Degradation of microcystin-RR in water by chlorine dioxide

Available online at www.sclencedirect.comJOURNAL 0F CHIA UNIVERSTYOFScienceDirectMINING &TECHNOLOGYEL SEVIERJ China Univ Mining & Technol 18 (2008) 0623 -0628Degradation of microcystin-RR in water by chlorine dioxideJI Ying, HUANG Jun-li, FU Jiao, WU Ming-song, CUI Chong weiSchool of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin, Heilongjiang 150090, ChinaAbstract: Due to the potent hepatotoxicity and tumor-promoting activity of microcytins, a successful removal of these toxinsduring drinking water ureatment processes is of increasing concern. The oxidation kinetics of MC-RR by chlorine dioxide (CIO2)was studied with HPLC and characterization of the reaction products was performed with UV-spectrometry, TOC and LC-MS. Ourexperimental results show that the oxidation process is a second order overall and a frst order with respect to CIO2 and MC-RR.The activation energy of MC- RR degradation by C1O2 is 53.07 kJ/mol. The rate constant k of the action can be increased by in-creasing temperature and decreasing pH value and ranged from 6.11x102 L(mol min) to 5.29x102 L(mol-min) at pH from 3.44 to10.41 at 10 C. Reaction products were determined to be organic and volatile, because they could be almost removed from aqueoussolution by heating for 15 min at 60 C. In addition, the main oxidation products have m/z values of 1072 and are identified as di-hydroxy isomers of MC-RR.Key words: microcystin-RR; CIO2; reaction kinetics; UV spectrometry; TOC; LC-MS1 Introductionare extensively found in drinking water reservoirs ofmany countries such as Australia, Japan, Argentina,Blooms of toxic cyanobacteria (blue-green algae)German, Scotland, Switzerland and Chinal8. Thehave become increasingly common in surface waterWorld Health Organization (WHO, 1998) has set aof the world. They potentially threaten human healthprovisional guideline value for MC-LR (L: lysine, R:and are increasing a world-wide interest in the healtharginine) of 1.0 μg/L in drinking water. Traditionalrisk associated with cyanobacterial toxins. Of thewater treatment methods of chemical coagulation,known toxins produced by cyanobacteria, the micro-flocculation and sand filtration are often effective incystins (MCs) form the most severe threat to humanremoving cyanobacterial cells but are not effective inand animal health'- 4.removing and destroying cyanobacterial toxins'" .MCs are a family of highly specific cyclic hepta-The potential for these toxins to impact adversely onpeptide hepatotoxins and show potent hepatotoxicityhuman and animal health requires that these toxins beand tumor-promoting activity through inhibition ofremoved from water supplies prior to use.protein phosphatases 1 and 2AR 5. They have aboutMany methods have been examined in order to70 congeners with the general structure of cyC-remove these microcystins from water. The use 0lo-(D-alanine1-X2-D-McAsp3-Z4- Adda5-D- glu-activated carbons (PAC and GAC) is a very typicaltamate6-Mdha7) in which X and Z are variable Lphysical method-. They are efficient but have aamino acids'*. Adda is a characteristic B-amino acidlimited lifetime and are expensivell8 21. Biological(2S, 3S, 8S, 9S)-3-amino-9-methoxy-2, 6, 8-trime-methods have a relative long reaction time and arethyl- 10-phenyldeca-4(E)，6(E)-dienoic acid and iscomplex products'Compared with these meessential for the characteristic biological activities ofthods, chemical oxidation appears an effective suita-microcystins, because the toxicity disappears com-ble way to destroy the microcystins. Up till now,pletely by ozonolysis of the Adda portion and theUV/H2O2/TiO2, UV-radiation, ozonation, iron(II)geometrical isomers of Adda do not exhibit biologicalchloride, permanganate and chlorinationhave mainlyactivitys. Possibly as a result of their cyclic structure,been investigated, but for degradation of microcystinsMCs are chemically very stable and are only slowlyduring preoxidation and disinfection processes, ClO2decomposed by acid, alkali or boilingcould be taken as a feasible option'Microcystins produced by cyanobacterial bloomsChlorine dioxide (CIO2), a strong oxidizing and中国煤化工Reccived 18 January 2008; accepted 11 March 2008Projects 50178022 supported by the National Natural Science Foundation of China andMHC N M H Gnd Delopmaca Pogamof ChinaCorresponding author. Tel: +86 451-86282702; E mail address: jiyingdoctor@126.com624Journal of China Universty of Mining & TechnologyVol.18 No.4sanitizing agent with broad and highly effective in awith 10 mL portions of deionized water, 5% methanolbiocidal way, has about 2.5 times the oxidation caand a 10% methanol aqucous solution and then driedpacity of chlorine and does not react with some or-under a nitrogen flow for half an hour. MC-RR wasganic contaminants to produce harmful byproductsthen eluted out from the catridge using 5 mL oflike trihalogenmethanel5S3I.HPLC-grade methanol under vacuum at a flow rate ofOf over 70 different analogues of microcystins,1 mL/min. The eluate was dried in ntrogen flow andMC-_LR, MC-RR and MC-YR are the most commondissolved with 0.2 mL HPLC-grade methanol beforemicrocystins in blooms of toxic cyanobacteria in sur-being analyzed. The concentration of MC-RR wasface water of the world58l. Although MC-LR degra-analyzed using a Shimadzu LC-10A HPLC (LC-dation by chlorine dioxide had been reported, there10ATVP Plus， SPD- 10AVP(UV-VIS)) detector,was no thorough investigation about degradation by(CLASS-VP Workstation, auto sampler, 50 μ山) afterCIO2 of other MCs'39 40. In our study, the degrada-being pre-concentrated by solid phase extractiontion of MC-RR by chlorine dioxide was investigated,(SPE) and a reversed-phase Hypersil BDS C18 col-kinetics parameters were measured, the effects ofumn (5 um, 4.6 mmx250 mm). The HPLC conditionstemperature and pH were explored and the reactionwere as follows: the UV absorbance detection was setproducts were characterized.to 238 nm, the mobile phase was methanol -0.1%trifluoroacetic acid (TFA) (70:30， v/v) with a2 Experimentalflow-rate of 1.0 mL /min, the column temperature washeld at 25 C.2.1 Chemicals2.3 Experiments of reaction kineticsMicrocystin-RR (mw 1038.2, C4gHzsN;O12, puri-The MC-RR stock solution was obtained by dis-ty295%) standard was purchased from ALEXIS Bio-solving the MC-RRstandard in H2O-MQ. Experi-chemicals (San Diego, USA). All other chemicalsments with chlorine dioxide degeneration of MC-RRused were HPLC-grade or analysis-grade. All the so-were performed in airtight amber glass bottles in alutions were prepared in H2O-MQ. Chlorine dioxidejark place in duplicate. The operating parameters(aqueous) (purity298%) was generated in our labora-such as chlorine dioxide dosage, pH value, tempera-tory according to the method described by Huangture and reaction time were changed, respectively.The reactions were quenched with a drop of sodiumChlorine dioxide (aqueous) preparationthiosulfate solution at predefined times and the con-A solution of 25% NaC1O2 was introduced bycentrations of residual MC-RR in the reaction mix-pumping it at a feed rate of 2-3 mL/min into ature were analyzed by the SPE-HPLC method.gas- generating bottle containing 25% H2SO4 Thisbottle was connected to a chlorine scrubber bottle2.4 Characterization of reaction productscontaining a 10% solution of NaClO2. The scrubber1) Spectroscopic analysiswas connected to a chlorine dioxide collection bottleIn order to obtain spectroscopic characteristics offilled with de-ionized distilled water. At the end of theintermediates or products formed during the reactions,series, an additional chlorine dioxide trap bottle withhe reaction solution was scanned by a Shimadzu10% KI was present to trap any remaining chlorineUV-2550 UV-Vis spectrophotometer. The solutiondioxide. The stock solution of chlorine dioxide was ofwas scanned in time at wavelengths ranging from 200an average purity of 98% and stored in a closed con-to 900 nm in order to monitor the changes in thetainer at 4 C in the dark to prevent light decomposi-spectrum over the course of the reaction.tion of the CIO2.2) TOC analysis2.2 Analytical methodsThe MC-RR solutions heated at 100 C were re-spectively detected at 0, 10 and 20 min by the Shi-Analysis of chlorine dioxide (aqueous)madzu TOC-VCPH