Accumulation of free and covalently bound microcystins in tissues of Lymnaea stagnalis (Gastropoda) following toxic cyanobacteria or dissolved microcystin-LR exposure

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Environmental Pollution xxx (2009) 1ndash7

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Environmental Pollution

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Accumulation of free and covalently bound microcystins in tissues of Lymnaeastagnalis (Gastropoda) following toxic cyanobacteria or dissolvedmicrocystin-LR exposure

Emilie Lance a Milla-Riina Neffling b Claudia Gerard a Jussi Meriluoto b Myriam Bormans a

a UMR CNRS Ecobio 6553 University of Rennes 1 Campus de Beaulieu 265 Avenue du General Leclerc 35042 Rennes Cedex Franceb Department of Biochemistry and Pharmacy Aringbo Akademi University Tykistokatu 6 FI-20520 Turku Finland

The study concerns accumulation and elimination of both free and boucyanobacteria or dissolved MC-LR

nd microcystins (MCs) in tissues of a gastropod exposed to MCs producing

a r t i c l e i n f o

Article historyReceived 3 June 2009Received in revised form28 September 2009Accepted 15 October 2009

KeywordsCyanobacteriaGastropodsCovalently bound microcystinsAccumulationDetoxification

Corresponding author Tel thorn33 0223235037 faxE-mail address emilielancelivefr (E Lance)

0269-7491$ ndash see front matter 2009 Elsevier Ltddoi101016jenvpol200910025

Please cite this article in press as Lance EEnviron Pollut (2009) doi101016jenvpol

a b s t r a c t

Accumulation of free microcystins (MCs) in freshwater gastropods has been demonstrated but accu-mulation of MCs covalently bound to tissues has never been considered so far Here we follow theaccumulation of total (free and bound) MCs in Lymnaea stagnalis exposed to i) dissolved MC-LR (33 and100 mg L1) and ii) Planktothrix agardhii suspensions producing 5 and 33 mg MC-LR equivalents L1 overa 5-week period and after a 3-week depuration period Snails exposed to dissolved MC-LR accumulatedup to 026 mg total MCs g1 dry weight (DW) with no detection of bound MCs Snails exposed to MCsproducing P agardhii accumulated up to 699 mg total MCs g1 DW of which from 177 to 667 werebound After depuration up to 153 mg g1 DW of bound MCs were detected in snails previously exposedto toxic cyanobacteria representing a potential source of MCs transfer through the food web

2009 Elsevier Ltd All rights reserved

1 Introduction

Massive cyanobacterial blooms in freshwaters worldwide havebecome a serious threat to human health and aquatic biota due tothe production of potent toxic metabolites (for reviews Wiegandand Pflugmacher 2005 Ibelings and Chorus 2007 Martins andVasconcelos 2009) The hepatotoxin microcystins (MCs) intracel-lular cyclic heptapeptides of which 80 structural variants have beenidentified (for review Dietrich and Hoeger 2005) can enter theaquatic food web through accumulation in various organismsincluding zooplankton macroinvertebrates and vertebrates (forreview Martins and Vasconcelos 2009) MCs are preferentiallytaken up by hepatocytes or by the digestive cells where they canspecifically interact with the target proteins protein phosphatases(Ppases) in a two-step mechanism involving a rapid and reversiblebinding (ie accumulation of free MCs) potentially followed bya covalent bound after several hours (ie accumulation of boundMCs) (Hastie et al 2005 Maynes et al 2006) Both covalent andnon-covalent MCsndashPpases interactions result in enzyme inhibition

thorn33 0223235054

All rights reserved

et al Accumulation of free200910025

reorganization of cytoskeletal components and disruption ofhepatic architecture leading to severe and irreversible damagesand potentially death (for reviews Zurawell et al 2005 Wiegandand Pflugmacher 2005)

Freshwater gastropods inhabit the littoral area where cyano-bacteria frequently form scums and can therefore be intoxicated byingestion of cyanobacteria producing intracellular toxins or expo-sure to toxins released after cell lysis into the surrounding waterMCs accumulation by gastropods has been demonstrated in thefield (Kotak et al 1996 Zurawell et al 1999 Ozawa et al 2003Chen et al 2005 Xie et al 2007 Zhang et al 2007 Gerard et al2008 2009) In the laboratory the consumption of MCs producingcyanobacteria (5 mg L1 referred to MC-LR equivalents (MC-LReq))induced MCs accumulation and severe impact on the life traits inthe pulmonate Lymnaea stagnalis and the prosobranch Potamo-pyrgus antipodarum (Lance et al 2006 2007 2008) Negativeeffects on life traits were also observed following exposure to dis-solved MC-LR at 33 mg L1 in both species but with minor accu-mulation (Gerard et al 2005 Gerard and Poullain 2005)

