Adda Moiety Research Articles

Microcystins with Modified Adda5-Residues from a Heterologous Microcystin Expression System.

Microcystins are hepatotoxic cyclic heptapeptides produced by some cyanobacterial species and usually contain the unusual β-amino acid 3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyl-4E,6E-decadienoic acid (Adda) at position-5. The full microcystin gene cluster from Microcystis aeruginosa PCC 7806 has been expressed in Escherichia coli. In an earlier study, the engineered strain was shown to produce MC-LR and [d-Asp3]MC-LR, the main microcystins reported in cultures of M. aeruginosa PCC 7806. However, analysis of the engineered strain of E. coli using semitargeted liquid chromatography with high-resolution tandem mass spectrometry (LC-HRMS/MS) and thiol derivatization revealed the presence of 15 additional microcystin analogues, including four linear peptide variants and, in total, 12 variants with modifications to the Adda moiety. Four of the Adda-variants lacked the phenyl group at the Adda-terminus, a modification that has not previously been reported in cyanobacteria. Their HRMS/MS spectra contained the product-ion from Adda at m/z 135.1168, but the commonly observed product-ion at m/z 135.0804 from Adda-containing microcystins was almost completely absent. In contrast, three of the variants were missing a methyl group between C-2 and C-8 of the Adda moiety, and their LC-HRMS/MS spectra displayed the product-ion from Adda at m/z 135.0804. However, instead of the product-ion at m/z 135.1168, these three variants gave product-ions at m/z 121.1011. These observations, together with spectra from microcystin standards using in-source fragmentation, showed that the product-ion at m/z 135.1168 found in the HRMS/MS spectra of most microcystins originated from the C-2 to C-8 region of the Adda moiety. Identification of the fragmentation pathways for the Adda side chain will facilitate the detection of microcystins containing modifications in their Adda moieties that could otherwise easily be overlooked with standard LC-MS screening methods. Microcystin variants containing Abu at position-1 were also prominent components of the microcystin profile of the engineered bacterium. Microcystin variants with Abu1 or without the phenyl group on the Adda side chain were not detected in the original host cyanobacterium. This suggests not only that the microcystin synthase complex may be affected by substrate availability within its host organism but also that it possesses an unexpected degree of biosynthetic flexibility.

Transformation of Algal Toxins during the Oxidation/Disinfection Processes of Drinking Water: From Structure to Toxicity.

With the increase of algal blooms worldwide, drinking water resources are threatened by the release of various algal toxins, which can be hepatotoxic, cytotoxic, or neurotoxic. Because of their ubiquitous occurrence in global waters and incomplete removal in conventional drinking water treatment, oxidation/disinfection processes have become promising alternative treatment options to destroy both the structures and toxicity of algal toxins. This Review first summarizes the occurrence and regulation of algal toxins in source water and drinking water. Then, the transformation kinetics, disinfection byproducts (DBPs)/transformation products (TPs), pathways, and toxicity of algal toxins in water oxidation/disinfection processes, including treatment by ozonation, chlorination, chloramination, ultraviolet-based advanced oxidation process, and permanganate, are reviewed. For most algal toxins, hydroxyl radicals (HO•) exhibit the highest oxidation rate, followed by ozone and free chlorine. Under practical applications, ozone and chlorine can degrade most algal toxins to meet water quality standards. However, the transformation of the parent structures of algal toxins by oxidation/disinfection processes does not guarantee a reduction in toxicity, and the formation of toxic TPs should also be considered, especially during chlorination. Notably, the toxicity variation of algal toxins is associated with the chemical moiety responsible for toxicity (e.g., Adda moiety in microcystin-LR and uracil moiety in cylindrospermopsin). Moreover, the formation of known halogenated DBPs after chlorination indicates that toxicity in drinking water may shift from toxicity contributed by algal toxins to toxicity contributed by DBPs. To achieve the simultaneous toxicity reduction of algal toxins and their TPs, optimized oxidation/disinfection processes are warranted in future research, not only for meeting water quality standards but also for effective reduction of toxicity of algal toxins.

