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Oct 28, 2008 - Degradation Mechanism of Cyanobacterial Toxin Cylindrospermopsin by Hydroxyl Radicals in Homogeneous UV/H2O2 Process. Xuexiang He , Ges...
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Environ. Sci. Technol. 2008, 42, 8877–8883

Unveiling New Degradation Intermediates/Pathways from the Photocatalytic Degradation of Microcystin-LR MARIA G. ANTONIOU,† JODY A. SHOEMAKER,‡ ARMAH A. DE LA CRUZ,‡ AND D I O N Y S I O S D . D I O N Y S I O U * ,† Department of Civil and Environmental Engineering, University of Cincinnati, 765 Baldwin Hall, Cincinnati, Ohio 45221-0071, and Office of Research and Development, NERL, U.S. Environmental Protection Agency, 26 West Martin Luther King Drive, Cincinnati, Ohio 45268

Received June 13, 2008. Revised manuscript received August 14, 2008. Accepted September 2, 2008.

Mass spectrometry was utilized for structural identification of the intermediates formed during the photocatalytic degradation of the cyanotoxin, microcystin-LR with immobilized TiO2 photocatalysts at neutral pH. Most of the intermediates reported herein have not been found in prior studies. Results indicate that MC-LR degradation is initiated at four sites of the toxin; three on the Adda amino acid (aromatic ring, methoxy group, and conjugated double bonds) and one on the cyclic structure (Mdha amino acid). Several intermediates gave multiple peaks in the TIC (m/z ) 1011.5, 1029.5, 1063.5), which were deduced to be geometrical or constitutional isomers. This is the first study that reports the hydroxylation of the aromatic ring and the demethoxylation of MC-LR with TiO2 photocatalysis. The most targeted site was the conjugated diene bonds because of their location in the MC-LR structure. Isomerization at the C4-C5 and C6-C7 of the diene bond of the Adda chain was a direct result of hydroxyl radical addition/substitution. Based on the above, we concluded that oxidation and isomerization of the diene bonds of MC-LR occurred simultaneously. Other steps included hydroxyl substitution, further oxidation, and bond cleavage. As the reaction time progressed, simultaneous oxidation of the Adda chain and the cyclic structure occurred.

were linked to cyanobacterial contamination, both biologically and chemically (5-8). Analysis of human serum from exposed patients revealed that cyanotoxins can be present in blood samples up to 50 days following initial exposure (5). Thus the need to investigate the fate of cyanotoxins in exposed individuals emerged. In the past two decades, significant research efforts have focused on finding appropriate treatment technologies for the detoxification of water contaminated with cyanobacteria and cyanotoxins (1, 9). Among them, TiO2 photocatalysis (9-13), an advanced oxidation technology (AOT), has received significant attention because it demonstrated promise for the detoxification of water contaminated with cyanotoxins (10, 11). The phototransformation (14, 15) and degradation (10-13) of MC-LR with AOTs has been studied as well. Phototransformation of MC-LR does not occur under visible or UV-A radiation (λ ) 350 nm) (15), however, UV-C radiation (λ ) 254 nm) causes isomerization of its diene bonds (14). Studies mimicking environmental conditions showed that cyanobacterial pigments (and humic acids (16)) can act as photosensitizers and primarily cause isomerization of MCLR to the 6(Z) nontoxic derivative, with some oxidation as well (15). The degradation of MC-LR with hydroxyl radicals (HO•) generated by TiO2 photocatalysis showed that two sites of the toxin (diene bonds of the Adda chain and the Mdha in the cyclic structure; Supporting Information Scheme S1) undergo transformation (10, 11), while studies with ultrasonic irradiation showed that the aromatic ring is oxidized as well (17). This study reports new degradation pathways and intermediates formed during the photocatalytic degradation of MC-LR with TiO2 photocatalytic films (13) and new insights on mechanistic aspects of HO• attack on MC-LR. MicrocystinLR is a relatively large molecule with diverse functional groups in various positions, each with their own susceptibility (second order reaction rate constants, kOH) for degradation. Though the kOH for many of these groups have been estimated in isolation (18), steric and hindering effects and competition between sites for the active species may inhibit the oxidation of “relatively” available for oxidation groups. This study investigated which determining factor (position or susceptibility) lead to the preferable site of degradation. The breakdown byproducts in water can also be used to detect the presence of the toxin and determine the derivative. In addition, elucidating the transformations of MC-LR can assist in understanding the fate of these metabolites in human serum since some of the active species generated by TiO2 photocatalysis are also found in blood (19).

