1

Kinetics of Microcystin-LR Oxidation by Ozone Ayman R. Shawwa and Daniel W. Smith Department of Civil and Environmental Engineering University of Alberta Edmonton, Alberta T6G 2M8 Abstract The oxidation kinetics of the reaction between dissolved ozone and microcystin-LR, a cyanobacterial hepatotoxins, in acidic and neutral buffer solutions over the temperature range of 10 to 30 oC was investigated. The reaction is very rapid and the stoichiometeric determination indicates that eight moles of ozone are required for complete conversion of each mole of microcystin-LR. The overall kinetics can be modeled as a second order, with first order in each concentration of ozone and microcystin-LR. At 20 o C, the overall rate constant ranged from 3.4x104 to 1.0x105 M-1s-1 as the solution pH changed from 7 to 2. As the temperature increased from 10 to 30 oC, the average overall rate constant increased from 5.8x104 to 1.6x105 M-1s-1 at pH value of 2. Introduction Recent research on secondary metabolites of cyanobacteria (blue-green algae) suggests that these compounds could cause potential health risks to water consumers if they were present in drinking water. The presence of these toxins in the natural environment is generally due to a release caused by the rupture of algal cells. This phenomenon may arise during the water management cycle, e.g. an algaecide treatment of the natural medium, pumping and conveyance of raw water and the effect of certain substances used during water treatment. The toxins produced by blue green algae includes hepatotoxins, which are primarily cyclic peptides that are relatively stable once released to water (Carmichael, 1992). The most commonly identified compound in this group is microcystin-LR, released by

2 Microcystis aeruginosa (Fawell et al., 1993). This toxin can cause irreversible damage to the liver and can lead either to rapid death or to long term pathologies such as liver cancer. Currently, the Canadian Guideline for Drinking Water Quality for microcystin-LR is at the approval stage with a proposed value of 1.5 µg/L. The toxicity of microcystin-LR coupled with its stability is of concerns for water supply utilities. Consequently, the removal of this compounds in water treatment processes warrants investigation. Few studies have been carried out with ozone to determine its effectiveness in removing cyanobacterial toxins from drinking water supplies (Keijola et al., 1988; Himberg et al. 1989; Fawell et al. 1993; Rositano et al. 1998). These studies showed that water treatment processes that incorporated ozonation, at dosages less than 1 mg/L, were the most effective in eliminating toxicity of microcystin-LR. In addition, these studies showed that pH of the water supply can influence the oxidation rate of microcystin-LR by ozone. The results of these studies indicated that the destruction of microcystin-LR was reduced at alkaline pH values because the rate of ozone decomposition is accelerated under high pH values. The previously mentioned studies showed that the reaction between dissolved ozone and microcystin-LR is very rapid. However, there are no specific rate constants for the reaction between ozone and microcystin-LR in the literature. Therefore, the present work was undertaken to investigate the kinetics of microcystin-LR oxidation by ozone and to determine the reaction rate constant under different pH and temperature conditions. Experimental Methods A PCI ozone generator was utilized to produce ozone gas using extra dry, high purity oxygen. The ozone gas was introduced into deionized distilled water for 30 minutes to oxidize any contaminant. Following the removal of any ozone residual, the preozonated water was used to prepare buffer solutions using sodium phosphate, sodium phosphate monobasic and phosphoric acid to control the solution pH in the range of 2 to 7. An aqueous solution of dissolved ozone was obtained by bubbling the ozone gas in the buffer solution for 20 minutes depending upon the desired initial concentration. Initial ozone concentration and residual ozone concentration were determined by the indigo method following Standard Methods (APHA, AWWA, WEF, 1992). An aqueous solution of microcystin-LR was prepared by dissolving an appropriate amount of pure microcystin-LR (purchased from SEGMA, St. Louis) in the buffer solution of a fixed pH value. The initial concentration of microcystin-LR was determined by performing solid phase extraction and high performance liquid chromatography (HPLC) analysis. The extraction method of Berg et al. (1987) was used in this study with few modifications. Each aqueous sample was filtered through a prewashed 0.45 µm Millipore membrane filter. The filtrate was then passed through a disposable 1 g C18 cartridge (Supelco) attached to a vacuum chamber. The cartridges were

