Recent advances in the sample preparation, liquid chromatography tandem mass spectrometric analysis...

Trends Trends in Analytical Chemistry, Vol. 24, No. 7, 2005

Recent advances in the samplepreparation, liquid chromatographytandem mass spectrometric analysisand environmental fate ofmicrocystins in waterSandra Perez, Diana S. Aga

This review article covers recent developments in the analysis of micro-

cystins (MCs), the natural toxins produced in cyanobacterial blooms that

occur in many eutrophic waters. We report applications of new extraction

methodologies, such as immunosorbents for sample preparation, and current

advances in liquid chromatography with tandem mass spectrometry for

detection and identification of new transformation products.

Due to the complex nature of MCs, there is a growing interest in analyzing

MCs and characterizing their transformation products. The widespread

occurrence of toxic cyanobacterial blooms in aquatic resources that are used

for drinking water supply has raised public health concern. In addition, the

presence of MCs in surface waters has resulted in poisoning animals and

killing fish worldwide.

For this reason, we review the fate of MCs in surface waters, and include

current knowledge about how various environmental conditions, such as pH,

temperature and sunlight, influence the biodegradation of these toxins under

natural conditions.

Lastly, we also discuss studies that investigate the potential removal

mechanisms of MCs from drinking water supplies, such as advance oxidation

processes.

ª 2005 Elsevier Ltd. All rights reserved.

Keywords: Biodegradation; Fate; Liquid chromatography-mass spectrometry; Microcystins;

Photodegradation; Stability

Sandra Perez, Diana S. Aga*

The State University of

New York at Buffalo, Chemistry

Department, 611 Natural

Sciences Complex, Buffalo,

NY 14260, USA

658

*Corresponding author.

Tel.: +1 716 645 6800x2226;

Fax: +1 716 645 6963;

E-mail: [email protected]

1. Introduction

In recent years, there have been bloomepisodes of toxic cyanobacteria in surfacewaters that have caused a variety ofwater-quality problems. Toxin production,odor, scum and possible unsafe water,leading to death of wild and domesticanimals worldwide, and potential threatsto human health are the main conse-quences of the blooms [1].

0165-9936/$ - see front matter ª 2005 Elsev

Cyanobacteria are common consti-tuents of waters with varied organic andionic composition and salinity. The condi-tions that induce their formation aresunlight, moderate-to-high nutrient concen-trations (notably phosphorous and nitro-gen), water temperature of 15–30�C andpH > 6 [2], as well as thermal stratifica-tion and water-column stability that reg-ulates the buoyancy of these species [3].Under favorable conditions, bacterialnumbers multiply rapidly, doubling in oneday or less, and the bloom can remain forseveral weeks. The algal bloom commonlydisappears with a drop in temperature inearly autumn [4], high solar irradiance, orthe combination of high solar irradianceand high concentration of metals (i.e.iron) in the water [3]. Algal bloomsare triggered by contamination fromhuman inputs, and, since there is noindication of any decline in humanactivities, the problems of cyanobacterialblooms on surface waters are set tocontinue.

The cyanobacteria can produce differentkinds of compounds that are classified ascyclic peptides (i.e. hepatotoxins), alka-loids (i.e. neurotoxic compounds), lipo-polysaccharides and other bioactivecompounds. Microcystins (MCs) are one ofthe most frequently reported hepatotoxinsin surface waters. They were named in1988 after cyanobacteria Microcystisaeruginosa, but they can also be produced

ier Ltd. All rights reserved. doi:10.1016/j.trac.2005.04.005

mailto:[email protected]

Trends in Analytical Chemistry, Vol. 24, No. 7, 2005 Trends

by other genera – Nostoc spp., Oscillatoria agardhii,Anabena flos-aquae, Microcystis viridis and terrestrialHapalosiphon [1,5]. MCs are present inside cyanobacterialcells and enter surrounding water after lysis. The factorsthat trigger the formation of MCs and their role in cyano-bacteria remain unclear, though they are supposed toact as protective compounds against grazing zooplank-ton [6]. Some authors have reported that change intemperature decreases the amounts of MCs, dependingon which kind of cyanobacteria formed them (highercontent of MCs in Anabena at 25�C and in M. aeruginosaat 20–24�C) [7,8].

MCs are cyclic heptapeptides with over 75 structuralvariants identified, varying largely in peptide sequence,the degree of methylation and toxicity. Some of them arepotent and specific inhibitors of protein phosphatases,

HN

N

Z

H3COA1

CH3 CH3

Adda (4):3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid

D-Glu (3, iso)

L-amino acid (5)

Microcystin X Z A1 A2

LA Leu (L) Ala (A) CH3 CH3

AR Ala (A) Arg (R) CH3 CH3

LF Leu (L) Phe (F) CH3 CH3

D-Asp-LR Leu (L) Arg (R) CH3 H

Dha-LR Leu (L) Arg (R) CH3 CH3

DMAdda-LR Leu (L) Arg (R) H CH3

[D-Leu1]-LR Leu (L) Arg (R) CH3 CH3

LR Leu (L) Arg (R) CH3 CH3

LW Leu (L) Trp (W) CH3 CH3

FR Phe (F) Arg (R) CH3 CH3

RR Arg (R) Arg (R) CH3 CH3

YR Tyr (Y) Arg (R) CH3 CH3

WR Trp (W) Arg (R) CH3 CH3

1 MW: Molecular weight

Figure 1. Structures of the most c

lethal to many kinds of aquatic organisms and damagingzooplankton, fish and the liver of higher animals [9].These cyanotoxins share the general structure (Fig. 1)depicted in the cyclo compounds [commonly DD-alanine(1), N-methyldehydroalanine (2), DD-glutamic acid (3), avariable LL-amino acid (R2) (5), b-linked DD-erythro-b-methylaspartic acid (6), a variable LL-amino acid (R1) (7)]and a unique amino acid, named Adda [(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-(4E),(6E)-dienoic acid] (4).

