ARTICLE IN PRESS

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Water Research 39 (2005) 1628–1638

www.elsevier.com/locate/watres

Kinetics of reactions between chlorine and the cyanobacterialtoxins microcystins

Juan L. Aceroa,�, Eva Rodrigueza, Jussi Meriluotob

aDepartamento de Ingenieria Quimica y Energetica, Faculdad de Ciencias, Universidad de Extremadura, Avda. Elvas s/n,

06071 Badajoz, SpainbDepartment of Biochemistry and Pharmacy, Abo Akademi University, P.O. Box 66, 20521 Turku, Finland

Received 14 September 2004; received in revised form 25 January 2005; accepted 26 January 2005

Available online 11 March 2005

Abstract

Blooms of cyanobacteria can give rise to the production of toxins which contaminate drinking water sources. Among

the oxidants and disinfectants typically applied in waterworks, chlorine has been found to be effective for the

degradation of microcystins. In the present study, unknown second-order rate constants for the reactions of

microcystin-LR (MC-LR), -RR and -YR with chlorine were determined over a wide pH range. It was found that an

increase of pH has a negative effect on the microcystin degradation rate.

Apparent second-order rate constant for the chlorination of MC-LR at 20 1C varied from 475M�1 s�1 at pH 4.8 to

9.8M�1 s�1 at pH 8.8. From these apparent second-order rate constants, rate constants for the reactions of MC-LR

with hypochlorous acid (HOCl) and hypochlorite (ClO�) were evaluated. Half-life times ranged from minutes at pH 6

to 1 h at pH 8 for a constant residual chlorine concentration of 1.0–0.5mg l�1, typical of oxidation pre-treatment and

final disinfection. Similar reactivity with chlorine was found for MC-RR and MC-YR. Therefore, chlorination is a

feasible option for microcystin degradation during oxidation and disinfection processes, and can be applied in drinking

water treatment in case of cyanobacterial toxin risk if the pH is kept below 8.

Published by Elsevier Ltd.

Keywords: Cyanobacteria; Microcystins; Chlorine; Chloramine; Rate constants; Drinking water chlorination

1. Introduction

Toxic cyanobacteria (blue-green algae) have been

reported on all continents (Chorus and Bartram, 1999).

Cyanobacteria are considered an important water

quality problem and a hazard in regard to their potential

to contaminate water intended for drinking, recreational

and agricultural purposes (Lawton and Codd, 1991;

Chorus and Bartram, 1999). Many blooms are highly

acutely toxic and have been responsible for the death of

e front matter Published by Elsevier Ltd.

atres.2005.01.022

ing author. Tel./fax: +34 924289385.

ess: jlacero@unex.es (J.L. Acero).

both wild and domestic animals, as well as human illness

and death resulting from exposure to blue-green algal

toxins (Soong et al., 1992; Turner et al., 1990; Frazier

et al., 1998; Jochimsen et al., 1998). Therefore, drinking

water authorities world-wide are dealing with the

challenge of treating contaminated water.

A number of genera of cyanobacteria can produce

lethal toxins, which have been classified in two diverse

groups, namely hepatotoxic cyclic peptides and neuro-

or cytotoxic alkaloids (Chorus and Bartram, 1999). In

addition, all cyanobacteria contain pyrogenic and

nausea-causing lipopolysaccharides in their cell walls

(Weckesser et al., 1979). Within the cyclic peptide group,

ARTICLE IN PRESSJ.L. Acero et al. / Water Research 39 (2005) 1628–1638 1629

microcystins are highly acutely hepatotoxic and they

also show tumor promotion activity through inhibition

of protein phosphatases 1 and 2A (PP1 and PP2A)

(Yoshizawa et al., 1990). Microcystins, the most wide-

spread cyanobacterial hepatotoxins, are produced

mainly by cyanobacteria belonging to the genera

Microcystis, Anabaena, Planktothrix and Nostoc (Spoof,

2004). At least 76 different microcystin analogues have

been found in natural blooms and laboratory cultures of

cyanobacteria. The most common variant is microcystin

LR (MC-LR), which has been identified together with

other microtoxin variants such as MC-LA, MC-RR and

MC-YR in natural water samples (Yoo et al., 1995;

Falconer et al., 1999; Spoof et al., 2003). The chemical

structure of all microcystins is very similar, they are

cyclic peptides consisting of seven amino acid residues

with two main variable residues. All microcystins

contain a variant of the Adda residue (3-amino-9-

methoxy-2,4,8-trimethyl-10-phenyldeca-4(E),6(E)-die-

noic acid), the structural unit considered crucial for the

toxicity (An and Carmicael, 1994). MC-LR is the most

toxic microcystin, with an LD50 value of 50mg/kg(Dawson, 1998). Because of the potent toxicity of