TOC detector that has a determi-A sequential iodimetric method was used to ana-nation limit of 5 ug/L. The reaction solution obtainedlyze the concentration of CIO2, Cl2, CIO2 and CIO3~from MC-RR acting with CIO2. completely heated atin water by changing pH value according to the me-60 C, were respectively detected at 0, 5, 10 and 15thod described by Huang Jun-1ilS- 36].min by the Shimadzu TOC-VCPH TOC detector.SPE-HPLC analysis of MC-RR3) LC-MS analysisThe samples should be pre- concentrated by solid-The MS analysis was accomplished using a Finni-phase extraction (SPE) due to the trace concentrationgan LCQ Deca XP plus MS (Thermo Fisher Scientif-of MC-RR. The SPE cartridges (Isolute SPE columns,C, |xed with an electro-1 g of C18 (EC)/6 mL) were conditioned with 10 mLspr中国煤化工The ESI cnditionsof methanol followed by 10 mL of Milli-Q water. TheC N M H Gas fllws: caillaysamples were applied via Teflon tubes at a flow ratetempclaule JuU L, siicaul gas l10W rate 35 (arbitraryof 3 mUmin. Then the cartidge was rinsed, in turn,unit); ESI source voltage 5 kV; capillary voltage 43 V,JYingetalDegradation of microcystin-RR in waler by chorine dioxide_625scan range 100 to 1200 m/z.relations between In(C/C) and reaction time; the de-gradation process is very well expressed with this3 Results and discussionpseudo-first- order rate equation. The order of MC-RRwere first order reactions and the slopes of the lines3.1 Kinetics of the reactions of CIO2 with MC-are the pseudo-first order rate constants. The calcu-RRlated kinetics parameters are presented in Table 1.3.1.1 Determination of order of reactions and reac-The results show that the corresponding correlationtion rate constants kcoefficients R' were all more than 0.99 and the pseu-Determination of reaction order was according todo-first rate constants of MC-RR degradation bythe method of the. law of isolated parameters, de-CIO2 increased along with the increase in concenta-scribed by Pilling41.tion of CIO2, but their half-lives decreased.The rate of oxidation of MC-RR by ClO2 can beTable 1 Parameters of pseudo-first-order kinetics at diferentwitten as Eq.(1)initial C1O2 concentrations at 10 C and pH 6.48r=-d[MC- RR]=k[MC- RR][ClQ,]' (1)[MC-RR]o[C1O)]o(min)(ug/L)(mg/L)where r is the reaction rate, k2 is the rate constant,1000.0705.80.9965[MC-RR] and [ClO2] are the concentrations of1015380.087379 0.994423070.1319s30.997MC-RR and CIO2, respectively. x and y are the orders2030760.17370.9959of the reaction with respect to each reactant. In thesituation where the concentration of ClO2 is excessive,In Fig. 2, the experimental data of k\ and [CIO2]Eq.(1) can be simplified to Eqs.(2) and (3)were fitted with a linear equation, the corresponding= k[MC- RR]' (2)correlation coefficient R' was 0.9998 and the rela-dttionship of k1-[CIO2]o was similarly linear, based onk = k,[ClO2]’(3)Eq.(3), kn=k2[ClO2}', so the order of CIO2 was a firstorder reaction and the total order was second orderEq.(2) can be witten as Eq.(4)reaction in the degradation process. The second orderreaction rate constant kz can be obtained from the=kdt(4)[MC- RR]*equation k2= =k/[CIO2]o. The slope of the line was theIfx is 1, then the integration of Eq.(4) leads torate constants kz = 5.83x102 L(mol-min).Eq.(5)0.2 rIn [MC-RR][MC- RR]°=kt(5)The initial concentration of MC-RR was 100 ug/L.0.1 tThe dosages of CIO2 were 8, 10, 15 and 20 mg/,s'which were over-doses in the degradation reaction.05 tand be considered as constants.6「[CIO2] -20 mg/L00.00015 0.0003 0.00045CIO2 (molL)Fig. 2 Determination of reaction order of C1O2 and;treaction rate constantsk2 at 10 c and pH 6.48[CiO2}=8 mg/L3.1.2 Detemination of activation energy En[CIO2]=10 mg/LThe reaction rate constants at 5, 10, 15, 20 and(ClO2] =15 mgL25 C were confirmed by the same methods and theactivation energy was determined by the linearized10 20304050 607(Arhenius Eq.