MCs are neutral or anionic in environmental waters.The physical properties of MCs were calculated experi-mentally by Rivasseau et al. [10]. They determined that,while the solubility for MC-LR was higher than 1 g/L, thehydrophobicity (logKow) for the three most frequentlyfound MCs in surface water were: MC-RR (logKow 4.4);

N

NHO

CH2

OO

O

O

H

NHX

A3

COOH

A2

COOA4

A5

D-erythro-MeAsp (6, iso)

L-amino acid (7)

Mdha (2):N-methyldehydroalanine

D-Alanine (1)D-Leu

A3 A4 A5 MW1 (Da)

CH3 H CH3 909.5

CH3 H CH3 952.5

CH3 H CH3 985.5

CH3 H CH3 980.5

H H CH3 980.5

CH3 H CH3 980.5

CH3 H CH2CH(CH3)2 980.5

CH3 H CH3 994.5

CH3 H CH3 1024.5

CH3 H CH3 1028.5

CH3 H CH3 1038.5

CH3 H CH3 1044.5

CH3 H CH3 1067.5

ommon microcystins (MCs).

http://www.elsevier.com/locate/trac 659


MC-YR (3.9); and, MC-LR (4.2). The logKow are quitehigh because the Adda moeity increases the hydro-phobicity of the whole MC molecule. However, since MCspossess polar groups, such as carboxylic acids, aminoand amido groups, they behave differently from non-polar organochlorine compounds. The ionization of theMC depends on the pH, with pKa of 3.4 for MC-YR and3.3 for MC-RR and MC-LR [10].

This review summarizes new analytical methods andthe fate of MCs in surface waters. It discusses the sam-pling procedures, sample handling and extractionmethodologies applied for the determination of MCs fromaqueous samples, and the application of liquid chroma-tography tandem mass spectrometry (LC–MS2) for sep-aration and detection of these compounds and theirdegradation products. The stability of the MCs in theenvironment under sunlight, advanced oxidation pro-cesses, and biodegradation, and the effect of pH andtemperature are also summarized.

Water

LC-MS, LC-MSCE-UV, E

Algae cell (MCIntracellular)

Filtration

SonicaLyophiliz

Freeze-th

SPE Solvent ex

Evapor

FiltratiCentrifug

Dissolved phase (MC Extracellular)

A

B

A, B, C

A, B, C

A

Figure 2. Schematic diagram of sample-treatment procedures ap

660 http://www.elsevier.com/locate/trac

2. Analysis and occurrence of MCs in waters

The methodologies for the analysis of MCs in watersencompass a variety of extraction and detectionmethods. In the following section, several methodologiesare described, showing the state-of-the-art in MCanalysis in water samples and addressing the novelanalytical methods recently reported in the literature.

2.1. Sampling and sample preparationThe various approaches commonly used for determiningMCs in waters are summarized in Fig. 2. Prior toextraction, the selection of sampling sites and techniquesis crucial. Sampling is normally performed after the algalbloom during late summer and early autumn becausethe MC are in higher concentrations then. The samplingpoints have to be representative of the whole water body,taking into account the horizontal and the verticalvariations due to thermal stratification that usually

sample

-MS, LC-UV,LISA

Total MCconcentration

tionationawed

traction

ation

on/ation

C

A, B, C

A, B, C

A, B, C

A, B, C

C

plied to the determination of microcystins (MCs) in waters.


results in inhomogeneous distributions of populations ofcyanobacteria. Moreover, the selection of the sites shouldbe done in areas where accumulations of cyanobacteriacan affect both humans and livestock, or they should betaken at the raw-water intake of drinking water reser-voirs. The sampling of MCs from scums and water withalgae should be performed with a glass bottle or grabsampler and for the analysis of the MC content in thealgae with a plankton net.

As MCs isomerize under the influence of UV light,samples contaminated with MCs should be collected inamber glass bottles and filtered or extracted immediatelyin the laboratory in order to avoid changes in concen-tration due to lysis of the algae.

Regarding sample manipulation, Hyenstrand et al.[11] reported losses of MC-LR from aqueous solution byadsorption in plastic devices. They recommendedavoiding unnecessary dilution of MCs with pure water,minimal transfer procedures with plastic pipette tips andrecognition of the potential losses of aqueous MC-LR toglassware and plasticsware.

2.2. Extraction and purificationThe sample pre-treatment of water samples containingMCs sometimes includes a filtration step to separate thealgal cell from the water in order to differentiate intra-cellular and extracellular concentration of MCs. If thetotal concentration of MCs is desired, the sample issubjected to ultrasonication, lyophilization and freeze-thawing to break the algae and obtain the free MCs,released from the disrupted cells [12–14].

Different solvents were tested for the extraction of MCsfrom algal cells. One of the main problems is that cyano-bacteria are protein-rich so, depending on the solventused for the extraction, the extract can have a highprotein content. Extraction with pure water resulted inthree times more protein extracted as compared with theuse of diluted acetic acid, whereas methanol extractionsuppressed water-soluble protein materials [15].

Although MCs are highly soluble in water, otherauthors tested mixtures of organic solvents and water orpure solvents. Dahlmann et al. [16] found that a meth-anol–water mixture (50:50, v:v) was effective forextracting six MCs from algal cells obtaining more than90% recovery in spiked filters. In another study [17],methanol was found to be the most versatile solvent forextracting MC-LR, MC-LY, MC-LW and MC-LF.