MC-LR, the World Health Organization (WHO) has

set a provisional guideline value of 1.0 mg l�1 for MC-LRin drinking water (WHO, 1998). This WHO guideline

value is used as a basis in some countries such as Spain

to set a drinking water standard for total microcystins of

1.0mg l�1 (BOE, 2003).MC-LR can be eliminated from natural waters

with the oxidants and disinfectants typically applied

in waterworks. Oxidation investigations using ozone

(Rositano et al., 1998, 2001), chlorine (Nicholson et al.,

1994; Nicholson and Rositano, 1997) and UV photolysis

in presence of TiO2 (Lawton et al., 1999) have shown

that MC-LR is readily oxidised to non-toxic degrada-

tion products under appropriate conditions. Chlorami-

nation, however, did not have any significant effect on

MC-LR (Nicholson et al., 1994). Chlorine was selected

in the present work since it is used worldwide in large

quantities for preoxidation treatment and final disinfec-

tion (Bull et al., 1995). Although the ozonation process

is faster (Rositano et al., 1998), Nicholson et al. (1994)

have reported that microcystins can be destroyed by

chlorine as long as sufficient dose is present, the

oxidation being a pH dependent process. Chlorine

residual of 0.5mg l�1 after 30min contact time was

effective at pHo8, whereas inadequate chlorine dosesand higher pH caused negative results. Tsuji et al. (1997)

have identified dihydroxymicrocystin as the main

degradation product, proposing a reaction mechanism

in which HOCl likely attacks to the Adda group of MC-

LR to form a chloronium ion at the conjugated diene of

Adda, followed by hydrolysis to form dihydroxymicro-

cystin. The chlorination of microcystin-LR is not

believed to lead to the formation of noxious products

(Tsuji et al., 1997). Moreover, degradation of micro-

cystin by chlorination of drinking water did not result in

an increased incidence of cancer in mice genetically

predisposed to the effect of genotoxic carcinogens

(Senogles-Derham et al., 2003).

Although the chlorination of microcystins has been

extensively examined, there has not been a thorough

investigation to determine chlorination rate constants

useful to predict degradation efficiencies during full-scale

treatment processes. Therefore, the objective of the

present study was the calculation of second-order rate

constants for the reaction of chlorine with MC-LR and

other variants such as MC-RR and MC-YR. The

influence of some operating variables such as pH and

temperature on chlorination rate constants was also

established. In addition, a reaction mechanism was

proposed allowing the calculation of individual rate

constants for the reaction of MC-LR with chlorine species

(hypochlorous acid and hypochlorite). Based on these

chlorination rate constants, the required oxidant exposure

(CT value) to reduce MC-LR concentration to 1mg l�1

was determined under several treatment conditions.

2. Experimental section

All reagents were of the purest available quality.

Solutions of analytical reagents, chlorine, and phosphate

buffers were prepared with ultra-pure water produced

from a MilliQ (Millipore) water purification system.

Stock solutions of chlorine were prepared by diluting a

commercial solution of sodium hypochlorite (4% active

chlorine, Aldrich) and standardized spectrophotometri-

cally in presence of excess of iodide to form triiodine

(e at 351 nm ¼ 25,700M�1 cm�1) (Bichsel and von

Gunten, 1999). Monochloramine solution was prepared

daily by adding HOCl to ammonium nitrate at pH 8

with a molar ratio of 1:1. Such solution was standar-

dized by the ABTS method (Pinkernell et al., 2000).

2.1. Toxin materials

Purified toxins: MC-LR and MC-RR to be used in the

oxidation experiments were purified from Anabaena

strain 90 (originally from the culture collection of

Prof. Kaarina Sivonen, University of Helsinki) accord-

ing to Meriluoto et al. (2000). In summary, the

cyanobacteria were extracted with 50% methanol. The

extracts were diluted with water and concentrated by

C18 (Bond Elut, Varian) flash chromatography. The

extracts were further fractionated by silica gel chroma-

tography using a 47� 25mm I.D. Si60 column (Merck)

and a mobile phase of 0.1% trifluoroacetic acid-90%

methanol in water. The toxins were then subjected to

preparative HPLC using a Hichrom Nucleosil 100–10

C18 column, 150� 21.2mm I.D., and a mobile phase of

ARTICLE IN PRESSJ.L. Acero et al. / Water Research 39 (2005) 1628–16381630

27% acetonitrile-73% ammonium acetate, 1 g l�1, in

water. Finally the toxins were desalted on C18 solid-

phase extraction columns. For calibration purposes on

the analytical HPLC instrument (method E in Meriluoto

et al., 2000) a further purified grade of MC-LR was

used. The additional purification was done using a

Supelco Discovery C18 column, 250� 10mm I.D., and a

gradient mobile phase of 0.05% trifluoroacetic acid in

water and acetonitrile.

Toxin extracts: Freeze-dried cyanobacterial material

from Sulejow reservoir, Poland, collected in 2002, was

extracted with 50% methanol, cleaned-up with C18 flash

chromatography, filtered, divided into aliquots and

dried in vials with argon gas. The microcystins in the

extract were determined by analytical HPLC and found

to be MC-LR, MC-RR and MC-YR.

2.2. Analytical methods

The chlorine and chloramine concentration was

analyzed by the ABTS method (Pinkernell et al.,

2000). Spectrophotometric measurements were per-

formed with a Unican Helios b spectrophotometer. pHmeasurements were carried out with a Crison electrode

and a Crison GLP-22 pH-meter, which was calibrated

with standard buffer solutions (Crison).

Concentrations of microcystins was determined by

HPLC (Waters Alliance 2695 Separation Module and

Waters 996 Photodiode Array Detector). A preconcen-

tration step was not needed with the MC concentration

of 1.5–3.5mg l�1 used in the experiments. The column

was a C18 end-capped Merck Purospher STAR RP-18e,

3mm particles, LiChroCART 55� 4mm I.D. and the

injection volume 10ml. Gradient elution (flow rate

1mlmin�1) of acetonitrile with 0.05% trifluoracetic acid

(A) and water with 0.05% trifluoracetic acid (B) was

used by varying the volume percentage of A from 25%

to 70% over 5min. The photodiode Array (PDA)

detector was set in the range 200–300 nm, being

238 nm the wavelength selected for quantification. The

concentrations of MC-RR and MC-YR were estimated

using the same detector response as for MC-LR.

