Oxidation of the Cyanobacterial Hepatotoxin Microcystin-LR by

Oct 12, 2004 - Institute of Occupational Health, Hämeenkatu 10,. 20500 Turku, Finland. Cyanobacteria are known producers of cytotoxins, hepatotoxins,...
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Environ. Sci. Technol. 2004, 38, 6025-6031

Oxidation of the Cyanobacterial Hepatotoxin Microcystin-LR by Chlorine Dioxide: Reaction Kinetics, Characterization, and Toxicity of Reaction Products T O M A S P . J . K U L L , * ,†,‡ P E T E R H . B A C K L U N D , †,§ KRISTER M. KARLSSON,† AND JUSSI A. O. MERILUOTO† Department of Biochemistry and Pharmacy, A° bo Akademi University, P.O. Box 66, 20521 Turku, Finland, Department of Biology, A° bo Akademi University, BioCity, Tykisto¨katu 6A, 20520 Turku, Finland, and Turku Regional Institute of Occupational Health, Ha¨meenkatu 10, 20500 Turku, Finland

Cyanobacteria are known producers of cytotoxins, hepatotoxins, and neurotoxins. The main toxins are microcystins, cyclic heptapeptide hepatotoxins, produced by strains of several cyanobacterial genera frequently found in eutrophied freshwaters. Due to the acute and chronic toxicity of microcystins, successful removal of these toxins in drinking water treatment processes is of increasing concern. In the present work the kinetics of microcystin-LR (MC-LR) oxidation by chlorine dioxide (ClO2) was studied with UV-spectrometry and high performance liquid chromatography (HPLC). Characterization of reaction products was performed with mass spectrometric (MS) analysis, while the toxicity of reaction products was tested with a protein phosphatase inhibition assay (PPIA). The main reaction products formed, dihydroxy isomers of MC-LR as identified by MS, were nontoxic according to the PPIA. The overall rate constant k for the reaction between MC-LR and ClO2 at 293 K and pH 5.65 was modest, k ) 1.24 M-1 s-1, suggesting that ClO2 is not a suitable oxidant for the degradation of microcystins in drinking water treatment processes.

Introduction Mass occurrences (blooms) of cyanobacteria have become an increasing problem in eutrophied waters worldwide. Besides aesthetic aspects such as unattractiveness to bathers, odor, and bad taste of treated drinking water, many species or strains of cyanobacteria also produce potent cytotoxins, hepatotoxins, and/or neurotoxins. These toxins affect large human populations via drinking water, and they also pose a threat to domestic and wild animals. Animal kills caused by cyanobacterial toxins are frequently reported. Human intoxications, in some cases lethal, have occurred (1). * Corresponding author phone: + 358 2 215 3315; fax: + 358 2 215 4748; e-mail: [email protected]. † Department of Biochemistry and Pharmacy, Åbo Akademi University. ‡ Department of Biology, Åbo Akademi University. § Turku Regional Institute of Occupational Health. 10.1021/es0400032 CCC: $27.50 Published on Web 10/12/2004