The pH also appears to be an important factor forthe extraction of MCs from algal cells. Because MCscontain two ionizable carboxyl groups and some ion-izable amino groups, different dominant species arefound at different pH. The relationship between pHand extraction efficiency has been not been studiedextensively and contradictory results have beenreported. Van der Westhuizen and Eloff [18] reported

that the best recovery of MC-RR was achieved at pH10, whereas the recoveries using more acidic mediatended to be lower. However, other authors [10]showed that the solubility of MCs increased in meth-anol acidified with 1% trifluoroacetic acid. There is notmuch information about the physical properties ofMCs; however, one work [19] investigated the changesof MC partitioning between octanol and water withvarying pH. They found that, at low pH (= 1), MC-LRtend to be highly hydrophobic. This can explain thelow recoveries found by van der Westhuizen and Eloff[18] as, at low pH, MC-LR becomes increasinglyhydrophobic and thus less soluble in acidic media.

For extracting dissolved MCs from waters, after dis-ruption of algal cells or from filtered waters, the mostcommonly used method is solid-phase extraction (SPE)because it provides a suitable method for the simul-taneous extraction and clean-up from the aqueoussamples. Various kinds of packing materials and solventshave been tested for this purpose; however, the octadecylsilica (C18) and methanol (or aqueous mixtures withmethanol) is the most frequently used combination,showing recoveries higher than 85% [20–22]. A fewstudies used acidified solvents, [i.e. employing methanolwith 0.1% trifluoroacetic acid (TFA)], Ortea et al. [14]resulting in satisfactory recoveries. Rivasseau et al. [10]showed that methanol–water (40:60, v:v) was suitablefor the extraction of MCs by SPE C18 cartridges, al-though MCs increased the solubility in methanol acidi-fied with 1% TFA.

However, Rivasseau et al. [10] reported that, forextracting from drinking and river water, neutral pHprovides satisfactory recovery (75–80%) with lessorganic matter extracted (co-extraction of fulvic andhumic acids takes place at low pH). Maizels and Budde[23] used pH 10 for extracting MCs from deionizedwater, obtaining recoveries of 75–98%.

Although SPE with conventional cartridges has beenused successfully, immunosorbents and molecularlyimprinted polymers (MIPs) have been recently developedto improve the selectivity of the extraction and clean-upof water samples containing MCs. Aranda-Rodriguezet al. [24] reported the comparison of the extraction andclean-up of six MCs between two different immuno-sorbents containing anti-MC-LR polyclonal antibodiesand current SPE performed with a macroporouscopolymer sorbent named HLB (hydrophilic–lipophilic-balanced) in cartridges. Recoveries for individual MCswith immunosorbents (>85%) were comparable to thoseobtained with SPE (>90%). The advantage of immuno-sorbent extraction versus conventional SPE was that theimmunosorbent extracts were free of interferences, andthat enabled better detection and identification of MCs.In another work [25], the extraction of MC-LR withMIPs was reported, showing recoveries over 66%. Thesenew extraction methodologies improve the selectivity of



the extraction and clean-up of the MCs from differentmatrices and permit rapid processing of water samples.

2.3. DetectionThe detection methods commonly used for the determi-nation of MCs in waters are summarized in Table 1.There are a variety of analytical techniques for MCs,including bioassays and chemical methods. Some bio-assays are highly specific and detect only a single toxinand that sometimes poses some drawbacks. For example,the mouse bioassay most widely used for monitoringMCs has limited sensitivity and poor reproducibility, inaddition to being an animal-based method. The MC-dependent phosphatase assay, another example of bio-assay that is used for the analysis of MCs, sometimesprovides false positives or does not allow detection of

Table 1. Methods for the analysis of microcystins in waters

Compound Matrix Extraction proc

RR, LA Lake water (filtered algal cells) Freeze-thawed(C18 cartridges

LR Lake water (algal cells) Freeze-drying,

LR, XR, LW Lake water (lyophilized algalcells)

Sonication

LR Lake water (lyophilized algalcells)

Sonication, SPE(C18 cartridges

[DD-Leua]-LR,a LR,YR, LA, WR FR

Lake water (lyophilized algalcells)

Solvent extract(C18 cartridges

LR, RR, YR, LW, LF Rivers, treated effluent fromWWTPb

SPE (C18-RPS d

LR, RR, YR River water (lyophilized algalcells) dissolved phase

Solvent extractSPE (C18 cartriand ISd)

LR, RR, YR River and reservoir water: filteredalgal cells, dissolved phase

Solvent extract

LR Water from a reservoir Sonication

LR, RR, YR, LA, LW,LF

Marine and lake waters (filteredalgal cells)

Sonication

LR, RR, YR, LW, LF,LY

Freshwater and brackish water(filtered algal cells)

Sonication

LR, DD-Asp-LR,a

Dha-LR,aDMAddaa-LR,M-glu-LR,a AR,LW, VF,LF, LY

Cell cultures Sonication, SPE(C18 cartridges

LR, RR, YR Spiked surface water SPE (SDB-RPSLR Spiked surface water Sonication, SPE

aDD-Asp-LR, Dha-LR, DMAdda-LR, M-glu-LR, [DD-Leu]-LR (see Fig. 1).

bWWTP: Waste water treatment plant.cSPE: Solid phase extraction.dIS: Immunosorbents.


very low concentrations. Furthermore, false negativesmay also occur using the mouse bioassay, as reported bySim and Mudge [26]. However, the bioassays are veryuseful for evaluating water safety, as they show theoverall toxicity of MCs.

The enzyme-linked immunosorbent assay (ELISA) hasalso been reported as a sensitive technique for MCdetection using both monoclonal and polyclonal anti-bodies. Fischer et al. [27] developed an ELISA usingpolyclonal antibodies and reported limits of detection(LODs) below 1 lg/L in drinking waters, which is thevalue proposed by the World Health Organization(WHO), without any sample preparation or preconcen-tration steps. However, the immunoassays do not pro-vide precise quantitative analysis of samples containingfamilies of MCs with different levels of single toxins.

edure Separation/detectionmethods

Detection limit Ref.