Introduction

Experimental Section

Cyanotoxins are emerging drinking water contaminants released from harmful strains of cyanobacteria blooms (cyano-HABs) (1). Numerous human and animal poisonings have been linked to cyano-HABs occurrence since the 19th century (2), however analytical limitations delayed the structural identification of cyanotoxins until the early 1980s (3). Microcystins, a group of cyclic heptapeptides (>80 derivatives) (4) with primarily hepatotoxic activity, are most commonly found during cyano-HABs, especially their derivative known as microcystin-LR (MC-LR) (5). Human fatalities from poisoning incidences in Brazil (1996, 2001)

Safety. Cyanotoxin exposure, via ingestion, inhalation, and skin contact can cause acute and chronic health effects. Consequently, all the experiments were conducted in an Advance Sterilchem GARD III Class II Biological Safety Cabinet (The Baker Co.) with full exhaust. Proper laboratory clothing, safety gloves, and protective goggles were essential when handling toxin solutions. Materials. Solid MC-LR (0.5 mg, 96.4%) was purchased from CalBiochem and stored at -20 °C. A 482 mg/L standard of toxin solution was prepared by the addition of 1 mL of autoclaved Milli-Q Synthesis A10 water (Millipore Corp., Billerica, MA). Preparation of Photocatalytic Films. Two methods were utilized for the preparation of photocatalytic films. For the 6.7 µm thick films, a stainless steel plate was dip-coated in a modified plain alkoxide sol with colloidal TiO2 particles,

* Corresponding author fax: (513) 556-2599; e-mail: dionysios.d. [email protected]. † University of Cincinnati. ‡ U.S. Environmental Protection Agency. 10.1021/es801637z CCC: $40.75

Published on Web 10/28/2008

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followed by calcination at 500 °C (20, 21). The 0.3 µm thin transparent films on glass were prepared with a sol-gel method modified using surfactants as templating materials (22, 23). Reactor Design. The photocatalytic films (thin or thick) were activated by two long wave 15W UV lamps (Cole-Parmer) with maximum peak at 365 nm and UV intensity of 35 µW/ cm2 at 6 cm. The films were placed in round reactor vessels (diameter 6 cm) that contained 5 mL of 20 000 µg/L of MCLR (without pH adjustments and unbuffered solutions, pHsq 5.7) (13). LC, MS, and MS/MS Analysis. The obtained samples were split in half and analyzed for MC-LR and reaction intermediates with liquid chromatography (LC)/UV and LC/mass spectrometry (MS). The LC was performed under reversephase conditions and analyzed with a gradient method based on Liu et al. (2003) (11). A Thermo Finnigan LCQ Deca ion trap mass spectrometer was utilized for the MS and MS/MS identification of the reaction intermediates. More details on the analytical methods are summarized in previous publications and in the Supporting Information (12, 13).

