-decadienoic acid (Adda), D-glu-

tamic acid (Glu), and N-methyldehydroalanine (Mdha), where

X and Z are two variable L-amino acids, with L as leucine (Leu),

R as arginine (Arg), Y as tyrosine (Tyr), and A as alanine (Ala).

UV photolysis of MCs leads to the isomerization of MCs at the

conjugated double bonds at the Addamoiety (Tsuji et al., 1994,

1995; Kaya and Sano, 1998). The primary reaction site for hy-

droxyl radical is at the Adda side chain, while theMdha double

bond is another important reaction site (Liu et al., 2003; Song

et al., 2006; Antoniou et al., 2008; Song et al., 2009). We

report herein the UV-254 nm and UV/H2O2 induced degrada-

tions of four common MC variants (MC-LR, MC-RR, MC-YR,

and MC-LA), which share five common amino acids (AAs)

including the Adda and Mdha groups and have two variable

AA subunits. The influence of the two variable amino acids on

degradation kinetics and transformation mechanisms was

specifically addressed. Detailed product studies were con-

ducted using liquid chromatography mass spectrometry for

UV direct photolysis and UV/H2O2 processes to help identify

the reaction pathways. The second-order rate constants of the

MCs with hydroxyl radical were determined and processes

utilizing UV/S2O2�8 and UV/HSO�

5 were also compared. The

degradation of cylindrospermopsin (CYN), another problem-

atic cyanotoxin which is readily degraded by hydroxyl and

sulfate radical as shown by its comparable second-order rate

wat e r r e s e a r c h 7 4 ( 2 0 1 5 ) 2 2 7e2 3 8 229

constants with these two radical species (Onstad et al., 2007;

Song et al., 2012; He et al., 2013, 2014a, 2014b), was

compared to that ofMCs. Our study is among the first to assess

the influence of variable amino acids on the degradation of

MCs by UV direct photolysis and hydroxyl radical reaction.

Overall, these detailed kinetic and product studies provide

important structure reactivity relationships and mechanistic

insight critical for modeling and applications of appropriate

oxidative treatment of problematic MC variants.

2. Materials and methods

2.1. Materials and sample preparation

Pure MC-LR (>95%, molecular weight (MW) ¼ 995.2 g mol�1),

MC-RR (>95%, MW ¼ 1038.2 g mol�1), MC-YR (>95%,

MW ¼ 1045.2 g mol�1) and MC-LA (>95%, MW ¼ 910.1 g mol�1)

were purchased from CalBiochem (Billerica, MA, USA). Pure

CYN (>95%, MW ¼ 415.4 g mol�1) was purchased from EMD

Biosciences (Gibbstown, NJ, USA). The toxins were used

without further purification. A stock solution was prepared by

adding 1 mL of autoclaved Milli-Q Water (Millipore Corp.,

Billerica, MA, USA). Hydrogen peroxide (50%, v/v) was pur-

chased from Fisher Scientific (Pittsburgh, PA, USA). Sodium

persulfate (PS, Na2S2O8, also known as sodium perox-

ydisulfate) and potassium peroxymonosulfate (PMS, KHSO5,

the active component of a triple potassium salt Oxone®, 2

KHSO5 �KHSO4 �K2SO4) were obtained fromSigmaAldrich (St.

Louis, MO, USA). All other chemicals were ACS grade reagents.

2.2. Analysis

MCs and CYN were determined quantitatively using an Agi-

lent 1100 Series high performance liquid chromatography

(HPLC) system equipped with a photodiode array detector

(PDA). For the analysis of single MC-LR or CYN, reverse-phase

chromatographic methods using a C18 column (Discovery HS,

2.1 � 150 mm, 5 mm, Supelco) were applied as reported pre-

viously (He et al., 2012, 2013). For the analysis of para-

chlorobenzoic acid (pCBA), other MCs and the MC mixtures,

an Agilent Eclipse XDB C8 column (4.6 � 150 mm, 5 mm) was

used, with a gradient elution of A (H2O þ 10 mM formic acid)

and B (methanol, MeOH), starting with 60% A for 2 min,

decreasing to 10% A in the next 16 min, keeping 10% A for

another 6 min, increasing to initial 60% A in the following

0.1 min, and maintaining 60% A until the analysis was

completed with a total run time of 38 min. The injection vol-

ume was 50 mL, the flow rate was 0.5 mL min�1, the column

temperature was 25 �C, and the detection wavelength was

238 nm for all compounds.

