Halogen Radicals Promote the Photodegradation of Microcystins in

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Halogen Radicals Promote the Photodegradation of Microcystins in Estuarine Systems Kimberly M. Parker, Elke Susanne Reichwaldt, Anas Ghadouani, and William A. Mitch Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01801 • Publication Date (Web): 22 Jul 2016 Downloaded from http://pubs.acs.org on July 25, 2016

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Environmental Science & Technology

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Halogen Radicals Promote the Photodegradation of

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Microcystins in Estuarine Systems

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Kimberly M. Parker1, Elke S. Reichwaldt,2 Anas Ghadouani,2 William A. Mitch1*

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Stanford, California 94305, United States

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Engineering, The University of Western Australia, 35, Stirling Highway M015, Crawley,

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Western Australia 6009, Australia

Department of Civil and Environmental Engineering, Stanford University, 473 Via Ortega,

Aquatic Ecology and Ecosystem Studies, School of Civil, Environmental and Mining

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AUTHOR EMAIL ADDRESS: [email protected]

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CORRESPONDING AUTHOR FOOTNOTE:

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William A. Mitch: Email: [email protected], Phone: 650-725-9298, Fax: 650-723-7058

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ACS Paragon Plus Environment

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Abstract The transport of microcystin, a hepatotoxin produced by cyanobacteria (e.g., Microcystis

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aeruginosa), to estuaries can adversely affect estuarine and coastal ecosystems. We evaluated

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whether halogen radicals (i.e., reactive halogen species (RHS)) could significantly contribute to

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microcystin photodegradation during transport within estuaries. Experiments in synthetic and

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natural water samples demonstrated that the presence of seawater halides increased quantum

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yields for microcystin indirect photodegradation by factors of 3-6. Additional experiments

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indicated that photoproduced RHS were responsible for this effect. Despite the fact that

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dissolved organic matter (DOM) concentrations decreased in more saline waters, the calculated

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photochemical half-life of microcystin decreased 6-fold with increasing salinity along a

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freshwater-estuarine transect due to the halide-associated increase in quantum yield. Modeling of

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microcystin photodegradation along this transect indicated that the timescale for RHS-mediated

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microcystin photodegradation is comparable to the timescale of transport. Microcystin

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concentrations decline by ~98% along the transect when considering photodegradation by RHS,

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but only by ~54% if this pathway were ignored. These results suggest the importance of

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considering RHS-mediated photodegradation in future models of microcystin fate in freshwater-

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estuarine systems.

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Introduction Harmful algal blooms (HABs) are a growing concern in freshwater, estuarine and marine

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ecosystems, particularly due to their potential to produce toxins.1-3 The deleterious impacts of

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coastal HABs to estuarine ecosystems has been well-documented.4,5 However, recent reports of

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estuarine contamination by hepatotoxic microcystins, produced by freshwater blooms of


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cyanobacteria (e.g., Microcystis aeruginosa), have brought attention to the transport of toxins

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produced by freshwater HABs to estuarine ecosystems.6-9 Furthermore, blooms of microcystin-

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producing M. aeruginosa have occurred within estuaries.10-12 Relative to other freshwater algae,

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M. aeruginosa is salt-tolerant (95%, Enzo Life Science) and microcystin-YR (>90%, Sigma

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Aldrich) were used as received. Suwannee River natural organic matter (SRNOM) was obtained

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from the International Humic Substances Society (IHSS). All salts used in this study were

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reagent-grade or higher.


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Natural water samples. Water samples were collected and filtered through 0.7-µm glass fiber

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filters from estuaries impacted by M. aeruginosa blooms: six sites along the Swan River estuary

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in Western Australia11,12 (Figure S1) and two sites in central California (San Francisco Bay,10

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and the Pajaro River,6 which discharges to Monterey Bay; Figure S2). Solution absorbance was

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measured using an Agilent Cary 60 UV-Vis spectrophotometer. Dissolved organic carbon (DOC)

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content in the filtered water samples was measured using a Shimadzu Total Organic Carbon

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TOC-L analyzer as a surrogate measure of DOM. Solution absorbance was normalized by DOC

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to calculate specific UV absorbance (SUVA) at 350 nm. Chloride and bromide concentrations

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were measured using a Dionex DX-500 ion chromatograph. Iron concentration was measured

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using a Thermo Scientific iCAP 6300 spectrometer. Nitrate concentration was measured using a

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WestCo SmartChem 200 discrete analyzer.