3 prepared by washing them with 10 mL of methanol and then 10 mL of pure water. The water sample was passed through the cartridge at a rate of approximately 8 mL/min. The cartridge was then washed with 10 mL of water and 10 mL of 10% methanol in water. The microcystin was then eluted with 5 mL of methanol. Methanol was evaporated to dryness at 40 oC under a stream of nitrogen. The residue was then redissolved in HPLC solvent (10 mM ammonium acetate:acetonitrile, 76:24). This then was filtered through a 0.45 µm filter paper. A 20 µL sample was then analyzed by HPLC. A standard curve for microcystin-LR was plotted against the peak height of the HPLC curve for different concentrations of the toxin. HPLC analyses were performed with a Shimadzu liquid chromatograph (Shimadzu Scientific Instrument) equipped with Shimadzu pumps (model LC-10AT) and a Shimadzu UV-VIS detector (model SPD-10A). The HPLC system used for determination of microcystin-LR is summarized in Table 1. Table I HPLC System Used for Microcystin-LR Analysis Parameter Column Solvent Solvent flow UV detection Injection Retention time

C18 µ Bondapak, 3.9 mm ID x 300 mm (Waters) 10 mM ammonium acetate: acetonitrile, 76:24 1.0 mL/ min 238 nm 20 µL 5.9 min

Experiments were conducted to determine the quantity of dissolved ozone required for complete oxidation of microcystin-LR. In each experiment, a solution containing excessive amount of dissolved ozone was mixed with a solution containing known quantity of microcystin-LR at room temperature. The ozone residual was determined by the indigo after two minutes of mixing the two reactants to allow complete depletion of the microcystin-LR. The concentration of microcystin-LR that was used in these experiments was 500 µg/L (5.0x10-7 M). These experiments were repeated several times at each ozone concentration. The kinetic experiment was conducted by using a stopped-flow spectrophotometer system (Tritech Dynamic Instruments-model II) equipped with data acquisition subsystem. The system is designed for studying fast reactions between two liquid reactants under isothermal conditions. At the start of the kinetic experiment, the two aqueous solutions (one contained microcystin-LR and the other contained dissolved ozone) of ideal pH were mixed rapidly in the stopped-flow spectrophotometer system. Following mixing (less than 1 ms) of the reactants the solution absorbance at various reaction times was recorded at a constant wavelength. The two reactants and mixed solution were maintained at a desired temperature by water circulation prior and during the experiment. The initial concentration of microcystin-LR that was used in these experiments was 500 µg/L (5x10-7 M). The concentration of dissolved ozone in the solution was

4 maintained at large excess with initial concentration varying from 0.1 to 2.0 mg/L (2.2x10-6 to 4.2x10-5 M). The maximum absorbance exerted by ozone and microcystin-LR in solution were at wavelengths 260 and 238 nm, respectively. For this work, the wavelength was set at 238 nm to allow significant contributions of both reactants to the total absorbance of the reacting solution. Analysis of Kinetic data The initial attack of microcystin-LR by ozone molecules and the self-decomposition of ozone are likely the major steps controlling the oxidation of microcystin-LR. Preliminary tests indicated that the decomposition rate of ozone is very slow when compared with that of microcystin-LR reaction. Therefore, the overall reaction can be expressed as: a O3 [1]

+

microcystin-LR

→

(C49H74N10O12)

P

(Products)

In accordance to the above reaction, “a” mole of ozone is required for conversion of each mole of microcystin-LR. The decomposition rate for ozone and microcystin-LR are, respectively: dCO3/dt [2]

=

dCMLR/dt [3]

=

a

CO3m

k

-

CMLRn

CO3m

k

CMLRn

where CMLR is the molar concentration of microcystin-LR, CO3 is the molar concentration of ozone, k is the overall ozonation rate constant, m is the reaction order with respect to ozone concentration and n is the reaction order with respect to microcystin-LR concentration. Since the kinetic experiments were carried out with ozone in large excess of microcystin-LR, Equation [3] can be simplified to: dCMLR/dt [4]

=

-

CMLRn

k′

where the apparent rate constant, k′, is related to the overall rate constant, k, as follow: k′ [5]

=

a

CO3(0)m

k

where CO3(0) is the initial concentration of ozone. The integrated rate equations for microcystinLR oxidation by ozone is: CMLR(t)/CMLR(0) [6]