Detection and identification of byproducts was carried out

using an Agilent 6540 ultra-high definition accurate-mass

quadrupole time-of-flight (Q-TOF) liquid chromatography

mass spectrometer (LC/MS). A sample volume of 30 mL was

injected onto an Agilent ZORBAX Eclipse XDB C18 narrow-bore

rapid resolution column (2.1 � 50 mm, 3.5 mm). The mobile

phase consisted of A (H2O þ 0.1% formic acid) and B

(CH3CN þ 0.1% formic acid), with a gradient elution starting

from 2% B for 1 min, followed by a linear increase to 95% B in

the next 4 min, and back to 2% B in the last 0.1 min. Mass

spectra (m/z 500 � 1300) were obtained in positive ion mode.

The obtained data were analyzed by Agilent MassHunter

B.04.00 software providing a list of possible chemical formulae

for targeting compounds based on mass to charge ratio (m/z,

with relative mass differences �5 ppm) and isotope distribu-

tion. Isomers were further distinguished by their different

retention times (RTs).

2.3. Photochemical experiments

Illumination of samples was performed in a bench scale UV

photochemical apparatus housed with two low-pressure Hg

UV lamps (ColeeParmer) with monochromatic light emission

at lmax ¼ 254 nm. The photochemical reactor consisted of a

Pyrex® glass petri dish and a quartz cover (60 � 15 mm). The

average UV fluence rate across the reaction solution volume

was 0.1 mW cm�2, determined by iodide/iodate actinometry

(Rahn, 1997) and verified by ferrioxalate actinometry (Murov

et al., 1993) and a calibrated radiometer analysis (Model IL

1700, XRD (XRL) 140T254 low profile germicidal probe, Inter-

national Light, Co., Newburyport, MA) (Bolton and Linden,

2003; He et al., 2013). A sample volume of 100 mL was taken

at pre-determined UV fluence intervals andmixed with 100 mL

MeOH (for MCs) or 100 mL 0.05 N sodium thiosulfate (for CYN)

prior to analysis by HPLC. For Q-TOF-LC/MS analysis, a sample

of 150 mL was diluted with 150 mL MeOH and filtered using a

syringeless filter (0.2 mm, PTFE, Whatman). Competition

studies were carried out to determine the second-order reac-

tion rate constants of MCs with hydroxyl radical, using pCBA

as a competitor (Huber et al., 2003).

3. Results and discussion

3.1. Degradation of MC-LR and CYN under darkconditions

The three oxidants studied have significant difference in their

chemical reactivity. H2O2 is generally considered to be non-

reactive and unable to satisfactorily degrade organic com-

pounds such as MCs without activation (Cornish et al., 2000;

Qiao et al., 2005). PS is also regarded stable under room tem-

perature (Tsitonaki et al., 2010). PMS, on the other hand, was

reported to be even less active than H2O2 in the spontaneous

decomposition in water (Betterton and Hoffmann, 1990).

However, PMS can degrade directly and effectively various

organic compounds through the formation of an epoxide by

transferring an electrophilic perhydroxyl oxygen atom to the

organic double bond; a cyclic intermediate can be subse-

quently formed with the cleavage of the asymmetric peroxide

OeO bond (HOeSO3�, Betterton and Hoffmann, 1990; Zhu and

Ford, 1991). Its strong oxidation and the pathogen inactivation

properties have prompted its application as an alternative to

chlorine in swimming pool water disinfection (Eleraky et al.,

2002; Anipsitakis et al., 2008).

The degradation of MC-LR under dark reaction conditions

with H2O2, PS and PMS is shown in Fig. 1(a). No significant

degradation ofMC-LR (1 mM)was observedwith H2O2 (1mM) in

990 min. With PS (1 mM), MC-LR was completely degraded in

Fig. 1 e (a) Degradation of MC-LR under room temperature

and dark reaction conditions; (b) degradation half-life (t1/2,

min). [MC-LR]0 ¼ 1 mM, [phosphate buffer]0 ¼ 5 mM with

pH ¼ 7.4.

Fig. 2 e Degradation of MC-LR and CYN by UV and UV-

AOPs. [MC-LR]0 ¼ 1 mM, [CYN]0 ¼ 3.27 mM,

[oxidant]0 ¼ 1 mM, [phosphate buffer]0 ¼ 5 mM with

pH ¼ 7.4.

Table 1 e Calculated UV fluence-based pseudo first-orderrate constant (kobs, cm

2 mJ¡1) of the degradation of MC-LRand CYN, in UV only and UV-AOPs in Fig. 2.[Oxidant]0 ¼ 1 mM, [phosphate buffer]0 ¼ 5 mM withpH ¼ 7.4.