, SPEc

)LC–UV Not reported [14]IT/MS (+ESI) 0.1 ng (abs)Protein phosphataseassay

SPE (C18) LC–MS (+ESI) Not reported [21]TOF-MS (+ESI)UVIT-MS (+ESI) 250 pg (abs) [62]

)LC–UV Not reported [28]ELISA

ion, SPE)

LC–MS2 (+ESI) Not reported [29]

ELISAProtein phosphataseassay

isk) LC–MS (+ESI) 0.5 ng (abs) [23]

ion LC–MS (+ESI), UV Not reported [32]dges CE-CEC-UV

ion LC–MS (+ESI) 6–72 pg (abs) [34]

LC–MS (+ESI) 100 lg/L [2 ng (abs)] [12]

LC–MS, MS2 (+ESI) 0.5–1.0 ng (abs) [16]

LC–MS, MS2 (+ESI) 0.2 lg/L [13]

ELISA

)CE-MS, MS2 (+ESI) 200 lg/L [33]

disk) LC–MS (+ESI) Sub-ng/L level [63](HLB) IT-MS (+ESI) 2.6 ng/L [20]


ELISA can be used only as a screening assay eliminatingnegative samples for further chemical analysis. An ELISAdeveloped for the detection of MCs using monoclonalantibodies reported variability of MC concentrationsduring eight months using the direct assay [28].

The chemical methods used for determining MCs inwaters are mostly liquid-based separations combinedwith an ultraviolet (UV) or MS detector. The UV detectorpresents some disadvantages over the MS, because UV isnot sufficiently sensitive and selective (many neutralinterferences absorb in the same UV region as the MCs);moreover, the 60 MCs have very similar UV spectra.However, MS allows for identification of MCs and theirdegradation products in waters, and both sensitivity andselectivity can be improved using selected ion monito-ring (SIM), selected reaction monitoring (SRM), ormultiple reaction monitoring (MRM). For example, Spoofet al. [13] improved sensitivity in the analysis of MCs inalgal cells [13] working with MS2 (+ ESI) and as didOrtea et al. [14] by using LC-ion-trap (IT)-MS (+ ESI).Whereas previous authors had reported LODs of 0.2 lg/Land 0.1 ng (abs) using MS2 for MC-LR in lyophilizedcells, higher LODs (100 lg/L) were reported byRuangyuttikarn et al. [12] using LC–MS (+ ESI) for thedetermination of MC-LR in the same matrix. The identi-fication of the demethylated MC-LR was performed byLC-triple quadrupole [MS2 (+ ESI)] [29] and the deme-thylated and didemethylated MC-RR were identified bySpoof et al. [13] also using triple quadrupole [MS2

(+ ESI)]. LC-triple quadrupole [MS2 (+ ESI)] was also re-ported as being useful for the analysis of previouslyunidentified MC species in environmental samples [22].

Yuan et al. [30] compared the fragmentation patternof seven MCs with LC–MS (+ ESI). The mass spectra of[M + H]+ ions of MCs revealed two different types offragmentation pattern, with differences between the Arg-containing compounds (MC-LR, -RR, -YR) and MC-LA,which had no Arg residue. The differences would be

Table 2. Characteristic fragment ions observed for different microcystins a

MC-LRa MC-RR

[M + H]+ 996 –[M + 2H]2+ – 521PhCH2CH(OMe)+ 135 135[Ala-(Tyr–C6H6O) + H]+ – –[C11 H14O + H]+ 163 163[Glu-Mdha + H]+ 213 213[Ala-MAsp-(Tyr–C6H6O) + H]+ – –[C11 H14O-Glu-Mdha + H]+ 375 375[C11 H14O-Glu-Mdha-Ala + H]+ 446 446[Mdha-Ala-Tyr-MAsp-Arg+H]+ – –[M-(Tyr–C6H6O) + H]+ – –

In the column of fragment ions above, ‘‘-’’ denotes chemical bond, ‘‘–’’ daRef. [22].bRef. [16].cRef. [33].

ascribable to the different protonation sites in [M + H]+

ions. While the site of protonation for MCs containingArg was in this residue, the methoxy group in the Addaresidue was protonated in MC-LA. Using LC–MS2 (+ ESI),Humert et al. [22] also found differences in the massspectra between MC-LR and MC-YR due to their differ-ences in structure. Besides detecting typical fragmentions of MCs in the mass spectra of MC-YR (m/z 135 andm/z 213), other fragment ions, such as m/z 141, m/z271, m/z 602 and m/z 952, were characteristic of MC-YR (Table 2). The (+)-ESI mass spectra of MC-RR, MC-YR, MC-LR and MC-WR generated on an LC-IT-MS(+ ESI) are shown in Fig. 3. In the MS2 spectra of theprotonated and deprotonated molecules of MC-YR, MC-LR and MC-WR, the same fragmentation pathways werepresent. The fragment ions observed for these three MCsarise predominantly from the loss of water and thenconsecutive cleavages of the amide bonds that provideinformation concerning the amino-acid sequences. MC-RR presented a different fragmentation pattern due tothe formation of [M + 2H]2+ instead of [M + H]+. Otherimportant fragment ions, including [Arg-Adda-Glu + H]+ with m/z 599, were found in each spectrum.

Welker et al. [31] described the application of matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF-MS) in the analysis of MCs inwaters. MALDI-TOF-MS proved to be a reliable, rapidtool to detect and to identify MC variants using post-source-decay fragmentation. For monitoring MCs inenvironmental samples, MALDI-TOF-MS can provideconsiderable support to HPLC by identifying MCvariants not available as purified standards, especially inPlanktothrix-dominated blooms, in which demethylatedvariants predominate.