Results and Discussion MC-LR Intermediates with Thin and Thick Photocatalytic Films. The degradation of MC-LR, under the same experimental conditions, was faster using thick films compared to thin films (Supporting Information Figures S1 and S2), mainly because of the higher amount of catalyst immobilized on the stainless steel plate (27 times more catalyst per cm2). Even with thick films, the degradation time of MC-LR and its intermediates of primary and secondary compounds was prolonged (t ) 8 h) compared to previous photocatalytic oxidation (PCO) studies (t ) 1 h) (10, 11). Based on the peak criteria discussed earlier, degradation using thick films yielded overall 14 observed [M + H]+ intermediates in the total ion chromatogram (TIC), while 21 [M + H]+ intermediates were observed in studies using thin films (out of the 25). Moreover, some of the observed ions, m/z ) 1011.5 and 1029.5, comprise multiple peaks for both thick and thin films in a large retention time window (14 < t < 20 min) (Supporting Information Table S1 and Figure S3) (13). Liu and co-workers, who studied the degradation mechanism of MC-LR with TiO2 in slurry systems (1.0% w/v) and hydrogen peroxide (42.8 mM H2O2) at acidic pH (pH 4.0), reported the formation of 10 reaction intermediates (11). Isolating higher number of intermediates in the current study may be due to differences in the experimental conditions (i.e., absence of additional oxidants like hydrogen peroxide) and sampling events. Also, the treatment pH reduced strong interaction between the toxin and the catalyst that otherwise impact the available sites of MC-LR for oxidation (13). During treatment with both types of films, formation of intermediates was recorded within 2 min of irradiating the catalyst. Two time-dependent sets of peak formation maxima were observed for both films. For the faster toxin-degrading thick films, the majority of the peaks exhibited maxima between 10 and 15 min of treatment while the same maxima were observed after 1 h of treatment with thin films. The remaining peaks gave maxima after 15 min and 2 h for the thick and thin films, followed by the disappearance of peaks after the completion of the second and sixth hour of treatment, respectively. The major intermediates, as collective peak area counts for both of the films, were the m/z 1011.5 and 1029.5 (Supporting Information Figure S3). Masses observed with the thick films were found with the thin films as well, except for the m/z 965.6 (peak no. 1) eluting at 22.8 min in the TIC (Supporting Information Table S1). Since the treatment with the thin films can provide a more detailed depiction (more peaks observed) of the transformations that MC-LR undergoes with TiO2 photocatalysis, herein the 8878

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degradation pathways and intermediates obtained using thin films will be discussed. In order to clearly differentiate intermediates based on MS peak area criteria (13), the initial toxin concentration was chosen to be significantly higher than that typically encountered in water media (24). Intermediates were selected according to their peak areas and elution times; the corresponding MS/MS data were obtained and analyzed for structure elucidation purposes as described in an earlier publication (13). Structural identification of the intermediates led to assigning four sites of MC-LR where the HO• preferably initiate toxin transformations (Supporting Information Scheme S1). Three sites are located in the hydrophobic chain of the Adda amino acid namely the aromatic ring, the methoxy group, and the conjugated diene carbon bonds. The fourth site is in the cyclic structure of MC-LR and involves the unsaturated bond of Mdha amino acid. Reactive species formed during TiO2 photocatalysis include the HO• being the main species, the superoxide radical anion (O2•-), the perhydroxyl radical (HO2•-) and the conduction band electron (ecb-) (25-27). The positive holes in the valence band of the catalyst formed after the photoexcitation of TiO2 are highly oxidizing species for compounds (i.e., water, organics) adsorbed on the surface of the catalyst (27). While the role of solvents in radical reactions (in general) is limited to hydrogen transfer, in this case water can be oxidized to form HO• and can also participate in radical reactions if the reactants are soluble (28). The role of oxygen in the formation of the active species is not restricted to the role of electron acceptor and preventing charge-carrier recombination; it can also participate in subsequent radical-based reactions that eventually lead to the formation of secondary oxidized species such as aldehydes and ketones (25, 26, 29). Lack of analytical standards for cyanotoxin reaction intermediates complicates identification of primary intermediates. Prior knowledge of mechanistic steps, followed by the photocatalytic oxidation include oxidation, oxidative cleavage, geometric and constitutional (structural) isomerization, reduction, condensation, and polymerization of the primary compounds (30) were used as a guideline, with structures verified by MS/MS data. Hydroxyl radical attack (detailed mechanistic aspects to follow), initiates oxidation of compounds by forming carbon center radicals via hydrogen abstraction (see Supporting Information Schemes S2 and S3). Several studies have shown that the reaction of R-aliphatic amino acids with HO• occurs with a preferential hydrogen abstraction at the R-carbon. However, as the size of the amino acids becomes bigger, other CsH bonds are also involved (31). This was also observed in the oxidative transformation of MC-LR: preferential degradation of some sites without excluding the possibility of reactions at additional sites. Site A: The Aromatic Ring. The aromatic ring undergoes hydroxyl substitution of an aromatic hydrogen to form the m/z 1011.5 intermediate (Supporting Information Scheme S2) (31, 32). The necessary mechanistic steps for the creation of m/z 1011.5 include the addition of a HO• in one of the aromatic double bonds and formation of a carbon-centered radical, which rapidly reacts with oxygen, to form a peroxy radical. The release of a perhydroxyl radical results eventually in the substitution of the hydrogen with a hydroxyl group. Hydroxylation of aromatic rings subsequent to HO• attack is a very common alteration of biomolecules. Studies on the modification of proteins with reactive oxygen species (ROS) and particularly HO• have shown that proteins containing aromatic amino acids have undergone hydroxylation at different positions (33). Moreover, the second order reaction rate constant of HO• attack on the aromatic ring compared to alkenes is slightly higher (18); therefore preferable if the