(6)1 (min)Ink, =lnA- E. /RT(6)Fig. 1 Pseudo first order kinetic plot for the oxidationof MC-RR by CIO2 at 10 Cand pH 6.48where kr is the rate constant, A the frequency factor,Ea the activation energy (kJ/mol), R the universal gasIn Fig. 1, the solid lines are linear regressions ofconstant 8 214 Vmn1/K _d T the absolute tempera-the data. Initial concentrations: [MC-RR]o= 100 ug/L.ture中国煤化工From Fig. 1, it can be seen that points lie satisfactori-: second-order rately around straight lines, which are linear regressionsconsTHCNMHGreaction shows aof the data of In([MC- RR]/[MC-RR]) as the depen-positive temperature dependence. There is a lineardent variable versus time. There are, therefore, linearrelation between n(k2) and (-1/RT), with a corres-Joural of China University of Mining & TechnologyVol.18 No.4ponding correlation cofficient R2 of 0.99. The slope14of the regression line is the activation energy, which120.was 53.07 kJ/mol. We conclude that CIO2 can remove1.0瓷0.MC-RR effectively under conditions of traditionalwater treatment.0.8马0.0} .g0.6|.0「200300400 500.5 t0.4Wavelength (nm).0 t02 |200300400500600700800900Fig. 5 Spectral changes during the oxidation of MC-RR50004-0.0004by CIO2 at initial MC- RR concentration of 2 mg/L-0.00and CIO2 concenration of 10 mg/L at 15 C.-1/RT (mol/D)3.3 TOC analysisFig.3 Determination of activation energy EIn Fig. 6 it can be seen that TOC (total organic3.13 Effect of pHcarbon) and TC (total carbon) of the MC-RR solu-In Fig. 4, pH was found to have a moderate effecttions and reaction solution, obtained from MC-RRon the reaction of MC-RR with CIO2. The reactionacting with CIO2, completely changed with the hea-rate slightly decreased with increasing pH. The rateting time.constant kz decreased from 6.11x10- L(mol-min) toThe 200 μg/L MC-RR solutions heated at 100 C5.29x102 L/(mol-min) at pH 3.44 to 10.41 at 10 C.were respectively detected at 0, 10 and 20 min by theThe results indicate that CIO2 can be effective over aShimadzu TOC-VCPH TOC detector which showswide range of pH values.that its determination limit is 5 ug/L. TOC and TC arealways equal in value and the IC (inorganic carbon)values are always 0, during the 20 min of heating.s 45TOC and TC did not change and remained equal con-stants, so we concluded that MC-RR was not re-moved from the water by heating for 20 min at是150After 200 ug/L MC-RR reacting completely with10 mg/L CIO2, the reaction solutions, heated at 60 C,phwere respectively detected after 0, 5, 10 and 15 min.Fig. 4 Effect of pH value on reaction rate constant krTOC and TC remained equal in value among all thesamples detected and no inorganic carbon was pro-3.2 Spectroscopic analysisduced in the oxidation reaction, i.e, the IC values ofThe changes in the spectrum during the oxidationthe reaction solutions detected before and after heat-of MC-RR with CIO2 were investigated. The spectraling were always 0. The TOC values of the reactionchange in the course of the reaction is presented insolution decreased with an increase in heating timeFig. 5. Obviously, line 1 is the curve of the MC-RRand after 15 min were below the detection limit whensolution UV scan with its characteristic peak at 240heated at 60 C. With these results, the reaction prod-nm for MC-RR and the characteristic peak of theucts were determined as organic compounds and veryreaction solution is at 360 nm. Because of the pre-volatile, because they could be almost removed fromthe aqueous solution by heating for 15 min at 60 C.sence of CIO2 from line 2, the adsorption intensity at360 nm decreased gradually as the reaction proceeded100and the concentration of CIO2 decreased as shown byR 80a TC (MC-RR)lines3,4,5and6,butinlines5and6thenewab-Q TC (reaction solution)sorbance peak can be observed in the region of日TOC (MC-RR)266- -292 nm. The absorbance increased as the reac-0 TOC (reaction solution)tion proceeded, at the end of the reaction. The only2peak in the region of 266 -292 nm can be seen in line7. We can conclude that the peak in the 266 -292 nm中国煤化工region is probably the characteristic peak of the reac-tion products. The peak disappeared after heating forFig. 