Capillary electrophoresis (CE) is not a commonly usedmethod for the analysis of MCs because it requires samplepreparation to achieve the required LOD. In one work,CE-UV was used for the analysis of MC-LR in waters [32].

nalyzed by LC–MS2 (ESI+)

b MC-YRa MC-LWc MC-LFc

1046 1026 987– – –135 135 135141 – –

– 163 163213 213 213271 – –

– 375 375– 446 446602 – –952 – –

enotes loss of substructure out of amino acid.


200 300 400 500 600 700 800 900 1000 1100m/z

200 300 400 500 600 700 800 900 1000 1100m/z

82

0

20

40

60

9

0

2

4

6

28

0

10

20

100

0

50

82

0

20

40

60

9

0

2

4

6

28

0

10

20

Rel

ativ

e A

bund

ance

100

0

50

453

440 887596506 905453 620329 595298 755 886731285 696621535411368 862

453

440 887596506 905453 620329 595298 755 886731285 696621535411368 862

5991027

10179161028

603 996984620 710585 833571

977915

599

553 866978964728 879865

5991027

10179161028

603 996984620 710585 833571

977915

599

553 866978964728 879865

1050599

939 10511040

625939643 1037710571 856 1068

1050599

939 10511040

625939643 1037710571 856 1068

(a)

(b)

(c)

(d)

Figure 3. (+ESI)-ion trap mass spectra of: MC-RR (a), MC-YR (b), MC-LR (c) and MC-WR (d). MS2 product ions of: MC-RR [M + 2H]2+

(m/z 520.5 fi 200–1100); MC-YR [M + H]+ (m/z 1045.5 fi 285–1100); MC-LR [M + H]+ (m/z 995.5 fi 270–1100); and, MC-WR [M + H]+

(m/z 1068.5 fi 290–1100).


CE was described as a powerful alternative for a fast andsimple determination of the compounds, and the lack ofsensitivity could be overcome by pre-concentrationmethodologies. Bateman et al. [33] reported the sameconclusions using CE-MS (+ ESI) for determining MCs inalgal cells. The efficiency of separation involved platecounts of 230,000, but, using CE-(+ ESI)-MS, the LODwas found to be higher by at least an order of magnitudecompared to that of LC-(+ ESI)-MS.

2.4. Occurrence of MCs in waters and waterworksThe concentration of MCs found in various types ofwaters from different countries is summarized in Table 3.Generally, the MCs detected in waters are MC-LR, MC-RR and MC-YR. The compound MC-LR is one of the mostcommonly found MCs in natural blooms, making up 23–94% of total MC concentration. Other MCs found inwaters are MC-LA, MC-AR, MC-FR, MC-WR, MC-LW andMC-LF.

Although the total concentration of MCs is frequentlybelow 10 lg/L, Barco et al. [34] reported the concen-tration of MC-LR at 270 lg/L in river waters.

The concentrations can differ, depending on the age ofthe bloom and nutrient conditions. Park et al. [35]demonstrated that the relatively high percentages of MCs


in filtered lake water at the end of blooms suggested thatrelease of MCs from cells occurred during the decompo-sition period of Microcystis cells.

A study of MC release performed under laboratoryconditions [10] showed that, for old cultures, the mainpart of MC-LR synthesized by Microcystis strains wasfound in water, clearly indicating a toxin release in themedium when the cells die.

Oh et al. [36] reported that the seasonal variation ofthe concentration of MCs in a Korean reservoir can beestimated and indirectly monitored by analyzing thefollowing:

� the phytoplankton number;� the chlorophyll-a concentration;� the ratio of particulate to dissolved forms of N

and P; and,� the particulate N/P ratio, when the dominant

genus is Microcystis.Shen et al. [28] investigated the influence of water

temperature to the MC levels in bloom samples. Theaverage concentrations were 24.5–97.3 lg/g in dryalgae from May to October, which increased with a risein water temperature. Temperature climbed higher fromJuly to September and the maximum MC concentrations(97.3 lg/g) were found in August and September.

Table 3. Concentrations of microcystins found in waters in different countries

Compound Matrix Country Total concentration Ref.

LR, RR, YR, Lake water (intracellular + extracellular) Finland 42.3 lg/L [13]LR, RR, YR Lake water (extracellular) USA 79.1 lg/L (%MC-LR:53) [64]LR, RR, LW, LF Lake water (intracellular + extracellular) USA Extracellular: 0.047–2 lg/L [45]

(%MC-LR:55–99)Intracellular: 5.54 lg/g(%MC-LR:60)

LR, RR, YR Lake water Japan Extracellular: 10.3–184 lg/L [35](intracellular + extracellular) (%MC-LR:40–0)

Intracellular: 30–1360 lg/gLR Lake water (intracellular) China 24.5–97.3 lg/g [28]LR, RR, YR, LA, WR, AR, FR Lake water (intracellular) Canada 54–185 lg/g (%MC-LR:2451) [24]LR, RR, YR River, Portugal 4.8–7.1 lg/mg (%MC-LR:46–49) [65]

Lake 1.6–6.8 lg/mg (%MC-LR:94–70)Reservoir (intracellular) 1.0–5.6 lg/mg (%MC-LR:80–82)

LR, RR, YR River Spain 13.2 lg/L (%MC-LR:12) [34]Reservoir (extracellular) 0.6–4.7 lg/L (%MC-LR 67, 94)

LR, RR, LA, WR AR, FR Surface water (extracellular) Canada 54–584 lg/L (%MC-LR:23-69) [24]LR, RR, YR Reservoir (extracellular) Germany 1.3 lg/L (%MC-LR:23) [22]


The MC concentrations found in drinking water sug-gest incomplete elimination of MCs or cyanobacteriaduring drinking water treatment. The waterworks haveto not only reduce the cyanobacterial cells, odor andcolor during treatment process, but also eliminate thetoxins produced by cyanobacteria.