Nevertheless these studies only considered free MCs and notbound MCs accumulation (Goldberg et al 1995 Maynes et al2006) Covalently bound MCs have been demonstrated in someorganisms [eg bivalves (Williams et al 1997bc Dionisio Pires

and covalently bound microcystins in tissues of Lymnaea stagnalis

E Lance et al Environmental Pollution xxx (2009) 1ndash72

ARTICLE IN PRESS

et al 2004)] and may be important for MCs transfer through thefood web To evaluate bound MCs in gastropods is essential sincethey are consumed by numerous invertebrates and vertebrates(eg crayfish leeches insects fish waterfowl) (for reviewMichelson 1957) which in turn are consumed by aquatic andterrestrial predators

Here a chemical method has been adapted to detect total (boundplus free) MCs content in snail tissues through the formation of2-methyl-3-methoxy-4-phenylbutiric acid (MMPB) as an oxidationproduct of MCs (Sano et al 1992 Harada et al1996 Williams et al1997bc Ott and Carmichael 2006) and its detection by liquidchromatography electrospray ionisation tandem mass spectrometry(LCndashESI-MSMS) Free extractable MCs in snail tissues were alsoassessed using both enzyme-linked immunosorbent assay (ELISA)and LCndashESI-MSMS We examine the accumulation of bound andfree MCs in adult L stagnalis exposed during 5 weeks to cyanobac-teria (Planktothrix agardhii PMC 75-02) producing several variantsof MCs (Yepremian et al 2007) [5 and 33 mg MC-LReq L1] or todissolved pure MC-LR (33 and 100 mg L1) These concentrations canbe observed in eutrophic waters (for review Chorus and Bartram1999) We assessed the percentage of various MCs variants in Pagardhii and snail tissues as well as the proportion of free and boundMCs in snail tissues For all experiments the intoxication period wasfollowed by a 3-week depuration in order to determine the potentialdecrease of both free and bound MCs in gastropods

The discussion focuses on

the comparison between bound and free MCs accumulationand elimination in snails according to intoxication pathways(ingestion of MCs producing cyanobacteria vs dissolved MC-LRexposure) the change in MCs accumulation in snails depending on the

proportion of different MCs variants produced bycyanobacteria and the consequences in terms of potential MCs transfer in the

food web

2 Material and methods

21 Biological and toxic material

Adult L stagnalis were obtained from a laboratory population in the Experi-mental Unit of the Institut National de Recherche en Agronomie (U3E INRA Rennes)Prior to the experiment L stagnalis (25 3 mm shell length) were isolated in glasscontainers of 35 mL (1 snailcontainer) acclimated to the experimental conditions(1212 LD 20 1 C) and fed on organic lettuce for 7 days

P agardhii (strain PMC 75-02) originating from the recreational watersport siteof Viry (Essone France) was cultured as described in Lance et al (2006) This strainhas been shown to produce various MCs variants (ie dmMC-LR and MC-YR)(Yepremian et al 2007) The P agardhii suspension was provided twice a week to thegastropods and contained a total concentration of 5 or 33 mg MC-LReq L1 measuredby high pressure liquid chromatography with UV diode array detection (HPLC-DAD)using the method described in Lance et al (2006)

The natural extract of cyanobacterial bloom used to spike controls was preparedas in Jurczak et al (2005) and Neffling et al (in this issue) Purified MC-LR (AlexisCorporation USA) was solubilised with MeOH (1 mL L1) in dechlorinated water forfinal dissolved MC-LR concentrations of 33 and 100 mg L1

22 Experimental set up

After acclimation snails were divided into various treatment groups accordingto diet and medium and were held in

dechlorinated water with lettuce ad libitum (CONTR) dechlorinated water containing 33 mg of dissolved MC-LR L1 (D33LT) or

100 mg MC-LR L1 (D100LT) with lettuce ad libitum two P agardhii suspensions P1 and P2 (strain PMC 75-02 cultured in same

conditions but in two independent cultures at 8-month interval) both dilutedin order to obtain two MCs concentrations

Please cite this article in press as Lance E et al Accumulation of freeEnviron Pollut (2009) doi101016jenvpol200910025

- 5 mg MC-LReq L1 without additional food (CYAN5) and with lettuce adlibitum (CYAN5LT) both with the P1 and P2 suspensions

- 33 mg MC-LReq L1 without additional food (CYAN33) with P1 and P2 andwith lettuce ad libitum (CYAN33LT) only with P2