 2004 American Chemical Society

The main cyanobacterial toxins are microcystins, cyclic heptapeptide hepatotoxins. They are produced by many strains of cyanobacteria belonging to the freshwater genera Microcystis, Anabaena, Oscillatoria/Planktothrix, and Nostoc. About 70 different analogues of microcystins have been identified, among which microcystin-LR (MC-LR) is the most abundant and also the most toxic microcystin (LD50 50 µg kg-1, mouse i.p.) (1). The general structure of microcystins is cyclo-(D-Ala-L-X-MeAsp(iso-linkage)-L-Z-Adda-D-Glu(isolinkage)-Mdha), in which X and Z are variable L-amino acids, MeAsp is D-erythro-β-methylAsp, Mdha is N-methyldehydroAla, and Adda is the unique 20 carbon β-amino acid 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid. For MC-LR X and Z are Leu and Arg, respectively (Figure 1). The toxicity of microcystins is related to the Addaglutamate region of the molecule, which interacts with and irreversibly inhibits eukaryotic serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A) (2, 3). Structural modifications in the Adda-glutamate region will, in many cases, render the molecule nontoxic (4-6). In mammals, acute exposure to high levels of microcystins will cause lethal liver hemorrhage or liver failure, while chronic exposure to low levels exhibits tumor promoting activity and is possibly carcinogenic (7-9). Due to the health hazards caused by microcystins, the World Health Organization (WHO) has proposed a guideline value for a maximum concentration of 1.0 µg L-1 for MC-LR in drinking water (1). Water bodies containing potentially toxic cyanobacteria are unfortunately often used for drinking water production. Although produced and retained within healthy growing cells, cyanobacterial toxins are released to the water phase at cell lysis, either caused by natural processes or artificially induced, e.g. through the use of an algicide. In case of a toxic bloom, typical dissolved (extracellular) toxin concentrations are 0.110 µg L-1, but the concentration can be much higher if a major bloom is breaking down (1). This situation can be problematic for water works with the mission to produce safe drinking water for the consumers, since conventional treatment methods, i.e., coagulation/flocculation, clarification, and sand filtration, are ineffective in removing dissolved microcystins and other cyanobacterial toxins (10, 11). Addition of a granular activated carbon (GAC) filtration step to the treatment process will substantially reduce the concentration of dissolved toxins (10, 11). The lifetime of the GAC filters are, however, limited, and the removing efficiency will decrease with time (12). This can be overcome by more frequent regenerations of the filters, by recurrent analyses of the purified water in order to avoid breakthroughs of toxins, by using additional physical treatment methods, e.g. powdered activated carbon (PAC) or reverse osmosis (RO), or by introducing a chemical oxidation step in the treatment process that will degrade the toxins. The oxidizing agents most commonly used in drinking water treatment are chlorine (Cl2, HOCl/OCl-), potassium permanganate (KMnO4), ozone (O3), chlorine dioxide (ClO2), and chloramines (NH2Cl, NHCl2). Among these, O3 is probably the most effective and fastest agent in degrading microcystins. In pure water at 293 K, pH 2 and at an applied dose of 2 mg L-1 O3, MC-LR at a concentration of 500 µg L-1 was reduced to a level below the detection limit within fractions of a second (13). The decomposition rate was found to be pH dependent, with alkaline conditions less favorable (13). This pH-dependence has been explained with the lower oxidation potential of O3 at higher pH (14). In pure water and at an applied dose of 2 mg L-1 KMnO4, > 90% of dissolved MC-LR at a concentraVOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Structural formula for MC-LR (MW 995.2, average mass). tion of 1 mg L-1 was degraded within 10 min (14). Chlorination has been reported to be effective in degrading microcystins at pH < 8 and a free chlorine residual of > 0.5 mg L-1 after 30 min contact time, while chloramination had negligible effects on microcystins (15). For ClO2 there is very limited information available (16), and no scientific paper has to our knowledge been published concerning oxidation of microcystins with ClO2. In Europe and many other parts of the world, ClO2 is frequently used both as a pre- and posttreatment step in drinking water purification. It is therefore important with respect to public health to be aware of the effects of ClO2 on microcystins in water. The aim of this work was thus to determine the overall rate constant for the reaction between MC-LR and ClO2 and to study its dependence on temperature and pH, to characterize the main reaction products formed, and to test the toxicity of the products with respect to their protein phosphatase 1 (PP1) inhibition.

Experimental Section Chemicals. Microcystin-LR (MC-LR) was extracted and purified from a Microcystis aeruginosa (PCC7820) culture according to ref 17 with minor changes. Chlorine dioxide (ClO2) was prepared according to the procedure described below. Water was purified with a Millipore Milli-Q plus PF system to 18.2 MΩ cm. Protein phosphatase 1 (PP1) (rabbit skeletal muscle, recombinant from E. coli) was purchased from New England Biolabs, U.S.A. Bovine serum albumin (BSA) was obtained from Sigma. Acetonitrile (ACN) and methanol (MeOH) were of HPLC grade. Trifluoroacetic acid (TFA) was of protein sequence analysis grade. Ascorbic acid, dithiothreitol (DTT), formic acid, HCl, KH2PO4, K2HPO4‚2H2O, MgCl2‚6H2O, MnCl2‚4H2O, NaBH4, Na2CO3, NaHCO3, pnitrophenyl phosphate (p-NPP), and tris-(hydroxymethyl)aminomethane (Tris) were of analytical grade. NaClO2 was of technical grade. Chlorine Dioxide Preparation. ClO2 was prepared by reacting 15 mL of 0.67 M HCl with 45 mL of 0.28 M NaClO2 in a magnetic stirred reaction vessel. Formed ClO2 gas was transferred with a stream of Ar gas, bubbled through the solution at 0.1 bar pressure, for 2 h into an ice-cooled gaswashing bottle containing 100 mL of Milli-Q water. The obtained ClO2 stock solution, containing ca. 1.5 mg mL-1 of ClO2, was portioned into 8 mL glass vials and stored at 277 K until used within 1 week after preparation. The concentration of ClO2 in the vials was determined spectrophotometrically at 358 nm prior to use, using a molar absorptivity of 1200 M-1 cm-1 (18). Caution: ClO2 is a strong oxidative agent and highly toxic and may under certain circumstances spontaneously explode. Preparation of ClO2 should be carried out in a fume cupboard with high safety precautions and by skilled laboratory personnel only. Reaction Kinetics by UV-Spectrometry. The experiments were carried out by using a thermostated Shimadzu UV3000 dual wavelength double beam spectrometer connected via a MacLab/4 analogue to digital converter (AD Instru6026