1 mM CYNa 3.27 mM CYN 1 mM MC-LR

UV only ~0 ~0 3.65 � 10�3

UV/H2O2 1.31 � 10�2 1.01 � 10�2 2.81 � 10�2

UV/PMS 5.16 � 10�2 4.42 � 10�2 9.53 � 10�2

UV/PS 2.54 � 10�1 1.04 � 10�1 2.00 � 10�1

a He et al., 2013.

wat e r r e s e a r c h 7 4 ( 2 0 1 5 ) 2 2 7e2 3 8230

575min. PMS at �50 mM greatly accelerated the degradation of

MC-LR which was completely degraded in less than 100 min.

The half-life (t1/2) values were also determined and are shown

in Fig. 1(b) demonstrating the strong reactivity of PMS in

degrading MC-LR. In fact, PMS has also been reported to be

much more effective than the other two studied oxidants for

the degradation of another cyanotoxin, CYN, under dark

conditions (He et al., 2013).

3.2. Degradation of MC-LR and MC mixtures by UV onlyand UV-AOPs

As shown in Fig. 2, there was limited direct photolysis of MC-

LR by UV-254 nm. The addition of oxidants significantly

accelerated the degradation process with an order of UV/

H2O2 < UV/PMS < UV/PS. CYNwas then used as a substrate for

comparison. As reported previously, this cyanotoxin is resis-

tant to UV-254 nm irradiation, with no apparent degradation

of 1 mM [CYN]0 at 80 mJ cm�2 (He et al., 2013). Therefore, no

further direct photolysis experiment was performed herein.

The UV-254 nm irradiation of H2O2, PS and PMS generates

oxidizing radicals with different quantum yields, as shown in

Eqs. 1e3 (Baxendale and Wilson, 1957; Mark et al., 1990; Guan

et al., 2011). Consequently, though CYN has a comparable

second-order rate constant with hydroxyl radical and with

sulfate radical (He et al., 2013), UV/H2O2 was again found to be

the least efficient among the three processes studied (UV/

H2O2 < UV/PMS < UV/PS) in degrading 3.27 mM CYN. The

degradation of MC-LR appeared to be faster than CYN at the

same initial molar concentration (except in UV/PS system as

previously reported by He et al., 2013) or organic carbon con-

centration of the toxins (as shown in Fig. 2). This is further

shown by the calculated UV fluence-based pseudo first-order

rate constants (kobs) for all the processes applied (Table 1). It

appears that MC-LR might be more prone than CYN to the

attack of radical species (which will be discussed in Section

3.3), therefore, making the use of AOPs promising for the

degradation of MCs in water.

H2O2 þ hv/·OHþ ·OH F ¼ 1:0 (1)

S2O2�8 þ hv/SO$�

4 þ SO$�4 F ¼ 1:8 (2)

wat e r r e s e a r c h 7 4 ( 2 0 1 5 ) 2 2 7e2 3 8 231

HSO�5 þ hv/SO$�

4 þ ·OH F ¼ 1:04 ðpH ¼ 7Þ (3)

In naturalwaters,MCsusually exist asmixtures of different

variants. To assess the applicability of the UV-AOPs in the

treatment of water contaminated with MCs, the degradation

of MC mixtures was evaluated by using four common MCs,

MC-LR, MC-RR, MC-YR and MC-LA. As shown in Fig. 3a, the

degradation of individual MC variant in mixtures was com-

parable using the same UV-AOP, while their removal followed

a similar kinetics trend to the single MC-LR experiment as

discussed above. When data are presented as total MCs

removal, the same order of degradation efficiency was

observed (Fig. 3b). It should be noted that no buffer solution

was added in this series of experiments. There was no signif-

icant change in pH for UV/H2O2 process during the reaction.

The reduced pH may, however, change the activation effi-

ciency of the oxidants and/or the speciation of hydroxyl and

sulfate radical for UV/PS and UV/PMS processes, leading to

different degradation efficiency of the contaminants (Criquet

and Leitner, 2009). Nevertheless, UV/H2O2 may be a better

choice in degrading cyanotoxins in field since the end

byproduct for H2O2 is water compared with SO42� for the other

two AOPs (Legrini et al., 1993; Dries et al., 1998). The following

results and discussionwill therefore focus on the performance

of the UV/H2O2 process in the degradation of MC variants.

Fig. 3 e Degradation of MCs in the MC mixtures in UV only

and UV-AOPs, (a) individual MC variant; (b) total MCs; [MC

variant]0 ¼ 1 mM, [oxidant]0 ¼ 1 mM, no buffer was added.