Each group consisted of 20 isolated individuals Cyanobacterial suspensions aswell as the medium of starved control and MC-LR exposed snails were renewedtwice a week Each treatment was maintained for 5 weeks after which all gastro-pods were maintained in dechlorinated water and fed solely on lettuce ad libitumfor a 3-week depuration period

23 Quantitative analysis of free MCs in exposed snails using ELISA

Four snails were randomly chosen every week in the groups CYAN33 andCYAN33LT exposed to P2 and at the end of the intoxication and depuration periodsin all other groups Snails were starved for ca 24 h to empty their gut contents(Carriker 1946) MCs extraction from tissues and analysis by immuno-assay wereperformed as described in Lance et al (2006) with an ELISA Microcystin Plate Kit(Envirologix Portland ME USA) which detects all of the 6 purified hepatotoxins ofcommon bloom-forming cyanobacteria especially MC-LR and MC-RR (Gilroy et al2000) MCs were expressed in mg MCs g1 dry weight (DW) of snail tissue from0020 mg MCs g1 DW threshold and to the nearest 0005 mg MCs g1 DW The valueswere calculated by taking into account extraction recovery and possible signal over-or underestimation due to matrix interference with the ELISA test because ofunspecific binding to the antibodies The recovery for the extraction and the matrixeffect (ie effect of snail tissue) were assessed as described in Lance et al (2006) Theaverage recovery was 680 39 and matrix effect was negligible (from 03 to 75of differences between matrix and methanol results with an average of 29 04)Similar average recovery and matrix effect were already observed for L stagnalis(Lance et al 2006)

24 Quantitative analysis of free MCs in exposed snail tissues by LCndashESI-MSMS

241 Sample preparation MCs extraction from snail tissuesFor extraction of free MCs 10 mg of freeze-dried tissue material sample was

extracted with a mixture of 5 butanol (Merck Germany) 20 methanol (HPLCgrade Rathburn UK) and 75 water (purified to 182 U cm with Milli Q Synthesispurification system Molsheim France) and sonicated (15 min of bath and 1 min ofprobe sonication) The extract was cleaned-up and concentrated by solid-phaseextraction Oasis HLB 30 mg cartridges (Waters Milford MA USA)

242 LCndashESI-MSMS analysisThe chromatographic separation was achieved with Agilent 1100 LC Purospher

Star C-18e 4 mm 30 mm column with 3 mm particles The gradient mobile phaseconsisted of A) 01 formic acid (analytical grade Merck Germany) in water and B)acetonitrile (HPLC S grade Rathburn UK) The gradient was from 25 B to 90 B over8 min with flow rate 05 mL min1 and the column re-equilibrated to 25 B for 4 minwith flow rate 1 mL min1 The compounds were detected with a Micromass QuattroMicro triple-quadrupole instrument with the single ion recording (SIR) and multiplereactant monitoring (MRM) on positive ion mode The relevant ion recordings (inspiked controls see Section 243 and snail samples) were mz [dmMC-RRthorn2H]2thorn

5128 [MC-RRthorn2H]2thorn 5198 [dmMC-LRthornH]thorn 9815 [MC-LRthornH]thorn 9955 [MC-YRthornH]thorn 10455 and on the MRM mode the transitions from these ions to fragmentmz 1351 [MthornH]thorn

243 MCs quantification and signal response calculationsThe MCs quantification was based on control tissues of snails spiked with

a natural bloom extract (Jurczak et al 2005 Neffling et al in this issue) andextracted in the same way as the tissues of treated snails The concentrations of MCspresent in the spiking mixture had been determined by HPLC-DAD analysis (Mer-iluoto et al 2000 Meriluoto and Spoof 2005) The matrix from the snail tissuescaused severe signal suppression and the signal response fluctuated during the snailsample series The effects were monitored and corrected for with external standardsa mixture of MCs in the natural bloom extract also used for the spiking experimentsto normalise the signal response The amount of free MCs was expressed inmg MCs g1 DW with a detection threshold of 01 mg MCs g1 DW for the variantsMC-RR and dmMC-RR and 02 mg MCs g1 DW for the variants MC-LR dmMC-LRand MC-YR

25 Quantitative analysis of total MCs in exposed snails using the MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) method and quality control of MCsmeasurement

The method shortly described in this paper is described more in details ina related publication (Neffling et al in this issue)

251 Sample preparation Lemieux oxidation of snail tissuesFor MMPB analysis the tissue sample was trypsinated (trypsin 10 solution