=

exp

[-k′t]

for

n

=

1

5 [CMLR(t)/CMLR(0)]1-n = 1 + (n-1) k′ CMLR(0)n-1t [7]

for n ≠ 1

As dictated by Beer-Lambert’s law, the absorbance of dilute mixture can be taken as the sum of the absorbance of individual component. Therefore, the following equation can be derived to relate microcystin-LR concentration with the solution absorbance measured in the kinetic experiment (Espensen, 1981): CMLR(t)/CMLR(0) [8]

=

[A(t)-A(∞)]/[A(0)/A(∞)]

where A(t) is the absorbance of the reacting mixture measured at time t, A(∞) is the asymptotic absorbance of the solution at the end of the reaction period and A(0) is the initial absorbance of the reacting mixture. By utilizing the relationships given in Equations [4] to [8], the order with respect to the microcystin-LR concentration, n, and the apparent rate constant, k’, can be determined by analyzing the absorbance data measured in the kinetic experiment. Results and Discussion Stoichiometry of Microcystin-LR Ozonation

600

1.0

500

0.8

400

0.6

300 0.4

200 100

0.2

0

0.0

Ozone residual (mg/L)

Microcystin-LR ( g/L)

Ozone, which has a standard oxidation potential of 2.07 V under acidic conditions, is considered one of the most powerful oxidants used in water treatment (Rositano et al., 1998). Experimental results showed that the oxidation of microcystin-LR by ozone was extremely effective. A solution containing 500 µg/L microcystin-LR was oxidized to below than the detection limit of the HPLC system (1 µg/L in this study) at an applied ozone dose of 0.2 mg/L, as shown in Figure 1. Figure 1 shows the concentration of microcystin-LR and ozone residual after 2 minutes of reaction time at different initial ozone concentrations.

0.0 0.1 0.1 0.2 0.3 0.5 0.7 1.0 Initial ozone concentration (mg/L) Figure 1. Oxidation of microcystin-LR by ozone after 2 minutes of reaction time

6

Experiments were conducted to determine the stoichioimetric requirement for the complete destruction of microcystin-LR by ozone. The fraction of dissolved ozone consumed in these experiments is plotted against the initial ratio of moles of ozone to microcystin-LR, as shown in Figure 2. The figure indicates that the dissolved ozone was completely consumed for cases of eight moles of ozone per mole of microcystin-LR. On the other hand, if the ratio was higher than

Mole fraction of O 3 reacted

1.0 0.8 0.6 0.4 0.2 0.0 1

10 Initial molar ratio [O zone]/[Microcystin-LR]

100

Figure 2. Stiochiometric requirement for the ozonation of microcystin-LR eight moles then the residual amount of ozone was detected after the completion of the reaction. Therefore, the results of the experiments showed that 8 moles of ozone were required for complete degradation of each mole of microcystin-LR. This finding (a = 8 and b = 1) can be applied in Equation [5] to calculate the overall rate constants, k, for the ozonation reactions from the kinetic data, as discussed in the next section. The stoichiometry of the ozonation of microcystin-LR in this work tends to agree with the stoichiometry requirement that was determined from the results of Rositano et al. (1998). Kinetics of Microcystin-LR Oxidation by Ozone The stopped-flow spectrophotometer system was utilized to study the fast reaction between ozone and microcystin-LR in acidic and neutral buffered solutions containing excessive amount of ozone. Typical absorbance profiles are shown in Figure 3 for experiments carried out at 20 oC at a constant pH value of 2. The initial concentration of microcystin-LR, CMLR(0), in these runs was equal to 500 µg/L (5x10-7 M) and the initial concentration of ozone, CO3(0), ranged from 0.1 to 2 mg/L (2.1x10-6 to 4.2x10-5 M). The reaction of ozone with microcystin-LR was very fast, as

7 indicated by the rapid declines in the absorbance data. As shown in Figure 3, the half-life of the microcystin-LR was less than one second. Following complete destruction of the microcystinLR, the absorbance continued to decrease gradually due to slow decomposition of ozone.