Chlorination of microcystin-LR in natural water: Kinetics, transformation products, and genotoxicity

Microcystin-LR (MC-LR), a type of cyanotoxin commonly found in natural water bodies (sources of drinking water), poses a threat to human health due to its high toxicity. It is essential to successfully remove this cyanotoxin from drinking water sources. In this study, chlorine was used to oxidize MC-LR in Milli-Q water (MQ) (control test) and natural water collected from Lake Longhu (LLW) as a drinking water source. The removal efficiency, proposed transformation pathways, and genotoxicity were investigated. In the chlorine dose range investigated (4.0 mg L−1 - 8.0 mg L−1), the apparent second-order rate constants for MC-LR chlorination varied from 21.3 M−1s−1 to 31.9 M−1s−1 in MQ, higher than that in LLW (9.06 M−1s−1 to 17.7 M−1s−1) due to a faster chlorine decay attributed to the water matrix (e.g., natural organic matter) of LLW. Eleven transformation products (TPs) of MC-LR were identified in the two waters. The conjugated diene moieties and benzene ring of Adda moiety (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid), and the double bond of Mdha moiety (N-methyldehydroalanine) were the major susceptible reaction sites. Attacking unsaturated bonds by hydroxyl and chlorine radicals to generate monochloro-hydroxy-MC-LR was the primary initial transformation pathway, followed by nucleophilic substitution, dehydration, and cleavage in MC-LR. Chlorine substitution on the benzene ring was also observed. Based on the bacterial reverse-mutation assay (Ames assay), TPs in treated natural water did not induce genotoxicity/mutagenicity. These findings shed light on the role of chlorination in controlling the risk of cyanotoxins in drinking water treatment plants.

Rapid detoxification of Microcystin-LR by selective catalytic hydrogenation of the Adda moiety using TiO2-supported Pd catalysts

The hepatotoxicity of Microcystin-LR (MC-LR) is mainly caused by its Adda moiety. In this study, we used TiO2-supported Pd catalysts to selectively hydrogenate the CC bonds in the Adda moiety, achieving rapid detoxification of MC-LR in water under ambient conditions. MC-LR was removed within 5 min by catalytic hydrogenation on Pd(1.0)/TiO2 with a catalyst dosage normalized rate constant of 1.3 × 10−2 L mgcat−1 min−1, significantly more efficient than other catalytic treatment methods. The reactions proceeded in a highly selective manner towards catalytic hydrogenation at the CC bond of the Mdha moiety and subsequently the conjugated double bond of the Adda moiety, yielding two intermediates and one final product. Upon catalytic hydrogenation for 30 min on Pd(0.07)/TiO2, the toxicity of MC-LR (assessed by protein phosphatase 2A activity assay) drastically decreased by 90.8%, demonstrating effective detoxification. The influence of catalyst support, Pd content, initial MC-LR concentration, reaction pH, and catalytic stability were examined. Surface adsorption and the cationic Pd played a crucial role in the reaction kinetics. Our results suggest that catalytic hydrogenation is a highly effective and safe strategy for detoxifying MC-LR by selective reactions.

Ion exchange and vacuum UV: A combined approach for removing organic matter and microcystins from natural waters

UV based advanced oxidation exhibits promising potential for the degradation of cyanobacterial toxins from natural waters. However, degradation is hindered by the presence of natural organic matter (NOM), which absorbs both 185 nm and 254 nm photons and scavenges hydroxyl radical (•OH), affecting mechanisms of UV/vacuum UV (VUV) degradation and requiring higher fluences (exposure times) to achieve microcystin reduction. In addition to obstructing UV/VUV degradation, NOM is associated with the formation of harmful disinfection by-products (DBPs) with drinking and recycled waters. Hence, technologies that are capable of removing NOM are desirable. In this study, we investigated the synergistic application of ion exchange (IX) resins and VUV for the removal of NOM and Microcystin-LR (MCLR) from natural waters. A pre-treatment with 1 mL/L dosage of IX resins removed >80% of the dissolved organic carbon (DOC) and UV254 absorbing compounds. This increased the MCLR degradation rates (up to three folds) in the tested surface waters. A progressive analysis on the degradation products indicated subsequent degradation of the toxic ADDA moiety of MCLR confirmed by the formation of compounds with a specific mass to charge ratio (m/z) of 835.5 855.3 and 791.4. More importantly, pre-treatment with IX resulted in reducing VUV’s electrical energy per order (EEO) by up to four folds and the overall chlorine demand by eight folds for surface waters. Hence, the results of this study indicate that a combined IX-VUV will be highly effective at reducing microcystins in impacted natural waters.