25 g L1 SigmandashAldrich Chemie Germany) for two hours in pH 75 at 37 C and


E Lance et al Environmental Pollution xxx (2009) 1ndash7 3

ARTICLE IN PRESS

oxidised for three hours with solution containing 01 M KMnO4 (Merck Germany) and01 M NaIO3 (Merck Germany) in pH 90 at room temperature The reaction was endedwith sodium bisulphite solution (Merck Germany) and acidified with sulphuric acid(JT Baker the Netherlands) The solution cleaned-up and concentrated by solid-phaseextraction using Oasis HLB 30 mg cartridges The MMPB standard used was a kind giftfrom Wako Pure Chemical Industries Ltd (Osaka Japan) and Prof K-I Harada

252 LCndashESI-MSMS analysis and quantification MMPBInstrument mobile and stationary phase specifications are described above (in

242) The gradient was from 40 B to 70 B over 3 min and then rapidly taken to90 B for 1 min with flow rate 05 mL min1 and the column re-equilibrated back to40 B for 4 min with flow rate 1 mL min1 The MMPB molecule was detected withtransitions from mz 2092 [MMPB thorn H]thorn to mz 91 131 and 191

253 Bound MCs calculationQuantification of MCs with the MMPB method gives the total MCs content

present in the snail sample We inferred the bound MCs content in each homoge-nised snail tissue sample by differences from the expression bound MCs frac14 totalMCs free MCs

26 Data and statistical analysis

261 Index calculationA mean free MCs content (freeMCs) in snails from the cyanobacterial treatments

at the end of both intoxication and depuration periods was calculated by combi-nation of free MCs measurements from ELISA and LCndashESI-MSMS methods aftervalidation of each group by statistical comparisons (see Section 3221) We calcu-lated for each group of snails exposed to cyanobacteria

1) the percentage of free MCs elimination between the intoxication and thedepuration periods (elimfreeMCs) by the expression

elimfreeMCs [ 100 3 frac12freeMCsintoxication L freeMCsdepuration=freeMCsintoxication

2) the bound MCs content (boundMCs) in snails by the expressionboundMCs frac14 total MCs freeMCs

3) the percentage of bound MCs elimination between the intoxication and thedepuration periods (elimboundMCs) by the expression

elimboundMCs [ 100 3 frac12boundMCsintoxication

L boundMCsdepuration=boundMCsintoxication

The 8-week follow-up of the percentage of bound MCs in snails exposed to thesecond cyanobacterial suspension (P2 at 33 mg MC-LReq L1) with and withoutlettuce was calculated only from free MCs measured with LCndashESI-MSMS due todifference between LCndashESI-MSMS and ELISA measurements for 2 couples amongthe 16 couples of values (Section 3221)

262 Statistical analysisThe data that did not follow a normal distribution (according to the Kolmo-

gorovndashSmirnov test) were compared using the Kruskall-Wallis (KW) test and 2 by 2

Table 1Proportion (SD ) of free MC variants (dmMC-RR dmMC-LR MC-YR) in two MC-producconditions but in two independent cultures) and in Lymnaea stagnalis tissues after 5 wtreatment groups

P agardhii suspension P178 23 of dmMC-LR 274 26648 31 of MC-YR

MCs in snailtissues

dmMC-LR dmMC-RR

END OF TREATMENT PERIOD CYAN5 1180 791 1190 643CYAN5LT 1780 839 1200 284CYAN33 1150 426 1150 314CYAN33LT _ _

END OF DEPURATION PERIOD CYAN5 _ _CYAN5LT 000 000 374 274CYAN33 300 034 308 202CYAN33LT _ _

Snails were kept in two P agardhii suspensions P1 and P2 (strain PMC 75-02 cultureddmMC-LR dmMC-RR and MC-YR both for a total concentration of 5 mg MC-LReq L1 withconcentration of 33 mg MC-LReq L1 without additional feeding (CYAN33) and with lettupresented as mean standard deviation The MC variant present in the highest percentag4 measures per mix were assessed using LCndashESI-MSMS with SIR and MRM methods Validof each MC variant per treatment group


using 1) the MannndashWhitney U-test for free and bound MCs content in snail tissues(mg MCs g DW1) at the end of intoxication and depuration periods 2) the Chi2 testfor the percentage of each MCs variant accumulated in snails the boundMC theelimfreeMC and the elimboundMC A linear regression model of ELISA and LCndashMSndashMS analysis was applied on the free MCs content in snails from the 8-weekfollow-up experiment Data are reported as mean standard deviation (SD)Differences were considered as significant at P lt 005