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ments) to a computer equipped with the MacLab Chart v3.3 chart recorder program. The reaction between MC-LR and ClO2 was followed for 30 min by measuring the decrease in absorbance at 238 nm. The output signal from the spectrometer was recorded 20 times min-1 during the experiments. Reactions were carried out in a 3 mL 1 cm quartz cuvette with a Teflon lid at 7 different ClO2/MC-LR molar ratios (0, 17, 34, 68, 170, 340, and 680) at 293 K and pH 5.65 (pH of pure water in equilibrium with atmospheric CO2 (19)), at 3 different temperatures (283, 293, and 303 K) at a ClO2/ MC-LR molar ratio of 340 and pH 5.65, and at 4 different pH (5.65, 7.0, 9.0, and 10.0) at 293 K and a ClO2/MC-LR molar ratio of 340. For pH 7.0 a 10-3 M KH2PO4/10-3 M K2HPO4 buffer solution was used, for pH 9.0 a 10-3 M NaHCO3/10-4 M Na2CO3 buffer, and for pH 10.0 a 10-3 M NaHCO3/10-3 M Na2CO3 buffer. The initial concentration of MC-LR in the sample cuvette was 2.3 nmol mL-1 (2.3 µg mL-1), and the total volume in the cuvettes was 3000 µL. An equal amount (0-240 µL stock solution) of ClO2 was added to the reference and sample cuvettes prior to the addition of MC-LR (80 µL stock solution) to the sample cuvette. Mixing was achieved by inverting the cuvettes twice after addition of oxidant (reference and sample cuvettes) and reductant (sample cuvette). Recording of the output signal started when MCLR was added to the sample cuvette. The presence of 238 nm absorbing chromatophores in MC-LR that are not affected by ClO2 must be corrected for when converting the output signal or absorbance values to concentrations, otherwise the calculated rate constants will have incorrect values. This was achieved by setting the average output signal value at 25-30 min in the 680 molar ratio curve (plateau value) as zero concentration level for intact MC-LR, and combined with the output signal value for a 2.3 nmol mL-1 standard solution of MC-LR, a calibration curve was obtained from which the concentrations of intact MC-LR in the sample cuvette were calculated. Reaction Kinetics by HPLC. Reactions were carried out in 1.8 mL HPLC borosilicate glass vials with screw caps and septa in a thermostated water bath at 293 K, pH 5.65 and a ClO2/MC-LR molar ratio of 340 and 680. MC-LR (40 µL stock solution) was added to the vials prior to the addition of ClO2 (50 or 100 µL stock solution). Mixing was achieved by inverting the vials twice after each addition. The initial concentration of MC-LR in the vials was 2.3 nmol mL-1 and the total volume was 1500 µL. The reaction between MC-LR and ClO2 was stopped after 0, 1, 3, 5, 10, 15, 20, 25, and 30 min through the addition of ascorbic acid (50 µL stock solution) at a final ascorbic acid/ClO2 molar ratio of 7.35. The addition of ascorbic acid immediately consumed all remaining ClO2, so the reaction between MC-LR and ClO2 was instantly stopped (k ≈ 107 M-1 s-1 (20)). The remaining concentration of MCLR in the vials as well as in a control sample treated in the same way but without addition of ClO2 was analyzed by HPLC on an Agilent 1100 Series system (degasser, quaternary pump, auto sampler, column oven, UV/DAD detector) using a Merck Purospher Star RP-18e column (3 µm particles, 55 × 4 mm + 4 × 4 mm guard column) at 313 K with a linear gradient (eluent A: Milli-Q water + 0.05% TFA, eluent B: ACN + 0.05% TFA, eluent program: 0 min 25% B, 5 min 70% B, 6 min 70% B, 6.1 min 25% B, 9 min stop) and a flow rate of 1 mL min-1. Injection volume was 10 µL, and detection was performed at 238 nm. Toxicity of Reaction Products. The toxicity of the reaction products was tested with a colorimetric protein phosphatase 1 inhibition assay, slightly modified after (21, 22). The solutions used for the reaction kinetics studies by HPLC were diluted 1000 times with Milli-Q water. Ten microliters of each diluted solution was added to a well on a 96-well microtiter plate, mixed with 20 µL of PP1 solution (a 2.5 U* aliquot of PP1 dissolved in 1.5 mL of pH 7.4 buffer solution containing