Analysis of total microcystins and nodularins by oxidative cleavage of their ADMAdda, DMAdda, and Adda moieties

Microcystins (MCs) and nodularins (NODs) exhibit high structural variability, including modifications of the Adda (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid) moiety. Variations include 9-O-desmethylAdda (DMAdda) and 9-O-acetylDMAdda (ADMAdda) which, unless targeted, may go undetected. Therefore, reference standards were prepared of [ADMAdda5]MCs and [DMAdda5]MCs, which were analyzed using multiple approaches. The cross-reactivities of the [DMAdda5]- and [ADMAdda5]MC standards were similar to that of MC-LR when analyzed with a protein phosphatase 2A (PP2A) inhibition assay, but were <0.25% when analyzed with an Adda enzyme-linked immunosorbent assay (ELISA). Oxidative cleavage experiments identified compounds that could be used in the analysis of total MCs/NODs in a similar fashion to the 2R-methyl-3S-methoxy-4-phenylbutanoic acid (MMPB) technique. Products from oxidative cleavage of both the 4,5- and 6,7-ene of Adda, DMAdda and ADMAdda were observed, and three oxidation products, one from each Adda variant, were chosen for analysis and applied to three field samples and a Nostoc culture. Results from the oxidative cleavage method for total Adda, DMAdda, and ADMAdda were similar to those from the Adda-ELISA, PP2A inhibition, and LC-MS/MS analyses, except for the Nostoc culture where the Adda-ELISA greatly underestimated microcystin levels. This oxidative cleavage method can be used for routine analysis of field samples and to assess the presence of the rarely reported, but toxic, DMAdda/ADMAdda-containing MCs and NODs.

Ozonation of Microcystins: Kinetics and Toxicity Decrease.

The ozonation of six microcystins (MCs) (MC-LR, MC-RR, MC-LA, MC-LF, MC-YR, and MC-LW) was investigated with a focus on the kinetics and decrease in toxicity. Second-order rate constants for the reactions of the six MCs with O3 and •OH ( kO3,MC and k•OH,MC) ranged from 7.1 × 105 to 6.1 × 106 M-1 s-1 ( kO3,MC) and from 1.2 × 1010 to 1.8 × 1010 M-1 s-1 ( k•OH,MC), at pH 7.2 and 20 °C. The activation energies were calculated to be 21.6-34.5 and 11.6-13.1 kJ mol-1 for kO3,MC and k•OH,MC, respectively. The rate constants did not show an important pH dependency, except for kO3,MC-YR, which increased at pH > 7. A kinetic model using the determined rate constants and the measured exposures of O3 and •OH was able to precisely predict the removal of MCs in natural water. The hepatotoxicities of MCs were decreased by ozonation; the toxicities of the four MCs (MC-LR, MC-RR, MC-LA, and MC-LF) decreased nearly concurrently with decreases in their concentrations. However, MC-YR and MC-LW showed a gap between the concentration and toxicity as a result of the incomplete destruction of the Adda moiety (a key amino acid expressing the hepatotoxicity of MCs). A product study using liquid chromatography-mass spectrometry identified a number of oxidation products with an intact Adda moiety produced by the ozonation of MC-YR and MC-LW.

Elimination kinetics and detoxification mechanisms of microcystin-LR during UV/Chlorine process

Microcystin-LR (MC-LR), a toxin produced by cyanobacteria, is very toxic and poses a threat to public health when entering water treatment works. In this study, UV/chlorine process, as an advanced oxidation process (AOP), has been demonstrated for effective elimination of MC-LR levels and associated toxicity. At a chlorine dose of 3.0 mg L−1 and UV fluence of 125 mJ cm−2, MC-LR (initial concentration 1.0 μM) was reduced by 92.5%, which was much higher than 20.3% removal under UV irradiation alone and 65.1% removal during dark chlorination. Enhanced degradation was attributed by hydroxyl radicals (HO) and reactive chlorine species (RCS), mainly Cl2- and ClO. Increasing chlorine doses or lowering pH favored MC-LR removal. Increased bicarbonate and natural organic matter concentrations inhibited MC-LR removal, but bromide ions enhanced MC-LR removal instead. MC-LR elimination rates in natural waters were roughly two times smaller than those in ultrapure water. The reactive radicals promoted hydroxylation of both diene of Adda moiety and double bond of Mdha moiety in MC-LR. UV exposure enhanced the dechlorination of chloro-MC-LR via the cleavage of CCl bond. The toxicity was evaluated by a protein phosphatase (PP2A) inhibition assay. At a chlorine dose of 3.0 mg L−1 and UV fluence of 125 mJ cm−2, the toxicity of the treated water was reduced by 75.0%, which was also higher than 25.7% and 46.7% removal under UV irradiation alone and during dark chlorination, respectively. These results highlight UV/chlorine is an efficient AOP for MC-LR degradation and detoxification.