2.3. Rate constants of chlorine and monochloramine with

MC-LR

Chlorination experiments were conducted under

pseudo-first-order conditions where chlorine was at least

20 folds in excess. Ultra-pure water produced from a

MilliQ system was used to prepare the reaction samples.

The temperature was varied in the range 10–30 1C and

the pH between 4 and 9 with 10mM phosphate buffer.

In a typical experiment, a 5ml volume of buffered MC-

LR solution (1.5–2.5mg l�1, 1.5–2.5 mM) was preparedin a batch reactor (serum vial of 5.5ml), which was

capped with PTFE-faced silica septum and located in a

thermostatic bath. The experiments were started after

addition of an aliquot of the chlorine stock solution into

the reactor while stirring (for 5 s). Thus the initial

chlorine concentration ranged from 49 to 223 mM. Atfixed time intervals, 0.3ml of sample was rapidly

transferred with a syringe into a HPLC vial containing

2ml of thiosulfate (0.1M) to stop the reaction. Theconcentration of residual MC-LR was analyzed directly

by HPLC. All experiments were duplicated.

Similar experiments were performed with monochlor-

amine to determine the rate constant of its reaction with

MC-LR at pH 8 and 20 1C. In this case, and due to the

low reactivity of monochloramine, the influence of the

pH and the temperature was not investigated. The initial

concentration of oxidant ranged from 400 to 720 mM,high enough to observe an appreciable degradation of

MC-LR within several hours. The initial concentration

of toxin was 2.4mg l�1.

2.4. Rate constants of chlorine with MC-RR and MC-YR

The determination of the rate constants for the

reactions of HOCl with MC-RR and MC-YR was

performed by using a competitive kinetics model. Direct

measurements were not performed to minimize HPLC

analysis errors (MC-RR and MC-YR concentrations

had to be estimated as MC-LR equivalent). To control

the accuracy of analysis MC-LR was present in these

experiments. MC-LR, which rate constant was already

determined, was used as the reference compound. In

addition, MC-YR was not available as pure toxin. Then,

couples of target and reference compounds were selected

to determine unknown rate constants.

The experiments with MC-RR were carried out in

5.5ml serum vials at 20 1C and pH ranging from 6 to 8

(10mM phosphate buffer). The initial solutions contained

equal concentration of MC-LR and -RR (2.3mM). Afteraddition of chlorine (100mM) while vigorously stirring (for5 s), samples were withdrawn at fixed time intervals and

rapidly transferred into HPLC vials containing tiosulphate

to quench the reaction. Remaining concentrations of

target and reference compounds were analyzed by HPLC.

Rate constants of MC-YR chlorination could be

measured in a toxin extract from Sujelow Reservoir

(Poland), in which MC-YR was present together with

MC-LR and MC-RR. A similar experiment to those

described for MC-RR was performed with this toxin

extract. Again, MC-LR was selected as reference

compound and MC-YR as target compound.

3. Results and discussion

3.1. Kinetics of the reaction of chlorine with MC-LR

The rate constant for the reaction of HOCl with

MC-LR was investigated under pseudo-first-order

ARTICLE IN PRESS

0 50 100 150 200-1.2

-0.9

-0.6

-0.3

0.0

ln([

MC

-LR

]/[M

C-L

R] 0

)

time, s

Fig. 1. Pseudo-first-order kinetic plot for the chlorination of

MC-LR at 20 1C and pH 7.2. The solid line is a linear least-

squares regression of the data. Initial concentrations:

[HOCl]0 ¼ 49.2mM, [MC-LR]0 ¼ 2.35mM (’); [HOCl]0 ¼

95.7mM, [MC-LR]0 ¼ 2.30mM (&); [HOCl]0 ¼ 142.5mM, [MC-LR]0 ¼ 2.35mM (K); [HOCl]0 ¼ 222.8mM, [MC-LR]0 ¼

2.50mM (J); [HOCl]0 ¼ 96.2mM, [MC-LR]0 ¼ 1.49mM (m);

[HOCl]0 ¼ 94.6mM, [MC-LR]0 ¼ 1.91mM (B).

Table 1

Chlorination experiments performed at pH 7.2 and 20 1C

Expt. [MC-LR]0 (mM) [Cl2]t0 (mM) k0 (� 103 s�1)

1 2.35 49.2 3.49

2 2.30 95.7 7.21

3 2.35 142.5 9.60

4 2.50 222.8 16.2

5 1.91 94.6 7.50

6 1.49 96.2 7.27

J.L. Acero et al. / Water Research 39 (2005) 1628–1638 1631

conditions ([HOCl]b[MC-LR]) in a batch system

between pH 4 and 9. Previous investigations on

chlorination of phenolic compounds over a wide pH

range have demonstrated that the reaction between

chlorine and phenols could be described by second-order

kinetics, first-order in active oxidant (HOCl+OCl�) and

first-order in phenol (Rebenne et al., 1996; Gallard and

von Gunten, 2002a). Based on these studies and taking

into account the presence of a conjugated double bond

in the MC-LR molecule, it can be assumed that the

reaction of chlorine with MC-LR follows second-order

kinetics:

�d½MC-LR�

dt¼ kapp½MC-LR�½Cl2�t ¼ k0

½MC-LR� (1)

where kapp is the apparent second-order rate constant

(pH dependent as will be discussed later), [Cl2]t is the

total concentration of chlorine species (HOCl+ClO�)

and k0 is the pseudo-first-order rate constant. The total

chlorine concentration can be considered constant

during the experiment, thus being k0 ¼ kapp [Cl2]t0. The

integration of Eq. (1) leads to:

ln½MC-LR�

½MC-LR�0¼ �k0t (2)

Therefore, if the assumption of first-order kinetics

with respect to MC-LR is correct, the representation of

the first term of Eq. (2) versus time must lead to a

straight line for each experiment. Fig. 1 presents the

results from experiments performed at a range of

MC-LR and chlorine concentrations and at pH 7.2. As

can be observed, points lie satisfactorily around straight

lines, whose slopes are the pseudo-first order rate

constants. After linear regression analysis (r240.99),the calculated values of k0 are summarized in Table 1

together with the initial conditions in these experiments.

The rate of MC-LR degradation increases with higher

initial chlorine dose and is not dependent on the initial

MC-LR concentration. Fig. 2 illustrates a plot of k0

values versus the initial concentration of chlorine. A

linear plot could be drawn (r240.99), which confirmsthat the rate of MC-LR degradation is also first-order

with respect to chlorine concentration. The slope from

linear analysis of data in Fig. 2 yields an apparent

second-order rate constant at pH 7.2 of 72.0M�1 s�1.

The rate of MC-LR degradation, therefore, is first-order

in the concentrations of chlorine and MC-LR.

The influence of the pH was investigated in the pH

range 4–9 in experiments performed with similar initial

concentration of both chlorine and MC-LR (95 and

2.3mM, respectively). Fig. 3 shows the first-order plotfor this group of experiments. Again, the results suit a

pseudo-first-order kinetic model. From these experi-

mental values of k0, kapp was determined at each pH,

being the obtained values represented in Fig. 4

(symbols). It can be observed that the degradation of

MC-LR is favored at low pHs. These results are

explained if HOCl is the major electrophilic species

and the reactivity of OCl� is negligible. In addition, the

increase in the reaction rate in acidic solutions (pHo4)suggests that the reaction is subject to acid catalysis.

This pH-dependence was described by considering the

speciation of chlorine (reaction 3, pKa3 ¼ 7.5 (Albert

and Serjeant, 1984)), the acid-catalyzed reaction of

HOCl with MC-LR (reaction 4), and the reactions of

MC-LR with HOCl (reaction 5) and OCl� (reaction 6).

HOCl3OCl� þHþ Ka3, (3)

MC-LRþHOClþHþ! products k4, (4)

MC-LRþHOCl! products k5, (5)

MC-LRþOCl� ! products k6. (6)

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0

2

4

6

8

10

12

14

16

18

0 50 100 150 200 250[Cl2]t0, �M

k', x

103

s-1

Fig. 2. Plot or the pseudo-first-order rate constant versus the

initial concentration of chlorine: calculation of the apparent

second-order rate constant at 20 1C and pH 7.2.

0 50 100 150 200-3.0

-2.7

-2.4

-2.1

-1.8

-1.5

-1.2

-0.9

-0.6

-0.3

0.0

ln([

MC

-LR

]/[M

C-L

R] 0

)

time, s

Fig. 3. Influence of the pH on the pseudo-first-order rate

constant at 20 1C (pH ¼ 4.8 (’); pH ¼ 5.6 (&); pH ¼ 6.3 (K);

pH ¼ 7.2 (J); pH ¼ 8.0 (m); pH ¼ 8.8 (B)). Initial concentra-

tions of MC-LR and chlorine were 2.3 and 95mM, respectively.The solid line is a linear least-squares regression of the data.

1

10

100

1000

4 6 8 10pH

kapp

, M-1

s-1

5 7 9

Fig. 4. pH dependence of the apparent rate constants for the

reaction of chlorine with MC-LR. The line is calculated

according to Eq. (9).

J.L. Acero et al. / Water Research 39 (2005) 1628–16381632

From this reaction set, the degradation of MC-LR

can be written as follows:

�d½MC-LR�

dt¼ ðk4½HOCl�½H

þ� þ k5½HOCl�

þ k6½OCl��Þ½MC-LR�. ð7Þ

Taking into account the speciation of chlorine

(reaction 3), Eq. (7) is transformed into Eq. (8):

�d½MC-LR�

dt¼

ðk4½Hþ� þ k5Þ

½Hþ�

½Hþ� þ Ka3

� �þ k6

Ka3

½Hþ� þ Ka3

� �� �

� ½MC-LR�½Cl2�t, ð8Þ

where [Cl2]t ¼ [HOCl]+[OCl�]. Finally, by comparing

Eqs. (1) and (8), the apparent second-order rate constant

at each pH, kapp, is given by Eq. (9)

kapp ¼k4½H

þ�2 þ k5½Hþ� þ k6Ka3

ðKa3 þ ½Hþ�Þ. (9)

The individual rate constants were determined by a

non-linear least-squares regression of the experimental

pH profile of kapp (Eq. (9)). The obtained values were

2.07� 107, 116 and 6.78M�1 s�1 for k4, k5 and k6,

respectively. Fig. 4 depicts the experimental and

theoretical pH profiles of the apparent second-order

rate constants for the reaction of MC-LR with chlorine.