There are a few studies investigating the efficiency ofremoving MCs and cyanobacteria. Hoeger et al. [37]investigated the elimination of MCs in two waterworksand reported that, after flocculation or sedimentation,sand filtration and chlorination, the removal was be-tween 13.1% and 61.9%; they were finding levels of MCsin the final effluent of <1.0 lg/L.

Jia et al. [38] also investigated the fluctuation of MCsand cyanobacteria with respect to commonly usedmechanisms in waterworks with advanced treatmentmethods. The study demonstrated that, during long-distance transportation of algae in raw water, the algaemay grow significantly even after preoxidation treat-ment. More importantly, coagulation/precipitation andfiltration may increase MCs in drinking water if operatedimproperly, because they trap the cyanobacteria, thusallowing their growth and the continuous release ofMCs.

Although disinfection can reduce the levels of MCs tosome degree, it is usually designed for inactivation ofbacteria rather than for MC degradation specifically.However, Jia et al. [38] demonstrated that commonadvanced processes, such as ozonation and granularactivated carbon, can remove MCs left in drinking waterafter conventional treatment processes. Shephard et al.[39] observed similar results and proposed the use ofadvanced photooxidation methods for the detoxificationof natural waters to be used as drinking waters instead ofusing chlorination or ozonation. Whereas the use of

chlorination or ozonation on cellular materials cancontribute to the release of toxin from algal cells and alsothe formation of non-desired by-products, the use ofadvanced processes appears to be the most effectivemethod for effectively eliminating MCs from waters.

3. Environmental fate of MCs in water

While the occurrence of MCs in waters has been docu-mented in many parts of the world, the fate of the MCs inthe aquatic environment has not been well studied. Inthe next section, we review the investigations conductedon the persistence of MCs in waters and the descriptionof the new degradation products identified by MS tech-niques reported in the literature.

3.1. BiodegradationStudies investigating the biodegradability of MCs in theaquatic environment have covered sewage effluent [40],surface waters [41] and sediments [42]. In naturalwaters, Jones and Orr [43] showed that MCs persisted inan embankment of a recreational lake for nine days afteran algaecide treatment before degradation commenced;then, rapidly, the MCs almost disappeared (90–95% in3 d). They also reported that, in the open lake, theMCs released from the cyanobacteria were diluted toundetectable levels during the first 24 h.

Investigations on the persistence of MC-LR in a lake inFinland compared the concentrations of MCs in dissolvedphase with those in the particulate matter [41]. It wasshown that dissolved MC-LR was detectable in the lakewater during decomposition of a Microcystis bloom andremained in low concentrations even weeks after thebloom had disappeared. Persistence of dissolved MC-LR



was longer (30 days) than the persistence of MC-LR inparticulate material (15 days) during natural decompo-sition of cyanobacteria.

In laboratory settings, Cousins et al. [44] reported thatthe primary degradation of MC-LR in surface water witha bed of sediment from a reservoir occurred rapidly witha half-life of 3–4 days. Perez et al. [45] compared thebiodegradation of MC-RR and MC-LR in two differentlake waters under laboratory conditions (Fig. 4). A watersample from Lake Erie (New York, USA) was spiked withMC-LR and MC-RR (Fig. 4), whereas a second samplefrom a lake in Nebraska (USA) contained natural MCfrom a recent algal bloom (Fig. 4). In the sample fromLake Erie, the two MCs presented similar behavior. Aftera lag phase of nine days, their concentration decreasedrapidly within one week to a non-detectable level. Bycontrast, primary degradation of the two MCs in thecontaminated lake was almost completed within twodays. Differences in the length of the adaptation phasecould be traced back to differences in the nature of thewaters.

Most of the studies on the microbial degradation ofMCs employed isolated bacteria. One work [46] re-ported the degradation rates of MC-LR, MC-RR andMC-YR with a new bacterium isolated from a hyper-trophic lake. The MCs were degraded in four days inthe presence of the bacterial strains and showed strongtemperature dependence with the maximum rate ob-served at 30�C. The authors showed that the bio-

0 5 10

0

2

4

6

8

10

40

60

80

100

Con

cent

ratio

ns [m

g L-1

]

Time

Figure 4. Biodegradation curves of two microcystins (MCs) studied in twoNew York (USA) spiked with MC-LR and MC-RR at 10 mg/L and another bnatural MCs from a recent algal bloom.


degradation also depended on concentration, and thehighest degradation rates of MC-RR and MC-LR wereobserved at concentrations of 13 and 5.4 mg/L/day,respectively.

Bourne et al. [47] reported on the enzymatic pathwayfor the bacterial degradation of MC-LR in cultures ofSphingomonas strains. They identified two intermediatesof MC-LR degradation by the LC–MS2 (+ ESI). The MSmolecular ions at m/z 1013 for product A and at m/z615 for product B and the MS spectra of these productsallowed identification of the compounds as a linearized(acyclo-) microcystin-LR [NH2-Adda-Glu (iso)-meth-yldehydroalanine-Ala-Leu-b-methylaspartate-Arg-OH]and a tetrapeptide [NH2-Adda-Glu(iso)-methyldehydro-alanine-Ala-OH]. These compounds originated from thehydrolysis of two amide bonds from amino acids 5 and 7(see Fig. 1) by at least three intracellular hydrolyticenzymes [47].

By contrast, Jones and Orr [43] suggested that thebiphasic degradation was due to the sequential inductionof two separate bacterial populations: one that canrapidly utilize MC-LR as a source of carbon and energy;and, a second one that co-metabolized the remaining lowconcentration of MC-LR.