3 Results

31 MC production by P agardhii

The P agardhii strain PMC 75-02 produced 3 MCs variants(MC-YR dmMC-LR and dmMC-RR) as identified and quantified byHPLC-DAD and LCndashESI-MSMS analysis For each of the two Pagardhii suspensions P1 and P2 cultured in the same conditions butat 8-month interval the proportions of MCs variant were stableduring the 5-week intoxication period However these proportionsdiffered between the suspensions P1 and P2 (Table 1) (ie duringthe 5-week intoxication period mean of 648 31 of MC-YR for P1and 905 09 of dmMC-RR for P2) and therefore between the twoexposure experiments

32 Accumulation of free MCs in gastropods

321 Proportion of the different MCs variantsWhatever MCs exposure dose (5 and 33 mg MC-LReq L1) and

intoxication or depuration period the main MCs variant whichaccumulated in snails significantly differed depending on the mainMCs variant produced in P agardhii suspensions (ie for bothexposure periods an average of 827 116 of MC-YR in all snailsexposed to P1 and 707 148 of dmMC-RR in all snails exposed toP2) (Table 1) Moreover the respective proportions of each MCsvariants in snail tissues significantly varied during the 3 weeks ofdepuration with an increase of MC-YR from an average of745 37 to 950 16 for all exposure doses and from 27 09to 144 94 in snails respectively exposed to P1 and P2 (Table 1)

322 Quantitative analysis of MCs3221 Comparison between ELISA and LCndashESI-MSMS methods Nofree MCs were detected with ELISA nor with LCndashESI-MSMSmethods in starved and control snails after intoxication or depu-ration period After intoxication the free MCs content in snails

ing Planktothrix agardhii suspensions P1 and P2 (strain PMC 75-02 cultured in sameeeks of exposure to these suspensions and after 3 weeks of depuration in various

of dmMC-RRP agardhii suspension P269 08 of dmMC-LR 905 09 of dmMC-RR25 04 of MC-YR

MC-YR dmMC-LR dmMC-RR MC-YR

7630 plusmn 1342 1420 644 8180 488 405 1567020 plusmn 1930 1370 230 8440 240 186 1987700 plusmn 1162 1320 240 8460 272 222 074_ 1660 086 8080 104 259 132

_ 2190 1160 6370 1082 1430 3649620 plusmn 458 2860 1570 6780 1210 360 1609390 plusmn 379 2110 548 5220 736 2660 481_ 3970 156 4690 1254 1330 939

in same conditions but in two independent cultures) with different percentages ofout additional feeding (CYAN5) and with lettuce ad libitum (CYAN5LT) or for a totalce ad libitum (CYAN33LT only with the second P agardhii suspension) Values are

e in snail tissues is indicated in bold Four snail bodies per treatment were mixed andations of each treatment group and methods were combined to give the percentage



ARTICLE IN PRESS

exposed to 33 or 100 mg dissolved MC-LR L1 differed according tothe method respectively 007 002 and 026 006 mg g1 DWwith ELISA and no free MCs with LCndashESI-MSMS After depurationno free MCs were detected with both methods in those groups Thefree MCs content measured in L stagnalis held in cyanobacterialsuspensions was similar between the two methods at the end ofintoxication and depuration A regression model applied to the8-week follow-up of MCs content in snails exposed to thesuspension P2 (33 mg MC-LReq L1) revealed a linear relation(R2 frac14 069 slope frac14 122) between LCndashESI-MSMS and ELISAmethods Analysed per couple of values results from the 2 methodswere similar for each week except for the groups CYAN33 at week3 and CYAN33LT at week 4 for which LCndashESI-MSMS measurementwere higher than ELISA measurement (Fig 1)

3222 Comparison between treatment groups The free MCscontent in snails differed significantly between groups afterintoxication and depuration Snails exposed to 33 and 100 mg ofdissolved MC-LR L1 presented a significantly lower MCs content(see above) than snails exposed to P agardhii (Table 2) Comparisonbetween snails exposed to P agardhii with and without lettuce wasimpossible due to the lack of data (death of snails during theexperiment) (Table 2) For both MCs concentrations in suspensions(5 and 33 mg MC-LReq L1) and after intoxication and depurationconcentration of free MCs was superior in snails exposed to P1 thanP2 (ie up to 82 times superior for snails exposed at 33 mg MC-LReq L1 without lettuce after the intoxication) (Table 2)

33 Accumulation and percentage of bound MCs in gastropods

The total MCs content in L stagnalis exposed to dissolved MC-LR evaluated with the MMPB method always remained below thedetection limit Snails exposed to P agardhii presented boundMCs concentrations (maximum 375 79 mg g1 DW) in theirtissues at the end of intoxication and depuration periods (Table 2)Bound MCs concentrations were superior in snails exposed to Pagardhii extracts P1 than P2 (ie up to 73 times superior for snailsexposed at 33 mg MC-LReq L1 without lettuce after the intoxi-cation) (Table 2)