Tin porphyrin immobilization significantly enhances visible-light-photosensitized degradation of Microcystins: Mechanistic implications

This study demonstrates that tin porphyrin (SnP) loading on a silica substrate (SnP/silica) markedly accelerates the degradation of Microcystins (MCs) under visible light irradiation, despite a reduction of photosensitized singlet oxygen (1O2) production. A comparative study using Rose Bengal, SnP, and C60 aminofullerene suggested that the MC-RR decay rate was directly proportional to the photosensitizing activity for triplet state-induced oxidation, while it exhibited poor correlation to singlet oxygenation efficiency. This implies that electron transfer from MC to the triplet state of SnP (facilitated by favorable MC sorption on silica) contributes to the photosensitized MC oxidation. Experiments to examine sensitizers for the one-electron oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) showed the superiority of SnP/silica for photo-initiated electron transfer as a possible MC oxidation route. This was corroborated by the negligible effects of reagents that quench or facilitate singlet oxygenation (e.g., azide ion, D2O) on the MC-RR degradation rate. Despite MC-RR removal below detection levels, residual toxicity (indicated by a significant decrease in protein phosphatase inhibition activity) was observed. Tandem mass spectrometric analysis suggests that this residual toxicity may be ascribed to byproducts resulting from addition of a single oxygen atom to the Adda moiety.

Using the MMPB technique to confirm microcystin concentrations in water measured by ELISA and HPLC (UV, MS, MS/MS).

Microcystins have been detected in raw and finished drinking water using a variety of techniques, including assays (immunoassay, phosphatase inhibition) and HPLC (UV, MS/(MS)). The principal challenge to microcystin analysis is accounting for the over 150 variants that have been described. A confirmatory individual variant HPLC analysis is prone to under-reporting total microcystins due to method specificity. One method that allows for total microcystin quantitation is the MMPB technique. In this study, water samples with native microcystins were oxidized to cleave the Adda moiety, common to all microcystin variants. LC-MS/MS analysis was conducted on the subsequent MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) molecule and calibrated using a certified reference standard (microcystin-LR) and 4-phenylbutyric acid. Total microcystin concentrations from MMPB were compared to Adda ELISA and individual variant analyses (LC-UV, LC-MS/(MS)). Variants of microcystin, including [DAsp(3)]MC-RR, [Dha(7)]MC-RR, MC-RR, MC-YR, MC-LR, [DAsp(3)]MC-LR, [Dha(7)]MC-LR, MC-WR, MC-LA, and MC-LY were detected and quantified in samples. The individual variant analyses did not account for total microcystins present in samples, as indicated by ELISA and MMPB data. Results demonstrated the MMPB technique is a simple and valuable approach to confirm ELISA data when analyzing microcystins, with method detection limits of 0.05 μg L(-1) for total microcystins.

Oxidation of microcystin-LR in water by ozone combined with UV radiation: The removal and degradation pathway