3.3. Determination of second-order rate constant of MCswith hydroxyl radical

Table 2 presents literature data regarding the reaction of the

amino acids with hydroxyl radical, where the order is Tyr

(Y)>Arg (R) > Leu (L) >Ala (A). According to group contribution

method (GCM) based on the group additivity of the rate con-

stants (Minakata et al., 2009), by combining the rate constants

of the amino acids that are present in the structure of the MC

variants under study, the reactivity of the MCs towards hy-

droxyl radical is estimated as kʹ�OH/MC-YR

(3.4 � 1010 M�1 s�1) > kʹ�OH/MC-RR (2.4 � 1010 M�1 s�1) > kʹ�OH/MC-

LR (2.2 � 1010 M�1 s�1) > kʹ�OH/MC-LA (1.8 � 1010 M�1 s�1). How-

ever, due to factors such as structure-reactivity relationships,

steric hindrance and molecular conformation, the direct

summations from the variable amino acidsmay not represent

the actual rate constant of the MCs with hydroxyl radical

(Song et al., 2009). Accordingly, competition kinetic experi-

ments were carried out to determine the second-order rate

constants of MCs with hydroxyl radical (Huber et al., 2003).

The concentration of H2O2 used in the competition study

was 1 mM, the presence of which did not influence the

average UV fluence rate. The following equation was subse-

quently applied for the calculation of the rate constants

(Shemer et al., 2006),

kOHðsÞ ¼�k0obsðsÞ � k0

dðsÞ�

�k0obsðref Þ � k0

dðrefÞ

�� kOHðref Þ (4)

where, k0obsðsÞ and k0

obsðrefÞ (cm2 mJ�1) are the observed degra-

dation rate constants of the substrate and the reference,

respectively, in UV/H2O2 process, while k0dðsÞ and k0

dðrefÞ(cm2 mJ�1) are those in UV only process; kOH(s) (M

�1 s�1) is the

second-order rate constant of the MC to be determined, while

kOH(ref) (M�1 s�1) is that of the reference, i.e., pCBA at

5� 109M�1 s�1 (Buxton et al., 1988). Due to overlap of the peaks

for pCBA and certain MCs in HPLC quantification analysis, the

second-order rate constants of MC-LR and MC-YR were

determined by competing with MC-RR. The results are pre-

sented in Table 3, with k�OH/MC-YR (1.63 � 1010 M�1 s�1) > k�OH/

Table 2 e Reported second-order rate constant (M¡1 s¡1)of amino acids and calculated theoretical second-orderrate constant of MCs with hydroxyl radical.

Amino acid variables Other components of MCs

Reported second-order rate constant of amino acids with �OH

Leucine (Leu, L)a 1.7 � 109 Aspartic acid (at Asp)a 7.5 � 107

Arginine (Arg, R)a 3.5 � 109 Acrylic acid (at Mdha)b 1.5 � 109

Tyrosine (Tyr, Y)a 1.3 � 1010 Glutamic acid (Glu)a 2.3 � 108

Alanine (Ala, A)a 7.7 � 107 Benzene (at Adda)b 7.8 � 109

Butadiene (at Adda)b 7.0 � 109

Theoretical second-order rate constant of MCs with �OH according

to GCM

L þ R as in MC-LR 5.2 � 109 Overall MC-LR 2.2 � 1010

R þ R as in MC-RR 7.0 � 109 Overall MC-RR 2.4 � 1010

Y þ R as in MC-YR 1.7 � 1010 Overall MC-YR 3.4 � 1010

L þ A as in MC-LA 1.8 � 109 Overall MC-LA 1.8 � 1010

a Sharma and Rokita 2012.b Buxton et al., 1988.

Table 3eDetermined kobs (cm2mJ¡1) in UV only andUV/H2O2 processes and the calculated second-order rate constant (kOH,

M¡1 s¡1).