Recently, Harada et al. [48] isolated the intact Adda asa degradation product of MC-LR using the same isolatedbacteria (Sphingomonas) as Bourne et al. [47]. To identifythe degradation products by LC–MS (+ESI), high-resolution fast atom bombardment MS (HR-FAB-MS)

15 20 25 30

(days)

MC - RR (Native)

MC - LR (Native)

MC - RR (Spiked)

MC - LR (Spiked)

different lake waters. One bioreactor settle with water from a lake inioreactor settle with water from a lake in Nebraska (USA) containing


and 1H nuclear magnetic resonance (NMR) and 13CNMR were used.

Although some authors reported that MCs releasedfrom cells remained detectable at low concentrations inlakes for long periods of time, fate studies conducted insurface waters reported that the complete biodegrada-tion of some MCs occurred in less than two weeks.

3.2. PhotodegradationMCs decompose to a very limited extent under naturalsunlight. Tsuji et al. [5] demonstrated that, although theMCs remain stable under natural sunlight, the additionof natural pigments for photosynthesis from cyano-bacteria (e.g. chlorophyll-a,b-carotene, mixoxanthophyll,phycocyanins, allophycocyanins and phycoerythrins)accelerated their decomposition.

It is known that MCs isomerize when exposed to UVlight, the isomerization rates depending on pigmentconcentration [5]. Tsuji et al. [5] identified by Frit-FAB-MS one geometrical isomer [i.e. (4E),(6Z)-Adda] and twounknown by-products with molecular weights of 1028,suggesting that two hydroxy groups were oxidativelyadded to the diene group of Adda.

Welker and Steinberg [49] determined the rates ofdegradation of microcystin-LR by natural sunlight in thepresence of photosensitizers (i.e. pure fulvic acids andnatural dissolved organic matter). The presence of purefulvic acids increased the rate of photosensitized degra-dation of microcystin-LR, while the rates of photosensi-tized degradation in natural waters were rather low.Estimates of in situ half-lives of MCs in the water columnwere 90–120 days/m depth.

Another study by Shephard et al. [39] compared thephotodegradation of MC-LR, MC-YR and MC-YA indistilled water and lake water using UV light, TiO2 andO2. The decomposition rates of the MCs in distilled waterunder the previous conditions were fast and dependedstrongly on the amount of TiO2 catalyst (0–5 g/L). Inthis study, the use of lake water, rather then distilledwater showed that advanced photodegradation is feasi-ble in natural waters, although increased levels ofcatalyst (up to 5 g/L) were required to achieve compa-rable decomposition rates.

Liu et al. [50] reported the structural characterizationof the 10 degradation products of MC-LR performingadvanced oxidation processes under laboratory condi-tions with UV light and TiO2 as a suitable semiconductorusing LC–MS (ESI+). Three of them, the geometricalisomer and the two dihydroxylated products, hadalready been suggested by Tsuji et al. [5]. Liu et al. [50]observed that the major destruction pathway of MCsforming non-toxic by-products appeared to be initiatedvia three mechanisms:

(1) UV radiation;(2) hydroxyl radical attack; and,(3) oxidation cleavage.

The UV radiation caused the geometrical isomeriza-tion of microcystin, converting the (4E), (6E) Addaconfiguration to (4E), (6Z) or (4Z), (6E) Adda configu-rations. The (4E), (6E) Adda configuration is thegeometry that is considered essential for the biologicalactivity, and the two isomers do not show toxicity. Thehydroxyl radical attack then destroyed the conjugateddiene structure to form dihydroxylated products, and thelast mechanism was the oxidation cleavage of thehydroxylated 4–5 and/or 6–7 bond of Adda.

3.3. Other elimination processesTo date, little research has been conducted on theadsorption of dissolved MCs from cyanobacteria ontosediments or soils. The adsorption of MCs onto sedi-ments from water-spiked samples at 5 lg/L and pH6.7 using LC–UV was reported by Rivasseau et al.[10]. Only 10% of MCs were adsorbed on particles and7% on sandy sediment in the time of residence of threedays. This was consistent with the physicochemicalproperties measured, namely water solubility andhydrophobicity in combination with the polar groupsof the MCs.

Morris et al. [51] investigated the scavenging ofMC-LR from water by fine-grained particles known tohave a high concentration of clay minerals (kaoliniteand montmorillonite). The results showed that morethan 81% of MC-LR can be removed from water by claymaterial. Since MC-LR is one of the more hydrophilicMCs and, with its evident affinity for clay surfaces, it ispossible that other members of this family of compoundswill show higher affinity to the clay.

Miller et al. [52] established a methodology forinvestigating the adsorption of MC-LR to soil using batchtechniques and also investigated the effect of pH andtotal dissolved solids on toxin adsorption. Similar toearlier findings [51], they suggested that the clay parti-cles are most likely to be the active binding componentsin soil, as opposed to the organic content.

With respect to the influence of pH, more adsorption ofMC-LR was observed at lower pH than at higher pH.Tsuji et al. [53], analyzed sediments from Japanese lakescontaining MCs. The results clearly indicated that theadsorption on sediments (by the hydrophilic part of themolecule) contributed to the detoxification of MCs undernatural conditions.

The effect of pH and temperature on the degradation ofMCs was reported by Harada and Tsuji [54]. Theydemonstrated that the half-life of MC-LR was about 10weeks under conditions mimicking natural conditions insummer (pH: 9–10, temperature: 40�C), and thattemperature was less influential than other factors. Theyalso showed that the structural analysis by LC–MS(ESI+) of the degradation products at 40�C suggestedthat the initial hydrolysis of the MC-LR occurred in one



of the amide bonds of the Mdha moiety (see Fig. 1) togive a linearized peptide.