Reasonable good agreement between the experimental

values (symbols) and the calculated values from Eq. (9)

(lines) confirms the proposed reaction set. In general,

kapp decreases continuously in the pH range from 4 to 9.

These results indicate that the main reaction pathway at

circumneutral pH range is the reaction between MC-LR

and HOCl (reaction 5). In effect, the acid catalyzed

reaction becomes important at pHo5 and the rateconstant for reaction 6 is 20 times smaller than the

corresponding rate constant for reaction 5. Conse-

quently, acid catalyzed reaction of MC-LR with HOCl

(reaction 4) as well as the reaction of MC-LR with OCl�

(reaction 6) can be considered as negligible during

chlorination of natural waters (pH region 6–8).

Based on earlier chlorination studies of phenols (Lee,

1967; Rebenne et al., 1996), the above mechanism would

not be valid for acidic pH if high concentrations of

chloride ions were present. Under these conditions, Cl2can be formed from HOCl and Cl�. Since Cl2 is more

electrophilic than HOCl, its presence could accelerate

MC-LR degradation (Rebenne et al., 1996). The low

Cl� concentration in our commercial hypochlorite

reagent did not apparently affect the rate of MC-LR

ARTICLE IN PRESS

0.0 -0.5 -1.0 -1.5 -2.0

-2.0

-1.5

-1.0

-0.5

0.0

ln([

MC

-RR

]/[M

C-R

R] 0

)

ln([MC-LR]/[MC-LR]0)

Fig. 5. Competition kinetics plot for the calculation of the

apparent rate constant for the reaction of chlorine with MC-

RR at pH ¼ 6 (’), pH ¼ 7 (&) and pH ¼ 8 (K). Experi-

mental conditions given in Table 2.

J.L. Acero et al. / Water Research 39 (2005) 1628–1638 1633

degradation since the reaction rates were first-order with

respect to MC-LR even at low pH.

This pH-dependence of MC-LR chlorination confirms

the results reported by Nicholson et al. (1994). They found

that aqueous chlorine as the chlorinating agent was a

more efficient oxidant for the degradation of MC-LR in

comparison to calcium hypochlorite or sodium hypo-

chlorite, which have a basic character. These results were

attributed to the effect of pH, being toxin degradation

significantly reduced above pH 8. Tsuji et al. (1997) also

reported that a free chlorine dose of 0.7–2.8mg l�1 was

enough to remove MC-LR from distilled water if the pH

was kept below 7.2. Therefore the pH of the natural water

must be adjusted to below 7.5–8 during the chlorination

process to ensure adequate toxin removal.

The temperature effect on the apparent second-order

rate constant was investigated between the range

10–30 1C in experiments performed with similar con-

centration of MC-LR (2.3 mM) and chlorine (100mM) atpH 7.2. The obtained values for the apparent second-

order rate constants were 53.4, 60.2, 72.0 and

92.6M�1 s�1 at 10, 15, 20 and 30 1C, respectively. The

activation energy was calculated to be 20.1 kJmol�1.

Although the reaction was reaction-controlled according

to the values of the rate constants, the activation energy

was slightly above the value of 20 kJmol�1 proposed by

Pilling and Seakings (1995) for diffusion-controlled

reactions. This activation energy is lower than

58.5 kJmol�1 proposed by Lee (1967) for phenol

chlorination. Therefore, temperature does not exert an

important effect of MC-LR chlorination.

3.2. Kinetics of the reaction of chlorine with MC-RR and

MC-YR

The rate constants for the reactions of chlorine with

MC-RR and MC-YR were determined by competition

kinetics. This method has been successfully apply for the

calculation of rate constants for the reactions of organic

compounds with several oxidants such as bromine

(Acero et al., 2005), ozone (Gurol and Nekouinaini,

1984), UV radiation (Zamy et al., 2004) and OH radicals

(Haag and Yao, 1992). This procedure consists of the

simultaneous degradation of mixtures of two organic

compounds with similar chemical structure. One organic

compound acts as the reference compound, whose rate

constant with the oxidant is known; and the second one

being the target compound, whose rate constant for the

reaction with the oxidant must be determined. In the

present study, the target compound was MC-RR or

MC-YR, and the reference compound was MC-LR. The

application of this competition kinetic methods leads to

the following equation:

lnMC-RR½ �

MC-RR½ �0

� �¼

kapp MC-RR

kapp MC-LRln

MC-LR½ �

MC-LR½ �0

� �, (10)

where kapp MC�RR and kapp MC�LR represent the

apparent second-order rate constants for the reactions

of chlorine with the target compound (MC-RR or MC-

YR) and with the reference compound (MC-LR).

According to this Eq. (10), a plot of ln([MC-RR]/[MC-

RR]0) against ln([MC-LR]/[MC-LR]0) should be a

straight line whose slope is the ratio of apparent rate

constants. As kapp MC�LR is known, kapp MC�RR can

then be determined. Fig. 5 shows this plot for experi-

ments performed with MC-RR at different pH and

20 1C. As can be observed, points lie satisfactorily

around straight lines, confirming the fulfillment of

Eq. (10). By least squares regression analysis, the slopes

kapp MC�RR/kapp MC�LR were determined, these values

being detailed in Table 2. The apparent second-order

rate constant for the chlorination of MC-RR (Table 2)

was determined from the values of kapp MC�LR at the

investigated pHs. From these results, it can be deduced

that the reactivity of MC-LR and MC-RR at circum-

neutral pH are very similar.