50 mM Tris-HCl, 1.0 mg mL-1 of BSA, 1.0 mM MnCl2, and 2.0 mM DTT), incubated 8 min, whereafter 200 µL of p-NPP solution (15 mM p-NPP in pH 8.1 buffer solution containing 50 mM Tris-HCl, 20 mM MgCl2, 0.2 mM MnCl2, and 0.5 mg mL-1 BSA) was added to each well. The content of the wells was then mixed by swirling the plate sideways. After incubation at 310 K for 2 h, the absorbance in the wells was read with a Wallac Victor 1420 multilabel counter at 405 nm. The PP1 inhibition was calculated according to the equation: PP1 inhibition ) (Apositive zero control - Asample)/ (Apositive zero control - Anegative zero control) × 100%. The positive zero control refers to 10 µL of Milli-Q water mixed with 20 µL of PP1 solution, while the negative zero control refers to 10 µL of Milli-Q water mixed with 20 µL of enzyme buffer without PP1 prior to mixing with 200 µL of p-NPP solution. *U ) unit of enzyme activity, defined by the manufacturer as the amount of enzyme that hydrolyzes 1 nmol of p-NPP (50 mM) in 1 min at 303 K in a total reaction volume of 50 µL. Characterization of Reaction Products. MC-LR (2.3 nmol mL-1) was oxidized by ClO2 at 293 K and a ClO2/MC-LR molar ratio of 340. The reaction was stopped after 0, 1, 3, 5, 10, 15, 20, 25, and 30 min through the addition of ascorbic acid at an ascorbic acid/ClO2 molar ratio of 7.35. The formation of oxidation products was recorded using a Micromass Quattro II Triple Quadrupole mass spectrometer (MS) equipped with a coaxial electrospray ion source, combined with a Shimadzu SIL-9A auto injector and a Shimadzu LC-10AT HPLC pump. Separation of the compounds was performed on a Merck Purospher Star RP-18e column (3 µm particles, 55 × 4 mm + 4 × 4 mm guard column) at 313 K using a linear gradient (eluent A: Milli-Q water + 0.5% formic acid, eluent B: ACN, eluent program: 0 min 25% B, 5 min 70% B, 6 min 70% B, 6.1 min 25% B, injection interval 12 min) and a flow rate of 1 mL min-1. The flow was split to 1/10 before introduction to the ion source. Injection volume was 10 µL. The instrument was operated in positive electrospray ionization mode with a capillary voltage of 3.6 kV, a cone voltage of 60 V, an ion source temperature of 383 K, and a dry gas flow of 250 L h-1. Selective ion recording (SIR) was used for analysis of the main oxidation products. An ion trap mass spectrometer (Agilent 1100 Series LC/MSD SL) with direct infusion was used initially to determine the m/z values of the reaction products. The m/z values found (1029 and 1047) were subsequently used for the SIR channels in the triple quadrupole instrument. Daughter ion recording of the m/z 1029 and 1031 ions was used for further identification of the products. The following ion trap instrument parameters were used: nebulizer gas pressure 15 psi, dry gas flow 4 L min-1, and dry gas temperature 598 K. Smart parameter settings (SPS) for m/z 1000 were used, and daughter scans were recorded and combined typically for 2 min. The fragments were identified according to ref 23, and the selective reduction of the R,β-unsaturated carbonyl group in the Mdha residue with NaBH4 was done according to ref 24.

FIGURE 2. 238 nm absorbance profile curves (a), and MC-LR concentration profile curves (b), for the reaction between MC-LR and ClO2 at 293 K, pH 5.65 and a ClO2/MC-LR molar ratio of 0-680 (top-bottom). Plot of pseudo-first-order rate constant k′ vs initial concentration of ClO2 (c).

TABLE 1. Pseudo-First-Order Rate Constants at Different Molar Ratiosa molar ratio

plot range (min)

r

t1/2 (min)

k′ (s-1)

0 17 34 68 170 340 680

0-30 0-30 0-30 0-30 0-20 0-15 0-10

0.095 -0.969 -0.987 -0.996 -0.998 -0.998 -0.998

-9378.5 209.1 121.0 59.4 24.5 11.7 4.4

-0.000001 0.000055 0.000096 0.000195 0.000471 0.000990 0.002609

a

Results The reaction between microcystin-LR (MC-LR) and chlorine dioxide (ClO2) in pure water was relatively slow. At 293 K, pH 5.65 and a ClO2/MC-LR molar ratio of 680 (107 mg L-1 of ClO2), MC-LR was fully oxidized after 20 min contact time. At a molar ratio of 17 (2.7 mg L-1 of ClO2), there was only a slight decrease in absorbance after 30 min contact time (Figure 2a). The corrected concentration profile curves are shown in Figure 2b. Assuming a negligible decrease in ClO2 concentration during the experiments, pseudo-first-order rate constants k′ for the oxidation of MC-LR by ClO2 are obtained from the slope in first-order plots, ln [MC-LR]/[MC-LR]0 vs t (25). At 293 K, pH 5.65, and a ClO2/MC-LR molar ratio of 17-680, the obtained k′ ranged from 5.5 × 10-5 s-1 to 2.6 × 10-3 s-1. The

T ) 293 K, pH 5.65.