After 5 weeks of intoxication the percentage of bound MCsamong total MCs (boundMC) varied from 177 42 to667 74 in all groups (Table 2) This boundMC globallyincreased during depuration from 478 186 (mean for allexposed snails at the end of intoxication) to 799 126 (mean for

0

1

2

3

4

5

6

7

8

9

10

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8

Fre

e M

Cs

cont

ent

in s

nail

tissu

es(micro

gg-

1 dr

y w

eigh

t)

Intoxication Depuration

Fig 1 Follow-up of free MC-LReq accumulated in Lymnaea stagnalis tissues(mg g DW1) (SE) fed on MC-producing (33 mg L1) Planktothrix agardhii (suspensionP2) without and with lettuce with MC measurement by LCndashESI-MSMS (respectivelyCYAN33 in black and CYAN33LT in white) or by ELISA (respectively CYAN33 in deepgrey and CYAN33LT in light grey) during 5-week intoxication and 3-week depuration


all exposed snails at the end of depuration) (Table 2) The 8-weekfollow-up of boundMC in L stagnalis exposed to suspension P2(33 mg MC-LReq L1) with and without lettuce (Fig 2) shows a rapidincrease of boundMC during the first week of intoxication witha maximum (ie 695 63) at the end of the second week Duringdepuration boundMC linearly increased in both groups up to909 38 in snails exposed to P agardhii without lettuce at theend of depuration

34 Percentage of elimination of free and bound MCs duringdepuration

Snails exposed to 33 and 100 mg dissolved MC-LR L1 eliminatedfree MCs from their tissues to below limit of detection during the 3-week depuration period The elimination of free MCs from L stag-nalis previously exposed to MC-producing P agardhii was far higherthan those of bound MCs (Table 3) The elimfreeMC was similar inall groups exposed to each suspension with an average of902 33 However the elimination of bound MCs was differentbetween the groups and higher for snails exposed to P1 vs P2 ieelimboundMC of 592 59 vs 04 01 respectively for theCYAN33 group (only one statistical comparison was performed dueto the lack of data) (Table 3)

4 Discussion

41 Detection methods of free MCs in gastropod tissues

The gastropod L stagnalis exposed for 5 weeks to 33 mg dis-solved MC-LR L1 accumulated a maximum of 007 mg g DW1 asmeasured with ELISA in accordance with Gerard et al (2005)whereas free MCs accumulation was below limit of detection whenmeasured by LCndashESI-MSMS ELISA measurements may give anoverestimation of toxin concentrations due to cross reactivity byimmunoaffinity with metabolised MCs (ie conjugated withglutathione and cysteine) which are non-toxic or less toxic (Metcalfet al 2000 Msagati et al 2006) However cysteine conjugateswere not detected by LCndashESI-MSMS in this study or in a study byDionisio Pires et al (2004) in mussels Moreover no free MCs andthus no false positives were detected with ELISA in starved andcontrol snails The absence of MCs in some by ELISA positivesamples when measured by LCndashESI-MSMS was probably due tothe higher detection threshold of this method When MCs contentsincreased in snails exposed to P agardhii extracts values weresimilar between the two methods therefore indicating that theELISA method provides a good report of the free MCs content in Lstagnalis tissues

42 Free MCs accumulation according to intoxication routes andMCs variants

After penetration in the cytoplasm of the host cells MCs can beexcreted after conjugation with detoxificationbiotransformationenzymes such as glutathione (Pflugmacher et al 1998 Cazenaveet al 2006) or interact with the catalytic subunit of Ppases (PP1 2A4 and 5) via a reversible (accumulation of free MCs) or a covalent(accumulation of bound MCs) binding (Hastie et al 2005 Mayneset al 2006) After 5 weeks of intoxication the gastropod L stagnalisexposed to MCs producing cyanobacteria (5 and 33 mg MC-LReq L1) exhibited stronger free MCs accumulation (ie maximumof 324 mg g1 DW) than snails exposed to 100 mg of dissolved MC-LR L1 (ie maximum of 026 mg g1 DW) In the laboratory the twointoxication routes were already known to induce differences infree MCs accumulation by L stagnalis ie 1300 times higher in Lstagnalis after MC-producing P agardhii (5 mg MC-LReq L1)