kobs(RR) R2 kobs(pCBA) R2

MC-RR þ pCBA þ UV only 5.85 � 10�3 0.96 NA NA

MC-RR þ pCBA þ UV/H2O2 2.92 � 10�2 1.00 8.06 � 10�3 0.98 kOH(RR) 1.45 � 1010

kobs(LA) R2 kobs(pCBA) R2

MC-LA þ pCBA þ UV only 8.48 � 10�3 0.94 NA NA

MC-LA þ pCBA þ UV/H2O2 2.76 � 10�2 0.99 8.69 � 10�3 0.98 kOH(LA) 1.10 � 1010

kobs(LR) R2 kobs(RR) R2

MC-RR þ MC-LR þ UV only 6.63 � 10�3 0.97 5.73 � 10�3 0.96

MC-RR þ MC-LR þ UV/H2O2 2.32 � 10�2 1.00 2.69 � 10�2 1.00 kOH(LR) 1.13 � 1010

kobs(YR) R2 kobs(RR) R2

MC-RR þ MC-YR þ UV only 5.14 � 10�3 0.93 5.94 � 10�3 0.95

MC-RR þ MC-YR þ UV/H2O2 1.94 � 10�2 1.00 1.86 � 10�2 1.00 kOH(YR) 1.63 � 1010

wat e r r e s e a r c h 7 4 ( 2 0 1 5 ) 2 2 7e2 3 8232

MC-RR (1.45 � 1010 M�1 s�1) > k�OH/MC-LR

(1.13 � 1010 M�1 s�1) > k�OH/MC-LA (1.10 � 1010 M�1 s�1). The

determined k�OH/MC-LR is comparable to the existing values

reported by other researchers (Onstad et al., 2007; Song et al.,

2009). The magnitude of 1010 M�1 s�1 indicates the strong

promising application of hydroxyl radical-based treatment

processes or technologies in treatingMCs. The order in the rate

constants of MC variants is also in agreement with the above

discussion on GCM, suggesting the significant influence of the

variable amino acids in the reaction kinetics in UV/H2O2 AOPs.

3.4. Byproduct determination by UV photolysis andhydroxyl radical reaction

In an attempt to identify the byproduct formation, a relatively

high MC concentration of 4 mg L�1 was used if not noted

otherwise. The identification of the byproducts was based on

the high sensitivity of the Q-TOF-LC/MS onmonitoring them/z

values. Due to the overlap of peaks, especially for the isomers,

it was difficult to obtain the true area of the peaks. Therefore,

the volume (which equals tom/z� RT� abundance of the ions

associated with the compound) as indicated by the Mass-

Hunter data analysis software was used in this study for the

product kinetics. Different MCs had different response signals

in LC/MS analysis. The abundance between MCs and the

derived byproducts could not be compared. Consequently, the

volume as suggested by the software was used only as a

reference. The structural assignments of the byproducts were

further discussed through fundamental reaction mechanisms

involved in UV-254 nm direct photolysis and hydroxyl radical

reactions.

3.4.1. UV photolysisThere were mainly two significant groups of byproducts

detected for all the four MCs studied, the isomerization

byproducts with the samem/z value but different RTs, and the

hydration byproducts with a þ18 Da increase in the m/z. The

formation and subsequent degradation of these byproducts

are shown in Fig. 4(a)e(d).

Under UV-254 nm irradiation, an electron at the unsatu-

rated diene double bonds can be excited to a p* orbital

resulting in the formation of an uncoupled p bond and sub-

sequently a s bond. The bond rotation can occur to minimize

the repelling force of the electrons in the 2p orbitals between

the carbon atoms, leading to the isomerization of the MCs

(Lawton and Robertson, 1999). The two isomers observed in

the current study were most likely 4(E)-6(Z)-Adda and 4(Z)-

6(E)-AddaMC-LR as shown in Fig. 5 (Tsuji et al., 1995; Kaya and

Sano, 1998). The same results can probably be applied to the

other three MCs since the primary isomerization site is at the

diene double bonds. The geometrical isomers 4(E)-6(Z)-Adda

MC-LR and 4(E)-6(Z)-Adda MC-RR, which could be present in

natural water (Tsuji et al., 1994), were found to be less hepa-

totoxic andweaker in tumor-promotion than their parentMCs

suggesting that the 4(E)-6(E)-Adda is essential to the toxicity of

the MCs (Tsuji et al., 1994). Both 4(E)-6(E)-MC-LR and 4(E)-6(Z)-

Adda MC-LR were reported to be resistant to fluorescent and

natural sunlight (>295 nm, Tsuji et al., 1994), while their

transformation could be achieved by applying higher energy

and shorter wavelength UV such as UV-254 nm (Tsuji et al.,

1995). It is noteworthy to point out that conversion of the

less toxic 4(E)-6(Z)-Adda MC-LR to the much more toxic 4(E)-

6(E)-MC-LR has also been reported (Tsuji et al., 1994, 1995). It is

thus important to apply more effective processes such as

hydroxyl radical-based UV/H2O2 AOP for a more complete

destruction of MCs.