SCHEME 1. Attack of Hydroxyl Radicals on Site A: The Aromatic Ring of Adda

aromatic ring is close enough to the surface to react with the surface generated HO•. Consequently, similar intermediates for MC-LR were expected to be detected (Scheme 1). The m/z 1011.5, intermediate though not detected in previous PCO studies (10, 11), was reported with other HRAOTs such as ultrasonic irradiation (17). The mass chromatogram of m/z 1011.5 in Supporting Information Figure S3 displays three major peaks (and multiple smaller ones) possibly corresponding to ortho, meta, or para substitution. Which of the three positions is more prone to electrophilic substitution is determined by the remaining moiety of the Adda amino acid attached to the aromatic ring. The chain of the Adda amino acid has several different groups; however, the one directly connected to the benzene ring is an alkyl group. This alkyl group inductively donates electrons to the ring through the σ-bonds (I+) and therefore acts as an ortho-para director for the second electrophilic substitution with HO• radicals (to give the m/z 1011.5 intermediates) (34). The first aromatic hydroxylation is followed by a second one which yields the m/z 1027.5 intermediate. The detection of m/z 1027.5 verifies the first substitution, since the hydroxyl group increases the electron density of the aromatic ring and thus, electrophilic reactions (as is the hydroxyl radical attack) proceed faster (17, 34). Both the alkyl group and the hydroxyl group activate the aromatic ring and act as ortho-para directors; however, since the hydroxyl group is a stronger activator than the alkyl group, it controls the position of the second hydroxylation. The second hydroxylation most likely occurs in the ortho position with respect to the hydroxyl group. Hindering effects also seem to take place which would explain why m/z 1027.5 did not exhibit multiple peaks (34). Site B: The Methoxy Group. The methoxy group of the Adda chain is the second site were degradation is initiated. It can be completely removed to give m/z 965.6, DmADDA, after the formation of the formic acid ester MC-LR derivative (m/z 1009.6) (Scheme 2). Two possible mechanisms for the formation of m/z 1009.6 are depicted in Supporting Information Scheme S3 and both begin with the abstraction of hydrogen by HO• and the formation of a carbon center radical. The adjacent oxygen in the methoxy group promotes the hydrogen abstraction of the CsH bond rather than C9sH of the ADDA chain because it increases the electron density of