Table 2Accumulation of free and covalently bound MCs in Lymnaea stagnalis tissues (mg g DW1) (SD) and percentage of covalently bound MCs among total MCs (boundMC) after5-week exposure to two MC-producing Planktothrix agardhii suspensions P1 and P2 (strain PMC 75-02 cultured in same conditions but in two independent cultures) and after3-week depuration

with P agardhii suspension P178 23 of dmMC-LR 274 26 of dmMC-RR648 31 of MC-YR


MCs (mg g1 DW) boundMC MCs (mg g1 DW) boundMC

free MCs bound MCs free MCs bound MCs

END OF TREATMENT PERIOD CYAN5 2460 548 922 269 2720 522 327 054 656 200 6670 738CYAN5LT 1190 168 257 093 1770 416 339 096 (only elisa) _ _CYAN33 3240 524 3750 786 5360 714 397 114 513 248 5630 796CYAN33LT _ _ _ 556 098 390 102 4120 558

END OF DEPURATION PERIOD CYAN5 193 030 (only elisa) _ _ 029 006 ltdl _CYAN5LT 093 122 172 022 6470 926 019 004 ltdl _CYAN33 528 201 1530 518 7430 1388 048 018 511 072 9130 758CYAN33LT _ _ _ 055 017 465 062 8930 417

Snails were kept in two P agardhii suspensions (strain PMC 75-02 cultured in same conditions but in two independent cultures) with different percentages of dmMC-LRdmMC-RR and MC-YR both for a total concentration of 5 mg MC-LReq L1 without additional feeding (CYAN5) and with lettuce ad libitum (CYAN5LT) or for a totalconcentration of 33 mg MC-LReq L1 without additional feeding (CYAN33) and with lettuce ad libitum (CYAN33LT) Four snail bodies per treatment were mixed and 4 measuresper mix were assessed using LCndashESI-MSMS and ELISA methods for free MCs assessment [mean free MC values per treatment calculated by combination of free MCmeasurements from ELISA and LCndashESI-MSMS methods after validations of each group by statistical comparisons (see Section 3221)] and using the MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) method and LCndashESI-MSMS for bound MC assessment Validations of each treatment group and methods were combined to give the mean MCaccumulation value per treatment group dl frac14 detection limit


ARTICLE IN PRESS

ingestion than after dissolved MC-LR exposure (33 mg L1) (Gerardet al 2005 Lance et al 2006) Moreover we recently showedusing an immunohistological method (Lance 2008) that theamount of MCs penetrating the cytoplasm of digestive gland cellswas significant after ingestion of P agardhii (producing dmMC-LRdmMC-RR and MC-YR) by L stagnalis whereas negligible afterexposure to dissolved MC-LR (MCs contained into lysosomalvacuoles) Two hypotheses are then possible i) MCs penetration ingastropods is far higher by grazing toxic cyanobacteria than byuptake of dissolved toxins (eg via water ingestion) and ii) afterpenetration in the organism accumulation capacities differbetween structural MCs congeners While the first hypothesis hasalready been suggested in field studies (Kotak et al 1996 Zurawellet al 1999 Xie et al 2007 Zhang et al 2007) concluding thatgastropods accumulated free MCs mainly by grazing toxic phyto-plankton and to a lesser extent via uptake of dissolved toxins someresults of the present study seem also to support the secondhypothesis Indeed L stagnalis exposed to P agardhii producing33 mg MC-LReq L1 with 905 of dmMC-RR accumulated a lowamount of free MCs (ie maximum of 56 mg g1 DW) with a highproportion of dmMC-RR (829) On the other hand when exposedto the same cyanobacterial strain producing 33 mg MC-LReq L1 butwith 647 of MC-YR snails accumulated 82 times more free MCs(and 73 times more bound MCs) with a high proportion of MC-YR

0

1020

30

4050

60

70

8090

100

0 1 2 3 4 5 6 7 8

Weeks

Per

cent

age

ofbo

und

MC

sam

ong

tota

l MC

s in

sna

il tis

sues


Fig 2 Follow-up of the percentage () (SE) of covalently bound MCs among total(free thorn bound) MCs accumulated in Lymnaea stagnalis exposed to MC-producing(33 mg L1) Planktothrix agardhii (suspension P2) without (triangle) and with lettuce(square) during 5-week intoxication and 3-week depuration


(745) It thus appears that L stagnalis accumulated MC-YR toa higher extent than other variants involved in this study (dmMC-RR dmMC-LR and MC-LR) Moreover the proportion of MC-YR insnails exposed to the two P agardhii suspensions is higher after thedepuration period regardless of the proportion after the intoxica-tion period suggesting that MC-YR is less eliminated by snails thandmMC-RR In their review Dietrich and Hoeger (2005) suggest thatminor structural changes between MCs congeners may have majoreffects on uptake (eg different affinities with the organic iontransporters that allow MCs to penetrate across cell membranes)metabolization and excretion of MCs Thus MC-YR might i) moreeasily penetrates in digestive cells andor ii) more easily links toPpases andor iii) fewer links with detoxification enzymes thanMC-LR and the other MCs congeners produced by P agardhii(dmMC-LR and dmMC-RR) The differences in accumulation anddepuration between MCs variants suggested in this study wouldrequire further investigation