Compounds with e CH2 (i.e., 14 Da less in MW, data not

shown) were detected in all four initial samples of MCs. They

could be other MC variants, e.g., [D-Asp3] or [Dha7] MC-LR (m/z

981) and [D-Asp3] or [Dha7] MC-RR (m/z 1024) (Sivonen et al.,

1992). Similarly, it is also possible that m/z 1031 is [D-Asp3]

or [Dha7] MC-YR while m/z 896 and m/z 918 (M þ Naþ) is [D-

Asp3] or [Dha7] MC-LA. A second peak with the same m/z

formed later in the irradiated samples could be the corre-

sponding isomer of these MCs impurities. This group of

compounds (i.e., e CH2 and its reaction derivatives such as

þ16 hydroxylated byproducts) will not be considered or dis-

cussed further in this study.

The second group of byproducts with an increase ofþ18 Da

in m/zwas observed for the first time in this study. The enone

group at the Mdha moiety may be responsible for their for-

mation. As shown in Eq. (5), after excitation, a transition

dipolar structure could be formed. With the addition of water

at the electrophilic b-carbon, the proton exchange of the

subsequent zwitterion, and finally the tautomerization, a

photohydration byproduct b-hydroxyl ketone was then

Fig. 4 e Evolution of reaction byproducts by direct UV photolysis, (a) MC-LR; (b) MC-RR; (c) MC-YR; and (d) MC-LA;

[MC]0 ¼ 4 mg L¡1 (except for [MC-RR]0 ¼ 8 mg L¡1), [H2O2]0 ¼ 1 mM, no buffer was added. The number after the m/z value is

the corresponding RT in TOF scan. Note for the difference in the y-axes, with (a) and (c) sharing the same y-axis while (b) and

(d) sharing another same y-axis.

wat e r r e s e a r c h 7 4 ( 2 0 1 5 ) 2 2 7e2 3 8 233

formed (Cornell et al., 1979). In fact, the photohydration at the

olefinic bond was found to be efficient for enone containing

testosterone by UV-254 nm (Cornell et al., 1979) and trenbo-

lone acetate by solar light irradiation (Qu et al., 2013).

(5)

Beside the reaction at the enone moiety, photoinduced

hydration was also found to be a common process and has

been reported for alkenes, alkynes, and nucleic acids (Liu and

Yang, 1978; Wan et al., 1982; Vairapandi and Duker, 1994). The

unstable hydration at 5,6-double bond was proposed as an

important intermediate step for the deamination of 5-

methylcytosine, which was reported to be responsible for the

mutation of DNA under UV irradiation (Vairapandi and Duker,

1994). There were three hydrated byproducts detected in this

study for eachMCvariant, with an exception of four forMC-YR

(Fig. 4(a)e(d)). Though they may be preferably formed through

the photohydration at the enone of the parent MCs and their

corresponding isomers, the addition at the diene double bonds

cannot be excluded without a further MS/MS structural iden-

tification. Fig. 5 thus only shows one of the possibilities.

Aside from the isomerization and photohydration

byproducts, there were also other minor byproducts detected.

Due to their significantly low abundance, they are not

currently shown in the figures. Under UV irradiation of MCs,

an electron can be transferred fromMCs to molecular oxygen.

The formed superoxide radical anion can break down to yield

H2O2 which under UV irradiation will yield hydroxyl radicals

for the transformation of MCs (Eqs. 1 and 6e8, Legrini et al.,

1993; Lawton and Robertson, 1999). Therefore, in this study,

with an extended irradiation of the samples, hydroxylation

byproducts such as the þ16 Da were also observed in the UV

Fig. 5 e UV photolysis of MCs, isomerization and hydration.

wat e r r e s e a r c h 7 4 ( 2 0 1 5 ) 2 2 7e2 3 8234

only process. This study shows the comparable but slightly

different reaction mechanisms in UV only process with

different variable amino acids. Fundamental mechanism how

they impact the photolysis needs further investigation.

MCsþO2 þ hv/MC$þs þO$�

2 (6)

O$�2 þHþ4HO$

2 (7)

2 HO$2/H2O2 þO2 (8)

3.4.2. Hydroxyl radical reaction in the UV/H2O2 systemUpon illumination with UV-254 nm in the presence of H2O2,

the photohydration byproducts were not detected. The

isomerization byproducts were shown only for MC-LR and

MC-LA (i.e., m/z 995 and 910, respectively, as shown in Fig. 6).

Such observations might have been the result of competitive

absorption of UV photons between MCs and H2O2, as well as

the strong oxidative reactivity of hydroxyl radical. The LC/MS

analytical method used was not quite sensitive for MC-RR.