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Photochemical experiments. Photodegradation experiments under simulated sunlight were

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performed using a Q-SUN Xenon Test Chamber (Xe-1) solar simulator equipped with a

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Daylight-Q optical filter. The photon flux into the sample was calculated using p-

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nitroanisole/pyridine (PNA/pyr) actinometry.44,45 The total incident photon flux for the system

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from wavelengths 280-700 nm (Figure S3) was calculated to be 4.1 × 10-5 Einsteins L-1 s-1.

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Microcystin-LR and YR samples (initial concentration = 0.25 µM selected to facilitate analytical

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determination, initial volume = 5 mL) were stirred in quartz test tubes held at a 30° angle from

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the reactor bottom. Temperature was held at 20°C using a recirculating water bath. Aliquots (100

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µL) were periodically sampled from the test tubes.

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Natural sunlight experiments were performed in April 2015 in Perth, Western Australia.

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Irradiation was performed in unstirred quartz test tubes held at a 30° angle in a cooling water

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bath (approximately 20-30°C). The photon flux into the sample was calculated using p-


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nitroacetophenone/pyridine (PNAP/pyr) actinometry.44,45 Samples were collected at sunrise and

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sunset over a 4-day period. No degradation was observed in dark controls over the same time

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period.

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Analytical methods. Microcystin-LR and YR concentrations were measured using an Agilent

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1260 Infinity HPLC (simulated sunlight experiments) or a Waters Alliance 2695 HPLC (natural

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sunlight experiments) with a Supelco Ascentis RP-amide column. An isocratic mobile phase

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(65% trifluoroacetic acid (0.05% v/v) in MilliQ water and 35% trifluoroacetic acid (0.05% v/v)

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in acetonitrile)46 at 0.4 mL/min eluted microcystin at 6 minutes. UV absorbance at 238 nm was

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used to quantify microcystin concentration. Following elution of microcystins, the organic phase

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was increased to flush the column of organic contaminants prior to injection of the next sample.

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Additional methodology developed for product analysis is described in SI Text 3.1.

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Statistics. Error on pseudo-first order rate constants represents the standard error of the slope

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obtained by linear regression of duplicate sets of semi-log transformed kinetic data on

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microcystin degradation determined using Microsoft Excel. Differences in pseudo-first order

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rate constants were tested by Student’s t-test performed using GraphPad Prism and considered

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significant for P < 0.05.

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Results and Discussion

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Isolation of the effect of water chemistry parameters on microcystin indirect photodegradation.

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As freshwater containing microcystins discharges to estuaries, several water quality

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characteristics that may affect microcystin photodegradation rates change along these transects.

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Rising salinity results in higher ionic strength as well as increased concentrations of specific

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ionic constituents (e.g., halides, carbonates) that may affect photodegradation pathways.32,33

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However, DOM concentration and absorbance often are lower in estuarine waters than


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freshwaters,47 potentially decreasing indirect photodegradation rates of microcystin. Table 1

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provides water quality parameters for the natural samples collected for this study. The pH in all

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samples was >7.0, at which microcystins will be poorly sorbed to DOM.25 Using synthetic or

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natural freshwaters spiked with the constituents over the range measured in the natural samples,

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we identified water chemistry parameters important for the indirect photodegradation of

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microcystins along estuarine transects.

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To isolate the importance of DOM-sensitized indirect photodegradation,

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photodegradation of two microcystin analogues (MC-LR and MC-YR; structures in Figure 1A)

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were monitored in deionized water with (Figure 1) and without (Figure S4) 5 mg-C/L SRNOM.