Since pure MC-YR was not available, a similar

experiment was performed with microcystin extract

from Sujelow reservoir, in which MC-LR, -RR and

-YR were present. The initial toxin concentration ratio

in this extract was determined to be 1:0.9:1.2 measured

as MC-LR:MC-RR:MC-YR. This experiment was

performed at pH 7 and 20 1C with and initial chlorine

concentration of 100 mM and the initial toxin concentra-

tions given in Table 2. The plot of Eq. (10) lead a value

of the ratio kapp MC�YR/kapp MC�LR of 1.08 with

r240.99. Therefore, a value of 98.8M�1 s�1 is proposed

for the apparent second-order rate constant for the

reaction of chlorine with MC-YR at pH 7 and 20 1C.

The values of the chlorination rate constants are very

similar for the investigated microcystins. According to

ARTICLE IN PRESS

Table 2

Competition kinetics experiments for the determination of the apparent second-order rate constants for MC-RR and MC-YR

chlorination at 20 1C ([chlorine]0 ¼ 100mM)

pH [MC-RR]0 (mM) [MC-LR]0 (mM) kapp MC�RR/kapp MC�LR kapp MC�LR (M�1 s�1) kapp MC�RR (M

�1 s�1)

6.1 2.35 2.35 1.02 127.8 130.3

7.0 2.30 2.30 0.99 91.5 90.6

8.0 2.35 2.35 1.02 33.1 33.8

7.0* 2.51 2.17 1.08 91.5 98.8

*Experiment with MC-YR as target compound.

J.L. Acero et al. / Water Research 39 (2005) 1628–16381634

these results, chlorine likely attacks the conjugated

double bond of the Adda residue of microcystins. Since

the Adda moiety is the same in the three investigated

toxins, they show similar susceptibility to chlorine

oxidation at this point. In general, as the stereochem-

istry of toxic microcystins variants is similar in this part

of the molecule, it could be expected that all microcystin

species, including MC-LA, would react similarly with

chlorine. Similar behavior was observed for the ozona-

tion of microcystins (Newcombe et al., 2003).

Moreover, chlorine attack at the Adda group is

supported by the reaction products formed. Tsuji et al.

(1997) reported that the main degradation product was

dihydroxymicrocystin formed through the chloronium

ion at the conjugated diene of Adda, followed by

spontaneous hydrolysis. Other products may be its

stereoisomers and/or regioisomers, which were not

noxious.

3.3. Kinetics of the reaction of monochloramine with

MC-LR

The rate constant for the reaction of monochloramine

with MC-LR was also determined. In addition to the

importance of monochloramine as a final disinfectant, it

can be formed during the chlorination of ammonia-

containing waters (Vikesland et al., 2001). Therefore,

when the overall degradation of microcystin in a

chlorination process is to be investigated, reactions with

chlorine and monochloramine must be considered. For

that, the rate constant for the reaction with monochlor-

amine needs to be determined.

Experiments were performed at pH 8 and 20 1C in

presence of excessive monochloramine to observe an

appreciable degradation of MC-LR within several

hours. The rate of MC-LR degradation fits well

second-order kinetics, first order in the concentrations

of MC-LR and monochloramine. Since the degradation

of MC-LR was very slow, appreciable errors in MC-LR

concentration could be made. Moreover, the reaction

lasted several hours, time enough for monochloramine

self-decomposition (Vikesland et al., 2001), which makes

the consideration of constant monochloramine concen-

tration unlikely. Therefore, only an upper limit of

1.2� 10�2M�1 s�1 could be estimated for the second-

order rate constant for the monochloramination of

MC-LR. This result is in agreement with the weaker

oxidizing capability of monochloramine when compared

to both hypochlorous acid and hypochlorite (Glaze,

1990). Accordingly, monochloramine has been found to

be ineffective at oxidising microcystin in natural waters

(Nicholson et al., 1994; Hart et al., 1998). With this

insignificant rate constant, chloramine is not a suitable

oxidant for microcystin degradation from drinking

waters and plays an insignificant role in the degradation

of this group of toxins in the distribution system.

3.4. Significance of the reaction between chlorine and

microcystins in drinking water treatment processes

The efficiency of microcystin degradation during a

chlorination process depends on chlorine stability in the

water being treated, and on the reactivity of chlorine

towards the toxin. Chlorine stability varies with the

natural water quality parameters such as NOM,

ammonia and bromide concentrations and also with

the treatment conditions (pH and temperature) (Gallard

and von Gunten, 2002b). Therefore, to predict micro-

cystin degradation, in addition to the knowledge of

microcystin chlorination rate constants, chlorine decay

kinetics has to be characterized in each particular source

of water, for both oxidation pretreatment (high chlorine

demand) or final disinfection (reduced chlorine de-

mand). The chlorination treatment is deemed efficient

if microcystin concentration is reduced to below the

guideline value of 1mg l�1 (WHO, 1998).Based on the apparent second-order rate constants at

several pHs, the half-life times showed in Table 3 were

determined for MC-LR at a constant residual chlorine

concentration of 1mg l�1 (typical of the primary

disinfection) and 0.5mg l�1 (typical of the final disinfec-

tion). The degradation of MC-LR takes place in the

order of minutes at pH 6 and hours at pH48. As aconsequence, pH is a key variable in the chlorination

process and should be kept neutral or slightly acidic.