Even at a high MC-RR concentration of 8 mg L�1, it had the

least composition of byproducts. Compared to direct UV

photolysis, the oxidation byproducts from the hydroxylation

and the cleavage of double bond were found to be significant

for the MCs. Numerous byproducts were detected in this

study. Four groups can be derived and are discussed in the

following sections to reveal the influence of variable amino

acids on their distribution. Other byproducts that were

detected in this study but are not shown in Fig. 6 or 7 can be

formed through similar radical reaction mechanisms. There-

fore, they will not be further discussed.

Hydroxyl radical is known to react primarily through

hydrogen abstraction (H-abstraction), while the hydroxyl

addition to the double bond and the electron transfer are also

its potential reaction mechanisms. (1) The first group of the

byproducts was the hydroxylation byproducts. The most

likely targets were the aromatic ring and the diene-Adda

double bonds which have a much higher second-order rate

constant with hydroxyl radical than most other moieties

(Table 2, Buxton et al., 1988). Various hydroxylation

byproducts were detected such as monohydroxylation

(þ16 Da) (e.g., m/z 1011, 1054, 1061 and 926, for MC-LR, -RR,

-YR, and -LA, respectively) and dihydroxylation (þ32 Da) (e.g.,

m/z 1027, 1070, 1077 and 942) byproducts shown in Figs.

6(a)e(d) and 7. (2) Hydroxyl addition byproducts on the diene

double bonds were frequently reported by other researchers

(Liu et al., 2003; Song et al., 2006; Antoniou et al., 2008; Song

et al., 2009; Zong et al., 2013). However, these byproducts

were not detected or were only detected at a much lower

abundance (e.g.,m/z 1072, 1079 and 944, forþ34 DaMC-LR, -YR

and -LA, respectively; as well as m/z 1045, 1088, 1095 and 960,

for þ16 þ 34 Da MC-LR, -RR, -YR and -LA, respectively) by

current experimental and analytical methods. (3) The bond

cleavage byproducts, m/z 835, 885 and 750/772, as well as 795,

845 and 710, were found to be significant which could be due

to the oxidative cleavage of the double bond or the bond

cleavage of the dihydroxyl added vinyl diol (Song et al., 2006;

Zong et al., 2013). (4) The last group of hydroxyl radical reac-

tion byproducts, C48H70N10O13,m/z 995, was the elimination of

a guanidine exclusively for MC-RR. As reported by Ansari et al.

(1985) with the detection of norvaline during the degradation

of arginine by hydroxyl radical in UV/H2O2 process, the loss of

a guanidine moiety with a subsequent hydroxylation is thus

possible for MC-RR. It showed a strong involvement of hy-

droxyl radical resulting in more isomer varieties and higher

relative abundance of m/z 995 in UV/H2O2 than in UV only

system. Figs. 6(a)e(d) also show minor formation of m/z 965,

1008, 1015, and 880/902 for �30 Da MC-LR, -RR, -YR and -LA,

respectively. It could be attributed to the elimination of the

methoxy group at the Adda chain through H-abstraction at

the methyl group with a subsequent formation of m/z 1009

(Antoniou et al., 2008). This intermediate byproduct was not

detected in this study. Therefore, the mentioned byproducts

could be positively formed in UV/H2O2 system, but need

further verification and thus are not shown in Fig. 7.

A significant variation in the kinetic formation of these

byproducts was also observed for MCs. MC-LR had a compa-

rable formation of the isomerization, monohydroxylation and

dihydroxylation byproducts whose relative abundance were

higher than the double cleavage byproducts. MC-RR had a

Fig. 6 e Evolution of reaction byproducts by UV/H2O2, (a) MC-LR; (b) MC-RR; (c) MC-YR; and (d) MC-LA; [MC]0 ¼ 4 mg L¡1

(except for [MC-RR]0 ¼ 8 mg L¡1), [H2O2]0 ¼ 1 mM, no buffer was added. The number after the m/z value is the corresponding

retention time in TOF scan. Note for the difference in the axes.

Fig. 7 e Transformation of MCs in UV/H2O2 process.

wat e r r e s e a r c h 7 4 ( 2 0 1 5 ) 2 2 7e2 3 8 235

wat e r r e s e a r c h 7 4 ( 2 0 1 5 ) 2 2 7e2 3 8236

comparable formation of monohydroxylation, methoxy

elimination and guanidine elimination byproducts, while no

isomerization of parent MC-RR or the bond cleavage byprod-

ucts were detected. MC-YR, due to the presence of phenolic

tyrosine amino acid, had a significantly higher formation of

monohydroxylation byproduct than the other MC variants. As

for MC-LA, a comparable formation in the isomerization,

monohydroxylation and bond cleavage byproducts was

observed. The influence of variable amino acids in the

degradation of MCs by UV/H2O2 AOPs was therefore clearly

evident. Such influence can be further studied in sulfate

radical-based AOPs, which is beyond the scope of this study.