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Our natural samples collected from freshwater and estuarine sites featured DOC concentrations

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above 5 mg-C/L (Table 1), but with similar absorbance to these synthetic matrices (Figure S5).

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In all solutions, microcystin photodegradation exhibited first-order behavior, and the presence of

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SRNOM increased photodegradation rates, suggesting the importance of DOM-sensitized

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indirect photodegradation. In the freshwater system, the pseudo-first order degradation rate

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constant increased from 1.7(±0.1 standard error of the regression)×10-6 s-1 for MC-LR and

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1.9(±0.1)×10-6 s-1 MC-YR in the absence of SRNOM to 5.2(±1.0)×10-6 s-1 for MC-LR and to

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5.2(±0.4)×10-6 s-1 for MC-YR in the presence of SRNOM. Considering the fluence of the light

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source (UVR(280-400 nm) = 35 W/m2), the pseudo-first order observed rate constant for MC-LR

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in the presence of SRNOM is equivalent to 1.5(±0.3)×10-4 kJ-1 m2, in good agreement with

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previously reported values (0.5-9×10-4 kJ-1 m2)22,24 (see SI Text 3.2 for calculations).

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We evaluated the effect of increasing ionic strength on microcystin degradation by

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amending the synthetic and natural freshwater samples with sodium perchlorate (NaClO4) at 540

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mM, the concentration of halides in seawater. Seawater ionic strength increased the rate of diene


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isomerization in sorbic acid and domoic acid by energy transfer interactions with 3DOM*.32,33

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Despite the diene moiety present in the Adda amino acid side chain in microcystin, high ionic

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strength did not alter the photodegradation rates of microcystins sensitized by SRNOM in

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synthetic (Figures 2B and 2C) or natural (Figure 1D) freshwaters, and no isomerization products

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were observed (SI Text 3.1). While the reason for the lack of effect of ionic strength on

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microcystin photodegradation is unclear, one possibility is that the location of the diene adjacent

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to the microcystin ring inhibits energy transfer by close interaction of the diene with 3DOM*,

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decreasing the rate of isomerization relative to other degradation pathways.

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Next, we investigated the effect of halides spiked into synthetic water at seawater-

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relevant concentrations (540 mM sodium chloride (NaCl), 0.8 mM sodium bromide (NaBr)). In

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the absence of SRNOM, halides increased MC-LR and MC-YR photodegradation by a factor of

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3 relative to the control solution containing neither halides nor DOM (Figure S4). We suspect

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that this effect may be due to enhanced self-sensitization reactions or sensitization by

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contaminant algal pigments in the MC-LR standard in the presence of halides. However, the

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observed photodegradation rate remained 5-fold lower than in the presence of SRNOM. In

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synthetic water with SRNOM, halides increased the pseudo-first order rate constant to

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25.1(±1.5)×10-6 s-1 for MC-LR and 21.9(±1.0)×10-6 s-1 for MC-YR (Figure 1B-C), a factor of 4-5

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increase relative to the DOM-containing, halide-free water. In natural freshwaters, the addition

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of halides increased the photodegradation rate constant by factors of ~2.5-3 (Figure 1D). The

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addition of a complete matrix of major seawater ions (400 mM NaCl, 0.8 mM NaBr, 29 mM

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sodium sulfate (Na2SO4), 54 mM magnesium chloride (MgCl2•6H2O), 11 mM calcium chloride

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(CaCl2•2H2O), 10 mM potassium chloride (KCl), 0.35 mM boric acid (H3BO3), 1.8 mM sodium

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bicarbonate (NaHCO3) and 0.26 mM sodium carbonate (Na2CO3)) did not further affect the


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observed rate constant in the natural freshwater sample USA-1 (Figure 1E), indicating that

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halides exhibit the most significant ion-specific effects on microcystin indirect photodegradation

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among major seawater ions.