Therefore, when calcium or sodium hypochlorite are

ARTICLE IN PRESS

Table 3

Summary of MC-LR half-life values for chlorination at

different pHs. The initial concentration of chlorine (1 or

0.5mg l�1) was assumed to be constant

pH kapp (M�1 s�1) MC-LR half-life (min)

[chlorine]0¼ 1mg l�1

[chlorine]0¼ 0.5mg l�1

6 132.9 6.2 12.4

6.5 112.2 7.3 14.6

7 91.5 8.9 17.8

7.5 61.8 13.2 26.4

8 33.1 24.7 49.4

8.5 16.7 49.1 98.2

9 10.1 81.1 162.2

0

10

20

30

40

50

0 20 40 60

chlorine exposure (CT), mg l-1 min

[MC

-LR

], �g

l-1

Fig. 6. Degradation of MC-LR versus chlorine exposure.

Influence of the initial MC-LR concentration at pH ¼ 7 and

20 1C. [MC-LR]0 ¼ 50 mg l�1 (&); [MC-LR]0 ¼ 20mg l

�1 (J);

[MC-LR]0 ¼ 5 mg l�1 (B).

0

2

4

6

8

10

0 20 40 60 80

chlorine exposure (CT), mg l-1 min

[MC

-LR

], �g

l-1

Fig. 7. Degradation of MC-LR versus chlorine exposure at

pH ¼ 6 (&), pH ¼ 7 (J) and pH ¼ 8 (B). T ¼ 20 1C, [MC-

LR]0 ¼ 10 mg l�1.

J.L. Acero et al. / Water Research 39 (2005) 1628–1638 1635

used in high dose then the pH will need to be adjusted to

below 8 to guarantee the efficiency of the process.

Half-life values included in Table 3 were calculating

assuming a constant oxidant concentration during the

overall treatment process, which is not a realistic

situation. Instead, a parameter that includes the oxidant

decay within the reactor is the oxidant exposure.

Chlorine exposure (CT) is defined as the time-dependent

concentration of chlorine integrated over the reaction

time. To reach microcystin reduction in a chlorination

process, a certain chlorine exposure is needed. The

required CT value for achieving a certain degradation of

microcystin can be calculated from the following

equation in a batch process:

CT ¼� ln ½MC�=½MC�0

� �kapp

, (11)

where [MC] in the final concentration of microcystin

(guideline value of 1mg l�1), [MC]0 is the initial

concentration of microcystin and kapp is the apparent

second-order rate constant for the chlorination of

microcystin at a given temperature and pH. Note that

the widely used method for calculating CT10 has not

been applied since it leads to an underestimation of the

oxidant or disinfectant exposure in the reactor (Elovitz

et al., 2000).

The required CT value depends directly on the initial

concentration of microcystin in the natural water to be

treated or disinfected. Thus, Fig. 6 shows the degrada-

tion of MC-LR versus the CT value starting with

different initial concentrations of MC-LR at pH 7 and

20 1C. The initial microcystin concentrations were

selected within the range of MC presence in natural

waters. Spoof et al. (2003) reported microcystin con-

centrations up to 42.0 mg l�1 in Aland (SW Finland) in

2001. According to Fig. 6, in case of high toxin

concentration (410mg l�1) a chlorine exposure higherthan 30mg l�1min is required to match the guideline

value in the final water.

As discussed above, the pH exerts an important

influence on kapp, being the overall process favored at

low pHs. Fig. 7 shows the pH influence on the CT value

required for the degradation of 10 mg l�1 of MC-LR at20 1C. It can be observed that MC-LR is relatively easy

to eliminate at pH 6 or even 7. However, the required

chlorine exposure at pH 8 is above 80mg l�1min, which

can only be reached with high initial chlorine concen-

trations and elevated reaction times. In such conditions,

other problems such as trihalomethane and other

organic halogen formation have to be considered

(Gallard and von Gunten, 2002b).

Table 4 recompiles the CT values (calculated from

Eq. (11)) required for the degradation of MC-LR from

50 or 10 to 1mg l�1at several pHs (6–9) and temperatures(10–25 1C) in a batch or plug-flow reactor. Results

ARTICLE IN PRESS

Table 4

Chlorine exposure (CT values) for reducing microcystin

concentration to 1 mg l�1 at different pH and temperature in abatch or plug-flow reactor

pH [MC-LR]0(mg l�1)

CT-values (mg l�1min)

10 1C 15 1C 20 1C 25 1C

6 50 46.6 40.2 34.8 30.3

10 27.4 23.6 20.5 17.8

7 50 67.7 58.4 50.6 44.0

10 39.8 34.4 29.8 25.9

8 50 187.1 161.3 139.8 121.8

10 110.3 94.9 82.3 71.7

9 50 617.2 526.0 458.6 399.1

10 363.3 309.6 269.8 234.9

Table 5

Chlorine exposure (CT values) for reducing microcystin

concentration to 1mg l�1 at different pH and temperature in aCSTR

pH [MC-LR]0(mg l�1)

CT values (mg l�1min)