There is very limited literature information on the rate con-

stants of amino acids with sulfate radical. The aromatic

compounds have high reactivity towards sulfate radical via

electron transfer, which is shown by the second-order rate

constant of 3 � 109 M�1 s�1 for tyrosine and benzene (Neta

et al., 1988). It may indicate that the tyrosine moiety will

also play a significant role in the reaction kinetics and

byproduct formation of MC-YR. However, the amide form of

tyrosine in MC-YR could be less reactive towards electron

transfer compared to when it is a zwitterion in pH neutral

solution. Besides, the sulfate radical reaction may be influ-

enced by electrostatic and changes in the reactivity of ionized

functional groups (amino and carboxylate functional groups).

Therefore, the observation that the relative abundance of m/z

1061 was much higher than the other byproducts in UV/H2O2

system due to the presence of tyrosine may not be as signifi-

cant in sulfate radical systems. Further studies on the re-

actions of MCs with sulfate radical, where the suppression of

hydroxyl radical in sulfate radical-AOPs is essential, would be

interesting to explore such a hypothesis both kinetically and

mechanistically.

4. Conclusions

This study investigated the influence of variable amino acids

on the degradation of common microcystin variants, MC-LR,

MC-RR, MC-YR and MC-LA. The different chemical proper-

ties of the oxidants led to different rates of MC-LR removal

under dark and room temperature conditions, with HSO�5 >>

S2O2�8 >>H2O2. Limited direct photolysis of MCs by UV-254 nm

irradiation was observed, while the addition of the oxidant

significantly improved the removal efficiencywith the order of

UV/S2O2�8 > UV/HSO�

5 > UV/H2O2 at a 1 mM [oxidant]0, which

was attributed to the difference in the radical quantum yield

of the oxidants by UV-254 nm irradiation. The reaction of MC-

LR appeared faster than that of another cyanotoxin, CYN, at

either the same initial molar concentration of the toxin or the

same initial organic carbon concentration by UV/H2O2, which

was most likely due to the higher second-order rate constant

of MC-LR with hydroxyl radical compared with CYN. The dif-

ference in the variable amino acidswas found to correlatewell

with the determined second-order rate constant of all four

MCs with hydroxyl radical, at an order of k�OH/MC-YR

(1.63� 1010M�1 s�1) > k�OH/MC-RR (1.45� 1010M�1 s�1) > k�OH/MC-

LR (1.13 � 1010 M�1 s�1) > k�OH/MC-LA (1.10 � 1010 M�1 s�1). Both

isomerization and photohydration byproducts were observed

in UV only process for four MCs. In UV/H2O2, various

byproducts generated by isomerization, hydroxylation, hy-

droxyl addition or the oxidative cleavage of the double bonds

were detected; however, a difference in the composition and

relative abundance among the MCs was noted. The presence

of a tyrosine inMC-YR significantly promoted the formation of

monohydroxylation byproductm/z 1061;while the presence of

a second arginine allowed the elimination of a guanidine

group and the absence of diene-Adda cleavage byproducts for

MC-RR. Whether the presence of leucine in MC-LR and MC-LA

contributed to their exclusive formation of isomers in UV/

H2O2 needs to be further investigated. Nevertheless, the dif-

ference in the variable amino acids of MCs appeared to in-

fluence not only the degradation kinetics but also the reaction

mechanisms. This study provides important scientific data for

the treatment of the cyanotoxin MCs by UV-AOPs.

Acknowledgments

This work was funded by the Cyprus Research Promotion

Foundation through Desmi 2009e2010 which is co-funded by

the Republic of Cyprus and the European Regional Develop-

ment Fund of the EU under contract number NEA IPODOMI/

STRATH/0308/09. X. He is grateful for the University Research

Council of University of Cincinnati for a Summer Research

Fellowship and Graduate School of UC for a Dissertation

Completion Fellowship. D. D. Dionysiou acknowledges sup-

port from the University of Cincinnati through a UNESCO co-

Chair Professor position on “Water Access and Sustainabil-

ity”. T. Kaloudis, A. Hiskia and D. D. Dionysiou acknowledge

COST Action ES 1105 “CYANOCOST e Cyanobacterial blooms

and toxins in water resources: Occurrence, impacts and

management”.

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