1.0

o

Temperature = 20 C pH = 2

A(t)/A(0)

0.8

-7

[MLR]0 = 5x10 M

0.6 0.4 0.2

O3=2 mg/L O3=0.5 mg/L

0.0 0.0

0.1

O3=1 mg/L O3=0.1 mg/L

0.2 0.3 Reaction time, t (sec)

0.4

0.5

Figure 3. Absorbance changes during microcystin-LR ozonation According to Equation [8], the absorbance data can be converted to the dimensionless concentration of microcystin-LR after estimating the asymptotic absorbance at the completion of the ozonation reaction. Accordingly, the kinetic data can be analyzed utilizing Equations 5 to 8. The regression analysis of the absorbance data indicated that the oxidation of the microcystin-LR by ozone can be modeled as pseudo-first-order with respect to the concentration of microcystinLR, i.e. n=1, as suggested by Equation [6]. The assumption of pseudo-first-order kinetics yielded the best results with correlation coefficients higher than 0.98 for all runs. The results of the first-order kinetics can be illustrated by plotting the dimensionless concentration of microcystin-LR to the semi-logarithmic scale, as shown in Figure 4. This plot yielded a straight line where the slope of the line represents the apparent rate constant, k’, or the pseudo-first-order rate constant. The apparent rate constant could be also calculated directly from the regression analysis. In this study, at least five experimental runs were carried out, at given initial and operating conditions, to insure that the kinetic data are reproducible. For the average rate constants calculated and presented in this work, the maximum deviation was 9.2 % from the individual results. For the four runs shown in Figures 3 and 4, the apparent rate constants, k’, were 1.53, 3.72, 7.20 and 20.37 s-1 at the initial ozone concentrations of 0.1, 0.5, 1 and 2 mg/L, respectively. These results suggested that k’ increases with the initial concentration of dissolved ozone.

8

0.0

Reaction time, t (sec) 0.1 0.2 0.3

C M LR(t)/C M LR(0)

1.00

0.4

k'= 1.53 s

k'= 7.20 s

-1

-1

0.10 O3=2 mg/L O3=1mg/L O3=0.5mg/L

0.01

O3=0.1mg/L

k'= 20.37 s

-1

o

Temp.= 20 C pH= 2

Figure 4. Concentration profiles for microcystin-LR The apparent rate constants obtained from a series of experiments at fixed pH and at 20 oC were plotted against the initial concentration of ozone on a logarithmic scale, as shown in Figure 5. The plot yielded a straight line and the slope gave the order with respect to ozone concentration. As shown in Figure 5, the slope for each individual line at a given pH is very close to unity, which indicated that the reaction can be modeled as pseudo-first-order with respect to ozone concentration, i.e. m=1. The results of the reaction kinetics between ozone and microcystin-LR in the solution can be modeled as second order with respect to the concentration of both reactants. The effect of pH and initial ozone concentration on the apparent rate constant, k’, at 20 o C is illustrated in Figure 6.

9

o

Temp.= 20 C

-1

Apparent rate constant, k' (s )

100

10

pH=2 pH=4 pH=7

1 slope = 1 0.1 0

0.5 1 1.5 Initial ozone concentration, CO3 (mg/L)

2

Figure 5. Effect of ozone concentration on reaction rate

25

-1

k' (s )

20 15 10 5

2 4

0 0.1

0.5

7 1.0

pH

2.0

Initial ozone concentration (mg/L) Figure 6. Effect of pH and initial ozone concentration on apparent rate constant The overall rate constant, k, can be calculated from Equation 5 on the basis of the stoichiometeric requirement of 8 moles of ozone for complete conversion of each mole of microcystin-LR, i.e. a = 8 and b=1, which was verified in this study. At a constant pH, at least five experimental runs were carried out in each solution of different initial ozone concentrations in the temperature range of 10 to 30 oC. The overall rate constants for the individual runs were calculated and

10 averaged to yield the average k value at each pH and temperature. The maximum deviation of the individual average overall rate constants from the average value was 10 % for all rate constants reported in this study. Figure 7 shows a plot of the overall rate constants against the solution pH on a logarithm scale for all average rate constants calculated at different temperatures. At a fixed temperature, the rate constant increased rapidly with the decrease in the pH value. In the pH range investigated in this study (pH 2 to 7) a straight line drawn for rate constants at 20 oC yielded a slope of 0.2. This implies that the reaction rate decreased with the hydroxyl ion concentration of an exponent 0.2, i.e. [OH-]0.2. Similar trends were observed at different temperatures. These results are consistent with the fact that the oxidation potential of ozone is lower under alkaline conditions due to its rapid decomposition by the hydroxyl ions. At 20 oC, the average overall rate constant changed from 3.4x104 to 1.0x105 M-1s-1 as the pH decreased from 7 to 2. The effect of temperature on the reaction rate is also illustrated in Figure 7. As the temperature increased from 10 to 30 oC, the average overall rate constant increased from 5.8x104 to 1.6x105 M-1s-1 at pH value of 2.