the group and assists the electrophilic attack of the HO• (35). Following the formation of the center carbon radical, oxygen can react to form peroxy radical adducts which undergo radical termination reactions through dimerization (reaction with itself to form RO2sO2R unstable tetraoxide intermediate products) or react with the hydroxyl radicals to form polyoxides (36). In both cases, the removal of hydrogen peroxide results in the formation of the formic acid-(MCLR) ester derivative (m/z 1009.6). Formic acid is eventually released (direct oxidation from the positive holes (hvb+) of the catalyst, see below for explanation) and DmADDA is formed. Studies on the oxidation of dimethyl ether (CH3OCH3, a structure similar to the methoxy group of Adda chain) suggest a degradation path for the removal of the methoxy group through unimolecular decomposition and the formation of CH3O• and •CH3 (37). However, it is believed that the experimental conditions (UV-A intensity) of this study are not strong enough to cause this type of bond cleavage. Stefani et al. (2000) also proposed another mechanism for formation of the formic ester through the formation of alkoxyl radicals (formed after the removal of O2 from the tetroxide derivative and the formation of two alkoxyl radicals) (35). The m/z 1009.6 was not detected with other HO• based AOTs even though formate-compounds (another name for the formic esters) are the first oxidation products from the oxidation of MTBE with oxidants or bacteria (38, 39), and it should not be surprising that the same process was observed in MC-LR as well. DmADDA, on the other hand, was detected in surface, landing, and purified water following the treatment with iron(III) chloride (40). Toxicity studies showed that DmADDA did not cause acute toxicity when injected into mice (40). Site C: The Conjugated Double Bonds. The site most prone to HO•-oxidation is at the conjugated double bonds (11, 17) partially because of the high second order reaction rate constants (kOH ∼109-10 M-1s-1) (18), but mainly because of its position in the toxin molecule. Intermediates related to the modification of this site of MC-LR have been reported with previous HO• studies irrespective of the processes of radical formation (TiO2 photocatalysis, ultrasonic irradiation) (9, 11, 12, 17), and the experimental conditions (treatment pH, light source activation, addition of oxidants). HO• react with the diene bonds to produce hydroxyl adducts through two different mechanist steps: hydroxyl addition and hydroxyl VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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SCHEME 2. Attack of Hydroxyl Radicals on Site B: The Methoxy Group of Adda

SCHEME 3. Attack of Hydroxyl Radicals on Site C: The Conjugated Carbon Double Bonds of Adda

substitution. The m/z 1029.5 and m/z 1063.5 are intermediate products related to double and quadruple hydroxyl addition. Following the incorporation of the first HO• into the double bond and the formation of the allyl radical (RCH ) CHsR2C•), a second HO• reacts with carbon center (before O2 does, Supporting Information Scheme S2). Allyl (or allylic) radicals are more stable compared to the corresponding alkyl radical because of delocalization of the unpaired e- over the extended π-orbital (28, 34). The double hydroxylation (1,2 addition) can occur on any of the double bonds pairs (C4sC5 and C6sC7) (Scheme 3, m/z 1029 products A and B). In general, when conjugated dienes undergo electrophilic reactions, mixtures of products are formed (excluding stereoisomers) 8880

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(34), as observed in this study (Supporting Information Figure S3). This is caused by the different resonance configurations that the allylic radical can take, which eventually results in 1,2 (Scheme 3, m/z 1029 products A and B) or 1,4 additions (Scheme 3, m/z 1029 product C). The preference of which product will be formed is determined by the stability of the allylic radical: the higher the substituents of the carbon center radical, the more stable the allylic radical. Since in the conjugated dienes of MC-LR, all the carbons produce primary carboradicals (one R-group) (34), all the positions for hydroxylation and their corresponding stereoisomers can occur. Based on the above, it is believed the isomerization at the C4sC5 and C6sC7 of the diene bond of the Adda chain

SCHEME 4. Attack of Hydroxyl Radicals on Site C: The Conjugated Carbon Double Bonds of Adda

SCHEME 5. Attack of Hydroxyl Radicals on Site D: The Cyclic Structure of MC-LR

is a direct result of HO• addition mechanism (28, 31, 32) and not of the effect of UV-A radiation (13-15), as previously suggested (11). Following the m/z 1029.5, quadruple hydroxylated-MC-LR is formed (m/z 1063.5), were the hydroxylation occurs in the Adda chain alone, or in combination with the unsaturated group of the Mdha amino acid (Scheme 3). The second oxidation route in which initiated in the diene bonds yields the complete removal of the Adda chain (Scheme 4). Via hydroxyl substitution (Supporting Information Scheme S2), an OH-group substitutes the hydrogen of C7 and forms an enol-MC-LR (m/z 1011.5). The enol-MC-LR rapidly isomerizes to the more stable tautomer of ketone-MC-LR and follows a series of oxidative induced bond cleavage mechanistic steps, transforming to a ketone-derivative, m/z 835.4, followed by an aldehyde-derivative, m/z 795.4, and eventually to the hydroxyl-derivative m/z 783.4. The trans-