10 1C 15 1C 20 1C 25 1C

6 50 583.9 503.3 436.3 380.0

10 107.2 92.4 80.1 69.8

7 50 847.7 731.2 663.7 551.7

10 155.7 134.3 116.4 101.3

8 50 2347.5 2020.3 1751.8 1525.9

10 431.2 371.1 321.7 280.3

9 50 7731.1 6589.0 5740.9 4998.6

10 1420.0 1210.2 1054.4 918.1

J.L. Acero et al. / Water Research 39 (2005) 1628–16381636

indicate that low pH and low initial MC-LR concentra-

tion with elevated temperatures leads to the best CT

values. These CT values are comparable to those

required for achieving 99 percent (2 log) inactivation of

Giardia cyst and one to two order of magnitude higher

than those required for achieving 99.99 percent

(4 log) inactivation of viruses at pH 6 through 9 (CT

values for Giardia cyst (69mg l�1min) and viruses

(3–6mg l�1min) published by the USEPA (USEPA,

2003)). However, the pH influence is more pronounced

in MC-LR degradation than in bacteria or viruses

disinfection. As a consequence, degradation of micro-

cystins by chlorination at high pH must be carefully

controlled in order to meet the guideline value of 1mg l�1.The treatment of the reaction kinetics for the batch

experiments is mathematically equivalent to reaction

kinetics for an ideal plug-flow reactor (PFR), where

reaction time (batch reactor) and HRT (flow reactor) are

synonymous (Levenspiel, 1998). However, in drinking

water treatment practices, full-scale reactors have non

ideal flow behavior and can be considered hybrid of

PFR and completely stirred tank reactor (CSTR). The

PFR or CSTR represent the most and least efficient

reactor configurations, respectively (Levenspiel, 1998).

In the extreme case of ideal CSTR, the CT required to

eliminate microcystin can be calculated from Eq. (12)

CT ¼½MC�0=½MC�� �

� 1

kapp. (12)

Table 5 recollects the CT values required for the

degradation of MC-LR from 50 or 10 to 1mg l�1 atseveral pHs (6–9) and temperatures (10–25 1C) in a

CSTR. The CT values are almost one order of magnitude

higher than those corresponding with the PFR (Table 4).

These calculations are an example of the best and worst

case operating conditions for the degradation of MC-LR

and might serve for establishing safety chlorine dose in

oxidation or disinfection processes.

The chlorine dose needed to reach a certain CT value

in the reactor depends on the water quality parameters,

due to their influence on chlorine stability (Gallard and

von Gunten, 2002b). This is especially important in

surface water from rivers or eutrophic lakes, which

chemical/physical composition (e.g. temperature, pH,

DOM, ammonia, bromide) can change frequently

(Gallard and von Gunten, 2002b). In general, any

compound that reacts with chlorine (DOM, ammonia,

bromide) decreases the chlorine exposure, and therefore,

higher oxidant dose must be added to the water to reach

the required toxin elimination. Thus, higher chlorine

doses must be used in waters with high organic content

(Nicholson et al., 1994; Nicholson and Rositano, 1997).

In presence of ammonia, chloramines are formed, which

act as a chlorine sink and are not effective in microcystin

degradation. The presence of bromide is also important,

leading to the formation of hypobromous acid (HOBr)

which is a powerful oxidant, especially for phenolic

compounds (Acero et al., 2005). Kinetics of microcystin

bromination needs to be determined to assess the

importance of the bromination pathway in the overall

chlorination process. Therefore, it is necessary to

calibrate each water with respect to the rate of chlorine

depletion in order to determine the initial dose that leads

to the required CT value.

4. Conclusions

The reactions of MC-LR, -RR and -YR with chlorine

fit overall second-order kinetics over a wide pH range,

first-order with respect to both microcystin and chlorine.

It was found that an increase of pH has a negative effect

on the microcystin degradation rate. Apparent second-

ARTICLE IN PRESSJ.L. Acero et al. / Water Research 39 (2005) 1628–1638 1637

order rate constant for the chlorination of MC-LR at

20 1C decreased from 475M�1 s�1 at pH 4.8 to

9.8M�1 s�1 at pH 8.8. From these apparent second-

order rate constants, rate constants for the reactions of

MC-LR with aqueous chlorine species (hypochlorous

acid and hypochlorite) were evaluated. The results

indicate that the main reaction at circumneutral pH

range is the reaction between MC-LR and HOCl.

Similar reactivity with chlorine was found for MC-RR

and MC-YR, supporting that the main reactive site of

microcystin is the Adda moiety. These chlorination rate

constants can be utilized to determine the rate of

cyanotoxins degradation under typical water treatment

conditions. Half-life times ranged from minutes at pH 6

to hours at pH above 8 for a constant chlorine

concentration of 1.0–0.5mg l�1 (typical of oxidation

pre-treatment and final disinfection doses). Although

microcystin concentrations in our experiments were

much higher than expected for natural waters, the

chlorination rate constants determined in this study are

independent on the chlorine concentration and therefore

can also be applied in natural waters with much lower

microcystin concentrations.

The chlorine exposure (CT values) required to reduce

MC-LR concentration to 1mg l�1 in batch or PFR arecomparable to those required for achieving 99 percent

(2 log) inactivation of Giardia cyst, and one to two order

of magnitude higher than those required for achieving

99.99 percent (4 log) inactivation of viruses at pH 6

through 9. Chlorination is therefore a feasible option for

microcystin degradation during oxidation and disinfec-

tion processes if the pH of the water is kept below 8

during the treatment. However, monochloramine, which

can be formed during chlorination of ammonia-contain-

ing waters, is not capable of oxidizing microcystins.

These results are an important factor in process design

and the choice of the applied oxidants for the removal of

cyanobacterial toxins during drinking water treatment.

Acknowledgements

This work was supported by the European Commis-

sion under the Fifth Framework Programme, research

project TOXIC (EVK1-CT-2002-00107).

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