4. Bioaccumulation

From estimating the physico-chemical properties (i.e.logDow) of MCs, De Maagd et al. [19] expected a lowtendency to bioconcentrate, especially at the pH valuesbetween 6 and 9 that existed during bloom conditions.Another reason for expecting low bioconcentration is thelarge size of the MC molecules, which may prevent thepassive diffusion across the cell membrane.

Gastrointestinal uptake via food can be expected to bea more relevant route. Magalhaes et al. [55] reported thebiomagnification from phytoplankton to fish in samplesfrom a lagoon in Brazil. They observed a rapid transfer ofMCs from seston (material retained in filters after water-sample filtration) to fish via direct oral ingestion, proba-bly from picoplankton cyanobateria. They also reportedthe accumulation and persistence of MCs in Tilapiarendalli muscles, demonstrating a risk of consumption forhumans, since 71.7% of the muscle samples were abovethe total daily intake (0.04 lg/kg/day) recommended bythe WHO.

Xie et al. [56] examined the tissue distribution anddepuration of MC-LR and MC-RR from seston to silvercarp over a period of 80 days. The maximum MC-RRconcentration in the blood, liver and muscle of the fishwas 49.7, 17.8 and 1.77 lg/g, respectively. AlthoughMC-LR was detectable in the intestines of a concentra-tion of 115.3 lg/kg, no MC-LR was detectable in theother tissues. They proposed that MC-LR was trans-ported very fast to the intestines, while the depuration ofMC-RR concentrations was different. The eliminationoccurred more slowly than the uptake in blood, liver andmuscle, showing an order rate for depuration ofblood > liver > muscle. The same findings were reportedby Zhang et al. [57], indicating that MCs might accu-mulate in fish tissues rather than in fish muscle.

Using ELISA, Orr et al. [58] investigated the bio-accumulation of MCs in beef cattle consuming 1.42lg/kg/day from water contaminated with MC-LR, show-ing no detection of any MC-LR in liver and blood plasma.

However, previous studies performed with pigs hadshown that MCs were preferentially accumulated in theliver [59].

Ito et al. [60] reported on the distribution followingoral administration of MC-LR to mouse. MC-LR wastransported in the blood plasma to the liver, lung andheart, and finally reached the capillaries of the wholebody. The excretion of MC-LR was shown in the mucousfrom goblet cells in both the small and the large intes-tine. Although they were not able to detect MC-LR in theurine, the fact that it was present in the kidney indicatedthat the urinary tract was a likely route of extraction.


More studies are needed to understand the bio-accumulation of MCs in organisms in order to assess therisk of MCs to human health via the trophic chain.

Once the MCs are taken in by the organism, they cancause adverse effects. Poisoning of birds, fish and otheranimals by cyanobacterial toxins has been describedelsewhere [1,61]. The consumption of drinking watercontaminated with cyanobacteria produced gastro-enteritis, diarrhea, vomiting and, in some circumstances,the death of humans [61].

The underlying mechanism of MC toxicity is inhibitionof protein phophatases. It seems that MCs mediate theirtoxicity by the uptake into hepatocytes. It is also knownthat the toxicity of these toxins is related to the geo-metrical isomerization of Adda moiety, displaying thatthe conjugated diene in Adda and the glutamate areessential structural residues for the toxicity [47]. Forexample, the hydrogenation or ozonolysis of the dienesystem in the Adda unit or the stereoisomerization of thedouble bonds in the Adda moiety greatly reduces thetoxicity [40].

5. Conclusions

The occurrence of toxic cyanobacterial blooms is aserious environmental problem worldwide. Little isknown about the conditions that promote the MC pro-duction at ambient circumstances (i.e. nutrient status inthe cells, nutrient composition of the waters), althoughincreasing eutrophication of freshwater supplies due todischarges of large amounts of human wastes has beenshown to enhance the chance of cyanobacterial blooms.Whereas information on the fate of MCs and their degra-dation products under laboratory conditions has beenwell studied, these degradation products have not beenisolated from natural waters, indicating that the envi-ronmental fate of the MCs is still not clearly understood.

This review has covered the existing information onrecent developments of the analysis of MCs in waters,comparing the results obtained for the analysis of MCs inwaters with different novel extraction methods and ad-vanced detection techniques. Although there are someparameters in the extraction and clean-up protocols ofMCs in waters that have not been optimized, such as thestudy of best pH for sample extraction, the existingmethods provide a reasonable level of reliability andefficiency.

Regarding biological detection methods, the mousebioassay, which is the most widely used tool for moni-toring MCs, has a limited sensitivity and poor repro-ducibility. The MC-dependent phosphatase assay,occasionally employed for the analysis of MCs, is sus-ceptible to interferences that result in false positives andit is not sensitive enough to detect very low concen-trations. Lastly, ELISAs do not provide specific analysis of


each toxin in samples containing MCs with various typesof toxins. However, the bioassays can be used asscreening assays to eliminate negative samples beforefurther chemical analysis.

Regarding MS techniques, due to the higher LODsreported for LC–MS (+ ESI), it is necessary to apply MS2

techniques for unequivocal identification of MCs, inorder to minimize the effects of interferences and im-prove sensitivity.

LC-TOF-MS and LC-qTOF-MS are promising tools foridentifying unknown MCs as well as for characterizingthe structures of degradation products of MCs in environ-mental samples. These techniques have not yet beenexplored for MC analysis.

Acknowledgements

This material is based upon work supported by the Na-tional Science Foundation under Grant No. 0233700.Any opinions, findings, and conclusions or recommen-dations expressed in this material are those of theauthors and do not necessarily reflect the views of theNational Science Foundation. S. Perez acknowledges apost-doctoral fellowship from the Spanish Ministry ofEducation, Culture and Science (EX2003-0687).

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