10 oC 20 oC

slope = 0.2

-1 -1

Overall rate constant, k (M s )

1.0E+06

30 oC 1.0E+05

1.0E+04 0

2

4

6

8

pH

Figure 7. Effect of pH on microcystin-LR oxidation rate at different temperatures The temperature dependence of the rate constant is shown in Figure 8. In accordance to Arrhenius equation, a linear relationship exists in the semi-logarithmic plot of the overall rate constant versus the reciprocal of the absolute temperature. As shown in Figure 8, a straight line can be drawn among the rate constants obtained at a given pH. The individual lines at different pH values are nearly parallel and the slopes yielded average activation energy of 12.3 kJ/mole. Increasing the temperature enhanced the reaction rate, and as the temperature increased from 10 to 30 oC, the rate constant increased by a factor of 3.

-1 -1

Overall rate constant, k (M s )

11

1.E+06 Activation Energy = 12.3 kJ/mole

pH = 2 pH = 4 pH = 7

1.E+05

1.E+04 0.0032 0.0033 0.0034 0.0035 0.0036 o Reciprocal of absolute temperature (1/ K)

Figure 8. Effect of temperature on microcystin-LR oxidation rate

Conclusions The kinetics of the oxidation reaction of microcystin-LR by ozone was investigated in this study. The stopped-flow technique was applied to carry out the kinetic experiments in aqueous solutions of pH varying from 2 to 7 and varying temperature from 10 to 30 oC. The fast reaction could be modeled as second order in both reactants and pseudo- first-order for each ozone and microcystin-LR concentration. Microcystin-LR reacts much faster with ozone when the solution pH is in the acidic range. In the strongly acidic solution of pH 2, the overall rate constants varied from 5.8x104 to 1.6x105 M-1 s-1 in the temperature range of 10 to 30 oC. As the pH increased from 2 to 7, the reaction rate decreased in proportion to the hydroxyl ion concentration of the exponent 0.2. At 20 oC, the overall rate constant decreased from 1.0x105 to 3.4x104 M-1 s-1 as the pH increased from 2 to 7. The Arrhenius equation has been applied to correlate the temperature influence on the reaction rate yielding average activation energy of 12.3 kJ/mole. Acknowledgement The authors wish to thank Dr. Bob Jordan, Department of Chemistry, University of Alberta, for allowing the use of the stopped-flow spectrophotometric system. Also, the assistance of Ms. Maria Demeter who assisted with the microcystin-LR analysis and the kinetic experiments is gratefully acknowledged.

12 Key words Ozone; Cyanobacteria; Kinetics; Microcystin-LR; Ozonation Rate; Reaction Order; Toxins References American Public Health Association, American Water Works Association and Water Environment Federation, Standard Methods for the Eexamination of Water and Wastewater, 19th. Edition, (Washington, D.C., 1992). Berg, K., Carmicheal, W., Skulberge, O., Benestad, C. and Underdal, B., “Investigation of a Toxic Bloom of Microcystis Aeruginosa (Cyanophyceae) in Lake Akersvatn, Norway”, Hydrobiologia, 144:7-103 (1987). Carmichael, W., “Cyanobacteria Secondary Metabolites: the Cyanotoxins”, J. Appl. Bacteriol., 72:445-459 (1992). Espenson, J.H., Chemical Kinetics and Reaction Mechanisms, (New York, NY:Mc-Graw-Hill Book Co., 1981). Fawell, J., Hart, J., James, H., and Parr, W., “Blue Green Algae and their Toxins: Analysis, Toxicity, Treatment and Environmental Control”, Water Supply, 11:09-121 (1993). Himberg, K., Keijol, A., Hiisvirata, L., Pyysalo, H., Sivonen, K., “The Effect of Water Treatment Processes on the Removal of Hepatotoxins from Microcystis and Oscillatoria Cyanobacteria: A laboratory Study”, Water Res., 23:979-984 (1989). Keijola, A., Himberg, K., Esala, A., Sivonen, K., and Hiisvirata, L., “Removal of Cyanobacterial Toxins in Water Treatment Processes: Laboratory and Pilot Scale Experiments”, Toxicity Assessment, 3:643-656 (1988). Rositano, J., Nicholson, B. and Pieronne, P., “Destruction of Cyanobacterial Toxins by Ozone”, Ozone Science and Engineering, 20:223-238 (1998).