In this study, the performance of the combined UV/O3 process for the degradation of microcystin-LR (MC-LR) in water was investigated, and the possible degradation pathway of the toxin was elucidated according to reaction intermediates. Results demonstrated that the UV/O3 process was a more effective method for the removal and mineralization of MC-LR in water, compared with UV- and O3-alone processes. Increasing the solution pH (5–10) and dissolved organic carbon concentration (0–5.3mg/L) could inhibit the degradation of the toxin. Nine reaction intermediates of MC-LR were detected by LC/MS, and four initial reaction sites of the toxin were observed: the conjugated diene of Adda, the double bonds of Mdha and two free acid groups of MeAsp and Glu. The degradation pathways of the toxin during UV/O3 process involved isomerization, hydroxylation and oxidative cleavage of the Adda side chain, oxidation of Mdha and decarboxylation of MeAsp and Glu. The pathway of the oxidation and cleavage of Adda and Mdha could result from both HO and O3 oxidation, but the isomerization of Adda moiety and the decarboxylation of MeAsp and Glu mainly resulted from UV radiation. The yield trends of the intermediates indicated that the oxidation of Adda moiety was the dominant degradation pathway and that the combined UV/O3 process had a higher potential for the simultaneous degradation of MC-LR and its intermediates than the individual processes.

Further characterization of glycine-containing microcystins from the McMurdo dry Valleys of Antarctica.

Microcystins are hepatotoxic cyclic peptides produced by several cyanobacterial genera worldwide. In 2008, our research group identified eight new glycine-containing microcystin congeners in two hydro-terrestrial mat samples from the McMurdo Dry Valleys of Eastern Antarctica. During the present study, high-resolution mass spectrometry, amino acid analysis and micro-scale thiol derivatization were used to further elucidate their structures. The Antarctic microcystin congeners contained the rare substitution of the position-1 d-alanine for glycine, as well as the acetyl desmethyl modification of the position-5 Adda moiety (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid). Amino acid analysis was used to determine the stereochemistry of several of the amino acids and conclusively demonstrated the presence of glycine in the microcystins. A recently developed thiol derivatization technique showed that each microcystin contained dehydrobutyrine in position-7 instead of the commonly observed N-methyl dehydroalanine.

Sensitive determination of total microcystins with GC-MS method by using methylchloroformate as a derivatizing reagent

Various microcystins (MCs) produced by cyanobacteria pose a potential threat to water resources, therefore, the determination of the total MCs rather than the content of some specific types of MCs in water is of great importance. Oxidation of MCs causes cleavage of the Adda moiety of microcystins to 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB). In this paper, a new method based on the derivatization of MMPB with methylchloroformate is proposed for the sensitive determination of total MCs. The oxidation conditions as well as the esterification process were optimized through a series of batch experiments. With the optimized procedure, a detection limit as low as 0.56 μg L−1 was achieved for total MCs and the method was successfully applied to the determination of MCs in real samples. A comparison study by using the LC-MS method for direct quantification of the MMPB was also conducted to validate the proposed method. The results proved that the technique is highly sensitive and convenient for the quantification of total MCs in water samples. As an additional feature, the method does not require the use of toxic and expensive standards of MCs in the environmental monitoring.

Detoxification and degradation of microcystin-LR and -RR by ozonation

In the present study, two Microsystins (MCs) of Microcystin-LR and Microcystin-RR were degraded with different dosages of ozone (O 3). The possible degradation pathways were elucidated by analyzing their intermediates and end-products with liquid chromatography–mass spectrometry (LC–MS) method. The toxicity of the MCs ozonation products was also evaluated by assaying the protein phosphatase inhibition in vitro and acute toxicity in vivo. Results demonstrated that ozonation was a promising technology for removal and detoxification of the cyanotoxins. The MCs destruction was mainly involved in the attack of ozone on Adda side chain. First, the conjugated diene structure of Adda moiety was attacked by hydroxyl radical (OH ) to produce dihydroxylated products, then the hydroxylated 4–5 and/or 6–7 bond of Adda was cleaved into aldehyde or ketone peptide residues, and finally the residues were oxidized into the corresponding carboxylic acids. The fragmentation of the Mdha-Ala peptide bond of MCs also contributed positively to the oxidation process. Additionally, the attack on the benzene ring of Adda side chain was exclusively observed during MC-RR degradation. The toxicity evaluation of MCs ozonation products revealed that those end-products had no adverse effects in vivo and in vitro ozonation that could completely remove the MCs’ toxicity.