formation of m/z 795.4 to m/z 783.4 occurs via an intermediate oxidation step of the aldehyde to a carboxylic acid (35), which can be oxidized by the positive holes of the TiO2 (hvb+) and produce the m/z 783.4. In aqueous solutions, aldehydes are found in a hydrated structure [RCH(OH)2] and via HO•/O2 attack, the corresponding acid is formed (35). Carboxylic acids can adsorb on the catalyst’s surface and get oxidized directly from positive holes (27, 41), based on the mechanism shown below, and convert to hydroxyl-derivatives (m/z 783.4): + f R • + CO2 RCOO- + hvb

R• + HO• f ROH Under our experimental conditions the overall charge of the toxin is singly negative (the two free carboxylic groups are deprotonated and the guanidine group is protonated), VOL. 42, NO. 23, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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while the TiO2 has mostly zero charge (very close to the point of zero charge, PZC) (13). However, positive TiO2 centers, where the ionized carboxylic groups of the toxin can covalently attach, may still exist. The reaction intermediates m/z 795.4 and 783.4 have a mass difference of 12 Da and gave peaks that sequentially eluted (Supporting Information Table S1), therefore the structural differences of theses intermediates must be in close agreement. Site D: The Cyclic Structure, Modification of the Mdha. So far, the double bond of the Mdha amino acid is the only observed initiation site of oxidation in the cyclic ring of MCLR, based on the current study. Hindering effects caused from the functional groups of the other amino acids, as well as competition with the other three sites for radical utilization, make degradation of the cyclic structure less likely. In any case, consecutive oxidation steps, such as the double hydroxylation of the Mdha (m/z 1029.5, product E), its oxidation to aldehyde (m/z 1011.5, product D), and cleavage of the R2C-COR bond (m/z 1015.5), were isolated (Scheme 5). Intermediates, such as m/z 783.4 (product B), that correspond to alterations of the MC-LR structure in both sites, have also been observed. It is well established that during aerobic cell metabolism and via enzymatic processes, a variety of free radicals are formed (31). Starting from the formation of the superoxide radicals that are readily converted into hydrogen peroxide; the latter can be iron-catalyzed via the Haber-Weiss mechanism into HO• which can eventually attack organic compounds and lead to a chain of oxidation mechanisms. Garrison (1987), in his review on the reaction mechanisms of radiolysis of biochemical molecules such as peptides, mentioned that these techniques can be useful “in studying the physiological chemistry of unirradiated biological systems” (31). It is believed that TiO2 photocatalysis can be used as well for predicting part of the fate of the toxins in the body. Hilborn et al. (2007) used the MMPB method to determine the total microcystin concentration from blood serum samples (5). They also emphasize the need to understand and identify the nature of the serum bound microcystins (i.e., reaction with serum protein such as cystein-34 in human albumin), but since toxins in the serum persist for a long period of time, studies of the oxidized products of MCs should be accounted as well. To conclude, we studied the reaction intermediates formed during the PCO of MC-LR at neutral pH and we identified four sites where the degradation is initiated, with the conjugated diene bonds being the most affected site. The decisive factor for the preferential oxidation site was its position in the MC-LR structure rather than the kOH. The main HO• mechanistic steps were hydroxylation via substitution or addition with simultaneous isomerization, oxidation, and oxidative bond cleavage. Most of the identified intermediates had an intact cyclic structure; however, toward the end of the treatment, linear intermediates were isolated as well.

Acknowledgments This research was funded in part by the National Science Foundation through a CAREER Award (BES-0448117) to D.D.D., the U.S. EPA (RD-83322301), and the Center of Sustainable Urban Engineering (SUE) at the University of Cincinnati (UC). M.G.A. is grateful to Sigma Xi, The Scientific Society for a Grant-in-Aid of Research Fellowship, the Rindsberg Foundation of UC, and the University Research Council of UC for a Summer Research Fellowship. Disclaimer: Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy. 8882

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Supporting Information Available This material is available free of charge via the Internet at http://pubs.acs.org.

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