obtained k′ are summarized in Table 1, together with their corresponding correlation coefficients, r, and calculated halflife values, t1/2. Due to the very good correlations in the firstorder plots, the reaction between MC-LR and ClO2 can be considered to be of first-order in MC-LR concentration. The relationship between k′ and the initial concentration of ClO2 is given by the equation k′ ) 1.24 [ClO2]0 - 2.4 × 10-6 s-1 (r ) 1.000, n ) 6). The overall (second-order) rate constant k for the reaction between MC-LR and ClO2 at 293 K and pH 5.65 is equal to the slope of the regression line, i.e., 1.24 M-1 s-1 (Figure 2c). The reaction between MC-LR and ClO2 showed a positive temperature dependence. At pH 5.65 and a ClO2/MC-LR VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Pseudo-First-Order and Overall Rate Constants at Different Temperaturesa plot temp (K) range (min) 283 293 303 a

0-20 0-15 0-10

r

t1/2 (min)

k′ (s-1)

k (M-1 s-1)

-0.998 -0.999 -0.998

21.5 11.6 5.0

0.000538 0.000995 0.002302

0.68 1.25 2.90

ClO2/MC-LR molar ratio ) 340, pH 5.65.

TABLE 3. Pseudo-First-Order and Overall Rate Constants at Different pHa pH

plot range (min)

r

t1/2 (min)

k′ (s-1)

k (M-1 s-1)

5.65 7.0 9.0 10.0

0-15 0-15 0-15 0-15

-0.998 -0.997 -0.998 -0.998

12.1 13.3 16.3 18.1

0.000952 0.000870 0.000711 0.000637

1.20 1.10 0.89 0.80

a

T ) 293 K, ClO2/MC-LR molar ratio ) 340.

FIGURE 3. Decrease rate of intact MC-LR at 293 K, pH 5.65, and a ClO2/MC-LR molar ratio of 340 and 680 as determined by online UV-spectrometry and HPLC. molar ratio of 340, k′ increased from 5.4 × 10-4 s-1 to 2.3 × 10-3 s-1 as the temperature was raised from 283 to 303 K. The obtained k′ are summarized in Table 2, together with their corresponding correlation coefficients, calculated half-life values and overall rate constants. The preexponential factor A and the activation energy Ea in the Arrhenius equation k ) Ae-Ea/RT, determined by an Arrhenius plot, were 2.30 × 109 M-1 s-1 and 51.74 kJ mol-1, respectively (r ) -0.994, n ) 3). pH was found to have a moderate influence on the reaction between MC-LR and ClO2, and the reaction rate was retarded with increasing pH. At 293 K and a ClO2/MC-LR molar ratio of 340, k′ decreased from 9.5 × 10-4 s-1 to 6.4 × 10-4 s-1 as pH was raised from 5.65 to 10.0. The obtained k′ are summarized in Table 3, together with their corresponding correlation coefficients, calculated half-life values, and overall rate constants. At 293 K and a molar ratio of 340, the relationship between k and pH is given by the equation k ) -0.0927 pH + 1.73 M-1 s-1 (r ) -0.999, n ) 4). The reactions between MC-LR and ClO2 carried out in 1.8 mL HPLC borosilicate vials, quenched with ascorbic acid and analyzed by HPLC-UV/DAD, gave very similar results to those obtained with online UV-spectrometry. At a ClO2/ MC-LR molar ratio of 340 the curves were overlapping, while at a molar ratio of 680 there were minor differences in the decrease rate of intact MC-LR (Figure 3). k′ determined from the HPLC data were 1.01 × 10-3 s-1 and 1.94 × 10-3 s-1 at a molar ratio of 340 and 680, respectively, while the overall rate constant k obtained in a plot of k′ vs [ClO2]0 was 1.23 M-1 s-1 (r ) 0.999, n ) 2, regression line forced through the origin of coordinates). In the HPLC-UV chromatograms several new signals appeared at 1.9-2.8 min, and their intensity 6028