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In additional experiments conducted by spiking constituents into a natural freshwater

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sample (USA-1), we also evaluated the impact on microcystin photodegradation of iron and

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nitrate, which generate OH. The addition of nitrate and iron to freshwater sample USA-1 at

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concentrations ([NO3-] = 175 µM) and iron ([Fe3+] = 10 nM) relevant to those measured in our

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natural samples ([NO3-] = 1-175 µM; [Fe3+] < 20 nM (Table 1)) did not change MC-LR

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photodegradation rates with or without the addition of halides (Figure 1E). Therefore, DOM and

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halides were the primary constituents affecting microcystin photodegradation in fresh and

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estuarine waters.

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Contribution of RHS to microcystin photodegradation. We hypothesized that RHS were

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responsible for the halide-specific enhancement of DOM-sensitized microcystin

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photodegradation. Radical RHS are selective oxidants, targeting the most reactive constituents in

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natural waters including conjugated dienes (e.g., k(sorbic acid, Cl2-) = 6.8×108 M-1 s-1)40 and

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tyrosine (k(tyrosine, Br2-) = 2.0×107 M-1 s-1).48 MC-LR and MC-YR contain a conjugated diene

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in the Adda side chain, and MC-YR contains a tyrosine residue. To distinguish the contribution

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of selective radicals (e.g., RHS) from non-selective radicals (e.g., OH), we compared the

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reduction in MC-LR photodegradation rates in the presence of 25 mM tert-butanol vs. 250 mM

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isopropanol, conditions previously selected to demonstrate the role of RHS for domoic acid

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photodegradation.33 Both alcohols react with OH with nearly diffusion-limited second-order

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reaction rate constants (k(tert-butanol, OH) = 6.0×108 M-1 s-1, k(isopropanol, OH) = 2.3×109 M-

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1 -1 49

s ). However, their second-order rate constants with RHS span several orders of magnitude


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(k(tert-butanol, Br) = 1.4×104 M-1 s-1,50 k(tert-butanol, Cl2-) = 7.0×102 M-1 s-1,40 k(isopropanol,

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Br) = 6.6×106 M-1 s-1,50 k(isopropanol, Cl2-) = 1.2×105 M-1 s-1 40). While either condition (25 mM

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tert-butanol, 250 mM isopropanol) effectively scavenges OH, 25 mM tert-butanol is

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significantly less effective at scavenging RHS than 250 mM isopropanol.

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In the synthetic freshwater system containing 5 mg-C/L SRNOM, scavenging by 25 mM

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tert-butanol and 250 mM isopropanol slowed MC-LR photodegradation rates by 30% and 32%

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respectively (Figure 2). The similarity in the percentage reduction in photodegradation rate

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constants for these two scavenging conditions concurs with expectations that OH is the

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predominant radical oxidant in freshwater, while the absolute percentage reduction is in good

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agreement with the previous estimate of the contribution of OH to MC-LR photodegradation in

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a synthetic freshwater matrix (25%).25 In synthetic seawater, 25 mM tert-butanol and 250 mM

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isopropanol decreased MC-LR photodegradation by 19% and 72%, respectively (Figure 2). The

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difference in the effect of the two quenching conditions in seawater indicates the importance of

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RHS, while the increase in the magnitude of quenching by 250 mM isopropanol between the

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freshwater and seawater matrix demonstrates the greater role of radical-mediated reactions in

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halide-containing waters.

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Similar results were observed for experiments conducted using natural samples. The

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addition of aliphatic alcohols reduced the MC-LR photodegradation rate constants in the

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freshwater (USA-1; 25 mM tert-butanol, 21%; 250 mM isopropanol, 33%) and saline water

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(USA-2; 25 mM tert-butanol, 31%; 250 mM isopropanol, 71%) samples collected from

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California (Figure 2). In waters collected from Western Australia, the addition of aliphatic

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alcohols had no effect on the MC-LR photodegradation rate constant in the sample containing

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the lowest halide concentration (AUS-1), while the photodegradation rate constant in a more


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saline sample (AUS-5) decreased by 7% and 50% in the presence of 25 mM tert-butanol and 250

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mM isopropanol, respectively. Use of an optical filter to cutoff wavelengths

Halogen Radicals Promote the Photodegradation of Microcystins in

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