Radiolysis Studies on the Destruction of Microcystin-LR in Aqueous Solution by Hydroxyl Radicals

In this study, steady-state and time-resolved radiolysis methods were used to determine the primary reaction pathways and kinetic parameters for the reactions of hydroxyl radical with microcystin-LR (MC-LR). The fundamental kinetic data is critical for the accurate evaluation of hydroxyl-radical based technologies for the destruction of this problematic class of cyanotoxins. The bimolecular rate constant for the reaction of hydroxyl radical with MC-LR is 2.3 (+/-0.1) x 10(10) M(-1)s(-1) based on time-resolved competition kinetics with SCN-at low conversions using pulsed radiolysis experiments. The reaction of hydroxyl radical with MC-LR can occur via a number of competing reaction pathways, including addition to the benzene ring and diene and abstraction of aliphatic hydrogen atoms. LC-MS analyses indicate the major products from the reaction of hydroxyl radicals with MC-LR involve addition of hydroxyl radical to the benzene ring and diene moieties of the Adda side chain. Transient absorption spectroscopy monitored between 260-500 nm, following pulsed hydroxyl radical generation, indicate the formation of a transient species with absorption maxima at 270 and 310 nm. The absorption maxima and lifetime of the transient species are characteristic of hydroxycyclohexadienyl radicals resulting from the addition of hydroxyl radical to the benzene ring. The rate constant for the formation of hydroxycyclohexadienyl radical is 1.0 (+/-0.1) x 10(10) M(-1)s(-1) accounting for approximately 40% of the primary reaction pathways. Representative rate constants and partitioning of hydroxyl radical reactions were assessed based on the reactivities of surrogate substrates and individual amino acids. Summation of the individual reactivities of hydroxyl radical at the different reactive sites (amino acids) leads to a rate constant of 2.1 x 10(10) M(-1) s(-1) in good agreementwith the rate constant determined in our studies. The relative magnitude of the rate constants for the reactions of hydroxyl radical with the individual amino acids and appropriate surrogates, suggest 60-70% reactions of hydroxyl radical occur at the benzene and diene functional groups of the Adda moiety.

Further Investigation of Microbial Degradation of Microcystin Using the Advanced Marfey Method

It is known that microcystin (MC) is subject to microbial degradation to provide three types of products, linearized MCLR (Adda-Glu-Mdha-Ala-Leu-MeAsp-Arg), tetrapeptide Adda-Glu-Mdha-Ala, and Adda. They can be readily detected by the usual HPLC, because they commonly have an Adda moiety with a diene and an absorption maximum at 238 nm as the chromophore. However, no other degradation products without such a chromophore have been isolated to date. In this study, cell preparation of a bacterium B-9 that can degrade MC and detection of the degradation products were devised. First, we regulated the B-9 hydrolytic activity by washing with sodium chloride solution to obtain a desired cell preparation, which permitted an additional intermediate and the final products of MCLR to be obtained. Second, the resulting products could be firmly identified using the advanced Marfey method with the aid of log D. As a result of these experiments, the following degradation products were further identified: a tetrapeptide, Adda-Glu-Mdha-Ala, tripeptides Adda-Glu-Mdha, Glu-Mdha-Ala, and Arg-MeAsp-Leu, a dipeptide, Glu-Mdha, and amino acids Adda, Arg, and methylamine derived from Mdha. The present study expands the hydrolytic activity of the B-9 strain, which can hydrolyze not only cyanobacterial cyclic peptides but also MC to the intermediates and final products. The established characterization method composed of the advanced Marfey method and log D would be a standard technique for the structural characterization of a mixture of amino acids and peptides.

Oxidation of microcystins by permanganate: Reaction kinetics and implications for water treatment

A few genera of cyanobacteria produce toxins which contaminate drinking water resources. Microcystins (MC), widely reported cyanotoxins, cause acute and chronic toxicity effects in living beings including humans and warrant removal from drinking water. In the present study, unknown second-order rate constants for the reactions of microcystin-LR (MC-LR), -RR and -YR with potassium permanganate were determined at pH 6.2–8.2 and temperature 10–25 °C. The reaction of permanganate with MCs is second-order overall and first-order with respect to both permanganate and toxin. The second-order rate constant for the reaction of MC-LR with permanganate at pH 7 and 20 °C was 357.2±17.5 M −1s −1. The influence of pH on the oxidation process was not appreciable and the activation energy was 28.8 kJ mol −1. Slightly higher reactivity with permanganate was found for MC-RR (418.0 M −1s −1) and MC-YR (405.9 M −1s −1). According to the results obtained, permanganate likely attacks the Adda moiety of the MC molecule. The oxidation of MCs in a natural surface water was also investigated. A permanganate dose of 1–1.25 mg L −1 was enough to reduce MCs concentration below the guideline value of 1 μg L −1. Permanganate oxidation is therefore a feasible option for microcystin removal during preoxidation processes. However, the oxidant dose must be carefully optimized in order to remove extracellular MCs without causing cell lysis (due to chemical stress) and further release of MCs.