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FIGURE 4. Disappearance of the m/z 995 ion and appearance of the m/z 1029 and 1047 ions vs time at 293 K, pH 5.65, and a ClO2/MC-LR molar ratio of 340 as determined by LC-MS. The heights of the m/z 1029 and 1047 bar have been obtained by combining intensities of several peaks of isomers. increased with increasing reaction time. Their substantially shorter retention times compared to that of intact MC-LR (3.4 min) revealed that the oxidation products were of a more hydrophilic character. Their low absorptivity at 238 nm relative to that at 214 nm indicated that the conjugated diene system in the Adda residue had been destroyed. LC-MS analysis revealed that the main oxidation products had m/z values of 1029 and 1047, respectively. This strongly suggests that MC-LR, which has an m/z value of 995 in the positive ionization mode, is substituted with two hydroxyl groups (995 + 2 × 17 ) 1029) and further with one hydroxyl group and one hydrogen (1029 + 17 + 1 ) 1047). The SIRchromatograms of both products contained several peaks, suggesting that isomers were formed in the reaction. At 293 K and a ClO2/MC-LR molar ratio of 340, the reaction products with m/z 1029 appeared within 1 min contact time, while the products with m/z 1047 appeared after 3 min (Figure 4). Fragmentation of the m/z 1029 ion in the ion trap analysis revealed that the main attack of ClO2 was directed toward the conjugated double bonds in the Adda residue. Additional support to this conclusion was obtained through the reduction of the R,β-unsaturated carbonyl group in the Mdha residue with NaBH4 prior to ClO2 oxidation. This reduced form, MC-LRred, has an m/z value of 997 in the positive ionization mode, and thus the corresponding primary oxidation product will have an m/z value of 1031. Daughter ion recording spectra of the m/z 1029 and 1031 ions are shown in Figure 5, and the suggested fragment formulas for the main peaks are given in Table 4. In the protein phosphatase inhibition assay there was an immediate decrease in PP1 inhibition with increasing contact time at both ClO2/MC-LR molar ratios. At 293 K, pH 5.65 and a ClO2/MC-LR molar ratio of 680, there was only a slight inhibition effect left after 20 min contact time. At a molar ratio of 340, the inhibition effect had decreased by 3/4 after 30 min contact time. In a plot of PP1 inhibition vs remaining MC-LR concentration as determined by HPLC, a good correlation was found at both molar ratios (r340 ) 0.987, r680 ) 0.990, n ) 9) (Figure 6). In addition, when the relative % values of PP1 inhibition and remaining MC-LR concentration were plotted vs time, the curves correlated relatively well at both molar ratios.

Discussion Chlorine dioxide (ClO2) is used at low dosages in drinking water treatment processes, typically 1 mg L-1 or less in posttreatment of purified water. Following reaction with the natural organic matter (NOM) present in the water, the residual concentration of ClO2 will be very low. A thorough understanding of the reaction kinetics between ClO2 and hazardous pollutants such as microcystins is thus important.

FIGURE 5. Daughter ion recordings of the m/z 1029 (top) and 1031 (bottom) ions, the main products formed in the reaction mixtures of MC-LR/ClO2 and MC-LRred/ClO2, respectively.

TABLE 4. Suggested Formulas for the Daughter Ions of the m/z 1029 and 1031 Ions m/z

fragment (M ) MC-LR, Mred ) MC-LRred)

indicating...

1029 1031 1011 1013 993 995 900 902 765 767 682 684 633 633 615 615 553 555 504 504 486 486

[M + 2OH + H]+ [Mred + 2OH + H]+ [M + 2OH - H2O + H]+ [Mred + 2OH - H2O + H]+ [M + 2OH - 2H2O + H]+ [Mred + 2OH - 2H2O + H]+ [Mdha-Ala-Leu-MeAsp-Arg-Adda + 2OH + H]+ [Mdhared-Ala-Leu-MeAsp-Arg-Adda + 2OH + H]+ [Mdha-Ala-Leu-MeAsp-Arg-Adda + 2OH - 135 + H]+ [Mdhared-Ala-Leu-MeAsp-Arg-Adda + 2OH - 135 + H]+ [Glu-Mdha-Ala-Leu-MeAsp-Arg + H]+ [Glu-Mdhared-Ala-Leu-MeAsp-Arg + H]+ [Arg-Adda-Glu + 2OH + H]+ [Arg-Adda-Glu + 2OH + H]+ [Arg-Adda-Glu + 2OH - H2O + H]+ [Arg-Adda-Glu + 2OH - H2O + H]+ [Mdha-Ala-Leu-MeAsp-Arg + H]+ [Mdhared-Ala-Leu-MeAsp-Arg + H]+ [Arg-Adda + 2OH + H]+ [Arg-Adda + 2OH + H]+ [Arg-Adda + 2OH - H2O + H]+ [Arg-Adda + 2OH - H2O + H]+

addition of 2 OH addition of 2 OH to Adda addition of 2 OH addition of 2 OH to Adda addition of 2 OH addition of 2 OH to Adda addition of 2 OH addition of 2 OH to Adda addition of 2 OH addition of 2 OH to Adda no addition of OHs to Mdha or Arg no addition of OHs to Mdha or Arg addition of 2 OH to Adda addition of 2 OH to Adda addition of 2 OH to Adda addition of 2 OH to Adda no addition of OHs to Mdha or Arg no addition of OHs to Mdha or Arg addition of 2 OH to Adda addition of 2 OH to Adda addition of 2 OH to Adda addition of 2 OH to Adda