6JYHC N M H G.RR slumios beated15 min at 60 C as shown in line 8 and reactionat 100 C and the reaction solution of 200 ug/L MC-RR actingproducts are probably very voltile.with 10 mg/L CIO2 heated at 60 c versus timeJ Ying etalDegradation of microcystin-RR in water by chlorine dioxide6273.4 LC-MS analysisMC-LR and Cl2， dihydroxymicrocystin formedThe reaction products of MC-RR and CIO2 werethrough chloronium ion at the conjugated diene ofcharacterized by LC-MS and one oxidation product,Adda, followed by hydrolysis. Two hydroxyl groupswith a m/z value of 1072, could be determined. Manywere oxidatively added to the diene group of Adda'studies on the oxidation of MCs indicated that theThese are very similar to alkene or diene becausetwo double bonds of the Adda group of the MC-RRMCs almost show the same behaviour as that of al-structure.. were.. changed in the oxidation reac-kene or diene under the same oxidation conditions.tion'-” 9.42- 45. It may be the formation of MMPBThese results strongly suggest that the oxidationwhich contains the carboxylic acid function groupproducts, with an m/z value of 1072, are dihydroxyfrom. the MC-RR oxidation by potassium permanga-isomers of MC- RR and that MC-RR was added to thenatel50.45. Chlorine dioxide acting with the MC-LRtwo hydroxyl groups of the diene group of Addacould produce the dihydroxy isomers of micro-(1038+2x17=1072) whose structural formulas arecystin-LR by the attack of CIO2 toward the conju-presented in Fig. 7.gated double bonds in the Adda59. In the action ofHa pOoH HqH.pooH HqHOC HoH, HmL是a是d入rCooM。C00'R(a) MC-RR6) Dihydroxy isomer of MC-RR(c) Dihydroxy isomer of MC-RRFig. 7 Structural formulas of MC-RR and dihydroxy isomers products of MC-RR4 ConclusionsReferencesThe overall degradation process could be satisfac-[1] Tllett D, Ditmann E, Erhard M, et al. Structural or-torily fitted by second order kinetics. The rate con-ganization of microcystin biosynthesis in microcystisstant kr of the MC-RR action with CIO2 at 10 C andaeruginosa PCC7806: an integratecpeptidpolykketidepH 6.48 was 5.83x102 L(mol-min). The reactions forsynthetase system. Chemistry & Biology, 2000, 7(10):753- -764.CIO2 and MC-RR were both first order reactions; the[2] Tsuji K, Watanuki T, Kondo F, et al. Stability of mocro-activation energy of MC-RR degradation by C1O2cystins from cyanobacteria-IV. efect of chlorination onwas 53.07 kJ/mol. The rate constant kz was positivelydecomposition. Toxicon, 1997, 35(7): 1033- -1041.correlated to temperature and negatively with pH,[3] Mikhailov A, Harmil B A, Meriluoto J, et al. Produc-ranging from 6.11x102 U(mol-min) to 5.29x102tion and specificity of mono and polyclonal antibodiesagainstmicrocystinsconjugatedthroughL/(mol-min) for MC-RR at pH 3.44 to 10.41 at 10 C.N.methldehyydroalaninnine. Toxicon, 2001(39)39): 477- 483.Given the analysis of TOC and TC, the reaction[4] Shi HX, QuJ H, Wang A M, et al. Degradation of mi-products were determined as organic compounds. Incrocystins in aqueous solution with in situ electrogene-addition, we concluded that the reaction productsrated active chlorine. Chemosphere, 2005(60): 326 333.were very volatile because they could be almost re-[5] Harada K I, Imanishi S, Katob H, et al. lsolation of addamoved from the aqueous solution by heating for 15from microcystin-LR by microbial degradation. Toxicon,200(44): 107- -109.min at 60 C. The LC-MS analysis revealed that the[6] Lahti K, Rapala J, Fardig M, et al. Persistence of cyanomain oxidation products had m/z values of 1072 andbacterial hepatotoxin microcystin-LR inparticulate ma-were identified as dihydroxy isomers of MC-RR.terial andsolvedin lake water. Water Research,Based on these results, ClO2 can be accepted as an1997(31); 1005-1012.effective agent for removing MC-RR from drinking[7] HaradaK L, Tsuji K, Watanabe M F, et al. Stability ofmicrocystins from cyanobacteria-II. effect of pH andwater resources in traditional drinking water supplies.temperature. Phycologia, 1996(35): 83-88.[8] Hoeger S I, Shaw G Hitzfeld B C, et al. Occurrence andAcknowledgementselimination of cyanobacterial toxins in two australian中国煤化工C xicon, 2004(43): 639-Financial support by the National Nature Science[9C N M H Gf microcystins and itsFoundation of China (No.50178022) and the national863 projects (No.2006Z3022) is gratefully acknowl-uowoiupusiuon pIvuuLi . maner treatment process.Chemosphere, 1995, 31(7): 3635- -3641.edged.