Mechanistic studies of the photocatalytic oxidation of microcystin-LR: an investigation of byproducts of the decomposition process.

Microcystins (cyclic heptapeptides) are produced by a number of freshwater cyanobacteria and cause concern in potable water supplies due to their acute and chronic toxicity. The present study reports the structural characterization of the degradation products of the photocatalytic oxidation of microcystin-LR, so aiding the mechanistic understanding of this process. TiO2 photocatalysis is a promising technology for removal of these toxins from drinking water. However, before it can be adopted in any practical application it is necessary to have a sufficient knowledge of degradation byproducts and their potential toxicity. Liquid chromatography-mass spectrometry analysis demonstrated that the major destruction pathway of microcystin appears to be initiated via three mechanisms: UV irradiation, hydroxyl radical attack, and oxidation. UV irradiation caused geometrical isomerization of microcystin converting the (4E), (6E) of the Adda configuration to (4E), 6(Z) or 4(Z), 6(E). Hydroxyl radical attack on the conjugated diene structure of Adda moiety produced dihyroxylated products. Further oxidation cleaved the hydroxylated 4-5 and/or 6-7 bond of Adda to form aldehyde or ketone peptide residues, which then were oxidized into the corresponding carboxylic acids. Photocatalysis also hydrolyzed the peptide bond on the ring structure of microcystin to form linear structures although this appeared to be a minor pathway.

Total microcystin determination using erythro-2-methyl-3-(methoxy-d 3)-4-phenylbutyric acid (MMPB-d 3) as the internal standard

MMPB ( erythro-2-methyl-3-methoxy-4-phenylbutyric acid) from the Adda moiety of microcystin was determined by gas chromatography/chemical ionization mass spectrometry (GC/CI-MS) using MMPB-d 3 [ erythro-2-methyl-3-(methoxy-d 3)-4-phenylbutyric acid] as the internal standard. In the selected ion monitoring of GC/CI-MS, m/ z 223 for MMPB methyl ester and 226 for MMPB-d 3 were monitored; the retention times of the methyl esters of MMPB and MMPB-d 3 were almost the same. Methyl MMPB was determined from peak ratios of m/z 223/226. No peak interfered. The detection limit of methyl MMPB was 0.1 ng (0.43 pmol). Supposing the average of molecular weights of microcystin variants are 1000, the value (0.1 ng of MMPB) of the detection limit is equivalent to 0.43 ng of microcystin. These results indicated that this method is the most sensitive method for determination of total microcystin, and it could be applied to the monitoring of microcystins in drinking water, reservoirs and freshwater lakes. As an additional feature, this method need not use toxin (microcystin) standards.

Detoxification of Microcystins (Cyanobacterial Hepatotoxins) Using TiO2 Photocatalytic Oxidation

TiO 2 photocatalysis has been used to destroy microcystin-LR in aqueous solution. The destruction of this toxin was monitored by HPLC, and the disappearance was accompanied by the appearance of seven UV detectable compounds. Spectral analysis revealed that some of these compounds retained spectra similartothe parent compound suggesting that the Adda moiety, thought to be responsible for the characteristic spectrum, remained intact whereas the spectra of some of the other products was more radically altered. Six of the seven observed reaction products did not appear to undergo further degradation during prolonged photocatalysis (100 min). The degree to which microcystin-LR was mineralized by photocatalytic oxidation was determined. Results indicated that less than 10% mineralization occurred. Mass spectral analysis of the photocatalyzed microcystin-LR allowed tentative characterization of the reaction process and products. Reduction in toxicity due to the photocatalytic oxidation was evaluated using an invertebrate bioassay, which demonstrated that the disappearance of microcystin-LR was paralleled by a reduction in toxicity. These findings suggest that photocatalytic destruction of microcystins may be a suitable method for the removal of these potentially hazardous compounds from drinking water.