An overall rate constant of 1.24 M-1 s-1 obtained in this work for the reaction between microcystin-LR (MC-LR) and ClO2

at 293 K and pH 5.65 is modest, the calculated theoretical half-life for MC-LR is 10.5 h at a constant ClO2 concentration VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Plot of PP1 inhibition vs remaining MC-LR concentration as determined by HPLC. For each series, the data points represent (from left to right) 30, 25, 20, 15, 10, 5, 3, 1, and 0 min reaction time. Oxidation carried out at 293 K, pH 5.65, and a ClO2/MC-LR molar ratio of 340 and 680. of 1 mg L-1, suggesting that ClO2 is not a suitable oxidant for the degradation of microcystins in drinking water treatment processes. As a comparison, k for the reaction between MCLR and O3 at 293 K in pure water has been reported to be 1.02 × 105 M-1 s-1 at pH 2 and 3.80 × 104 M-1 s-1 at pH 7 (13). MC-LR is thus oxidized about 104-105 times faster by O3 than by ClO2 in pure water. Besides the reaction with MC-LR, it is also important to know how and to what extent ClO2 reacts with other compounds. In an extensive work by Hoigne´ and Bader (18), the kinetics of ClO2 consumption by a wide range of inorganic and organic compounds was studied. In all cases, the reaction was of first-order in ClO2 concentration as well as in reductant concentration. Depending on the substrate, the overall rate constant k varied from < 10-6 M-1 s-1 to > 108 M-1 s-1. The highest k were obtained for phenolic compounds, for which the reactions also were strongly pH-dependent. ClO2 often reacted 106 times faster with deprotonated species compared with their nondissociated forms. For substrates not changing speciation with pH, k showed only a slight increase with pH. In contrast to these findings, the reaction between MC-LR and ClO2 in our study was retarded with increasing pH. The half-life of MC-LR at pH 10.0 was 1.5 times higher than that at pH 5.65. Since MC-LR contains two free carboxyl groups and one free amino group, with pKa-values of 2.09, 2.19, and 12.48, respectively (26), the molecule has a net charge of -1 in the studied pH-range, although slightly more negatively charged at pH 10.0 (-1.003) than at pH 5.65 (-0.999). The reaction between MC-LR and ClO2 should thus not be retarded with increasing pH if the findings of Hoigne´ and Bader (18) are generally applicable. The discrepancy in pHdependence may partly be explained by the fact that Hoigne´ and Bader (18) determined the rate constants from the consumption of ClO2, while our calculations are based on the degradation of MC-LR. At alkaline conditions, ClO2 in reaction with OH- is known to form chlorite (ClO2-) and chlorate (ClO3-), leaving less ClO2 available for the reaction with MC-LR. Although the determination of the pseudo-first-order rate constants k′ with UV-spectrometry were done as single measurements, the reproducibility with the applied method was high, as evident from the 340 molar ratio measurements at 293 K and pH 5.65. In these three independent measurements, the absorbance profile curves were overlapping when combined, and there were only minor differences in the obtained k′: 9.79 × 10-4 ( 0.23 × 10-4 s-1 (av ( stdev, n ) 3). The obtained overall rate constant of 1.24 M-1 s-1 should thus be reliable. This is further strengthened by the overall rate constant obtained from the HPLC measurements, 1.23 M-1 s-1. The good correlation between the decrease rate of intact MC-LR as determined by UV-spectrometry and HPLC also shows that the correction made for the residual absorbance at 238 nm in the UV-spectrometry measurements 6030

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is necessary. The slight difference between the UV and HPLC curves at a ClO2/MC-LR molar ratio of 680 is probably due to the consumption of ClO2 by MC-LR in the sample cuvette during the UV-spectrometry measurements. Since ClO2 has a slight absorptivity at 238 nm, a decrease in ClO2 concentration will increase the transmittance in the sample cuvette compared to the reference cuvette, making the reaction look slightly faster than it actually is. At lower molar ratios this effect should not be as evident since less ClO2 is consumed in the sample cuvette due to the lower reaction rates. This also explains why k′ for the 680 molar ratio does not fit the regression line of the other pseudo-first-order rate constants, while k′ determined from the HPLC data (1.94 × 10-3 s-1) at the same molar ratio perfectly fits the regression line. Regarding characterization of the reaction products, the m/z 1029 ion detected and identified as dihydroxy MC-LR in the mass spectrometry analysis has also been observed in the oxidation of MC-LR by chlorine (27) and by TiO2 photocatalysis (28, 29). While dihydroxy isomers of MC-LR seem to be the main product formed in ClO2 and chlorine oxidation, further degradation of MC-LR takes place in TiO2 photocatalysis (29). The observed m/z 1047 ion in this work, formed in small quantities, might be a ring opening with the addition of one OH group and one hydrogen atom at the terminals. The concentration of this reaction product was, however, too low for further characterization. The MS analyses revealed no data suggesting that the R,β-unsaturated carbonyl group in the Mdha residue is affected by ClO2. The presence of the m/z 553, 555, 682, and 684 ions in the daughter ion recordings also indicates that the guanidine moiety in the Arg side chain remains intact. The protein phosphatase inhibition assay (PPIA) used in this work has an IC50 of ca. 2.25 pmol mL-1 MC-LR and a linear response range of ca. 0.1-4.0 pmol mL-1, while 100% inhibition is obtained at a MC-LR concentration of ca. 10 pmol mL-1. A 1000 times dilution of the samples was thus necessary in order to fit the MC-LR concentrations to the linear response range of the assay and avoid anomalies caused by the sigmoidal shape of the standard curve. As evident from Figure 6, the close to zero inhibition of PP1 at complete oxidation of MC-LR shows that the main toxicological mechanism of microcystins is destroyed in the reaction with ClO2. The data points should not approach the origin of coordinates if the main oxidation products formed, dihydroxy isomers of MC-LR, would have an inhibiting effect on PP1, since their concentrations increase as the remaining MC-LR concentration gets lower. Although we have shown in this work that the oxidation of MC-LR by ClO2 is effective at high ClO2 concentrations in pure water and that the main degradation products formed, dihydroxy isomers of MC-LR, are nontoxic according to the PPIA, the use of ClO2 at real water treatment conditions, using surface water as the raw water source and ClO2 dosages normally used in drinking water treatment (1 mg L-1 or less), will probably not give a sufficient protection against possible occurrence of microcystins. The high reaction rate between ClO2 and NOM in combination with the substantially higher concentrations of NOM compared to microcystins (mg L-1 vs µg L-1 level) will seriously impair the effects on microcystins. It is not recommendable to try to overcome this drawback by using ClO2 at higher dosages, since the main degradation products of ClO2, chlorite (ClO2-), and chlorate (ClO3-) may induce hematological effects (30).

Acknowledgments The authors thank Dr. Lisa Spoof for good advice concerning the protein phosphatase inhibition assay and the reviewers for valuable comments on the first version of the manuscript. Financial support by the Finnish foundation Maa- ja vesitekniikan tuki ry (MVTT) and the European Commis-

sion 5th Framework Program (contract number EVK1-CT2002-00107) is gratefully acknowledged.

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(16) Hart, J.; Stott, P. Microcystin-LR Removal from Water; Report FR0367; Foundation for Water Research: Marlow, 1993. (17) Meriluoto, J.; Lawton, L.; Harada, K. In Methods in Molecular Biology, Vol. 145: Bacterial Toxins: Methods and Protocols; Holst, O., Ed.; Humana Press: Totowa, NJ, 2000; pp 65-88. (18) Hoigne´, J.; Bader, H. Water Res. 1994, 28 (1), 45-55. (19) Stumm, W.; Morgan, J. J. Aquatic Chemistry: Chemical Equilibria and Rates in Natural Waters, 3rd ed.; Wiley: New York, 1996; p 161. (20) Kozlov, Yu. N.; Purmal’, A. P.; Uskov, A. M. Russ. J. Phys. Chem. 1985, 59, 930-932. (21) An, J. S.; Carmichael, W. W. Toxicon 1994, 32 (12), 1495-1507. (22) Rapala, J.; Erkomaa, K.; Kukkonen, J.; Sivonen, K.; Lahti, K. Anal. Chim. Acta 2002, 466 (2), 213-231. (23) Yuan, M.; Namikoshi, M.; Otsuki, A.; Rinehart, K. L.; Sivonen, K.; Watanabe, M. F. J. Mass Spectrom. 1999, 34, 33-43. (24) Meriluoto, J. A. O.; Nygård, S. E.; Dahlem, A. M.; Eriksson, J. E. Toxicon 1990, 28 (12), 1439-1446. (25) Atkins, P. W. Physical Chemistry, 4th ed.; Oxford University Press: Oxford, 1990. (26) De Maagd, P. G.-J.; Hendriks, A. J.; Seinen, W.; Sijm, D. T. H. M. Water Res. 1999, 33 (3), 677-680. (27) Tsuji, K.; Watanuki, T.; Kondo, F.; Watanabe, M. F.; Nakazawa, H.; Suzuki, M.; Uchida, H.; Harada, K.-I. Toxicon 1997, 35 (7), 1033-1041. (28) Lawton, L. A.; Robertson, P. K. J.; Cornish, B. J. P. A.; Jaspars, M. Environ. Sci. Technol. 1999, 33, 3 (5), 771-775. (29) Liu, I.; Lawton, L. A.; Robertson, P. K. J. Environ. Sci. Technol. 2003, 37 (14), 3214-3219. (30) Condie, L. W. J. Am. Water Works Assoc. 1986, June, 73-78.

Received for review January 5, 2004. Revised manuscript received July 14, 2004. Accepted August 2, 2004. ES0400032

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