43 Accumulation of bound MCs

Since MCs covalently bind to the Ppases and can not beextracted from the covalent complex by organic solvents detectionof MCs in animal tissues reported in several studies (for reviewsIbelings and Chorus 2007 Martins and Vasconcelos 2009) hasbeen limited to free MCs and probably also MCs conjugated withglutathione and cysteine The existence of bound MCs has beenoccasionally demonstrated in tissues of salmon (Williams et al

Table 3Percentage (SD) () of free and covalently bound MC elimination from Lymnaeastagnalis tissues after 3 weeks of depuration snails were previously exposed during5 weeks to two MC-producing Planktothrix agardhii suspensions P1 and P2 (strainPMC 75-02 cultured in same conditions but in two independent cultures) at 5 or33 mg MC-LReq L1 with (CYANLT) or without lettuce (CYAN)

P agardhii suspension P1 P agardhii suspension P2

free MC bound MC free MC bound MC

CYAN5 9212 872 _ 9110 416 _CYAN5LT 9223 582 3311 368 9444 682 _CYAN33 8374 838 5925 594 8793 554 039 012CYAN33LT _ _ 9012 708 No elimination



ARTICLE IN PRESS

1997a) and bivalves (Williams et al 1997bc Dionisio Pires et al2004) Consequently MCs accumulation by gastropods reported inprevious field (Kotak et al 1996 Zurawell et al 1999 Ozawa et al2003 Chen et al 2005 Xie et al 2007 Zhang et al 2007 Gerardet al 2008 2009) and laboratory (Zurawell et al 2006 2007 Lanceet al 2006 2007) studies were thus probably underestimated

Using an oxidation procedure adapted from previously devel-oped methods (Sano et al 1992 Harada et al 1996 Williams et al1997bc Ott and Carmichael 2006) and followed by a detection ofoxidation products by LCndashESI-MSMS we provided evidence for theexistence of covalently bound MCs in gastropod tissues On average44 of total MCs were bound in L stagnalis exposed to P agardhiiduring 5 weeks regardless of the MCs concentration (5 or33 mg MC-LReq L1) in the cyanobacterial suspension and thepresence or absence of a concomitant non-toxic food (lettuce)Moreover the proportion of bound MCs rapidly increased duringthe first week of exposure to P agardhii (33 mg MC-LReq L1) andreached the maximum of the intoxication period (ie 69 of totalMCs) at the end of the second week

In mammals and fish MCs are known to induce severe disor-ganization of the hepatic architecture during acute poisoning andhepatocyte degeneration during chronic exposure (for reviewsZurawell et al 2005 Malbrouck and Kestemont 2006 Ernst et al2007) In a study on the relationship between hepatotoxic injuryand MCs localisation in rainbow trout fed with toxic cyanobacteriaFischer et al (2000) showed that Ppase inhibition and hepatocytenecrosis appeared to be associated with the reversible interactionMCs-Ppases (ie free MC accumulation) whereas apoptotic celldeath resulted from the covalent interaction (ie bound MCsaccumulation) Using an immunohistological localisation of MCs intissues we recently showed a severe impact on the structure of thedigestive gland of L stagnalis exposed to P agardhii associated to thepresence of bound MCs (Lance 2008) The present study confirmsthat MCs are indeed both covalently bound and no covalentlybound in L stagnalis during a 5-week exposure to toxic cyanobac-teria and allow us to quantify bound MCs

44 Free and bound MCs elimination

During the depuration period the percentage of bound MCsincreased and reached 913 of the total MCs content This increaseis due to a high free MCs elimination [around 90 eliminated in allgroups in accordance with our previous study in which L stagnaliswas similarly exposed (Lance et al 2006)] associated to a lowerbound MCs elimination varying from 0 to 59 according to treat-ments As the covalent binding of MCs to Ppases is known to beirreversible and induces cell necrosis (for review Dietrich andHoeger 2005) their elimination might occur via degradation andconsequent elimination of damaged cells as observed for digestivecells of L stagnalis similarly exposed to P agardhii (Lance 2008)According to Williams et al (1997bc) mussels rapidly eliminatedbound MCs when transferred in untreated salt water the total MCscontent dropped from 337 mg to 11 mg MC g1 fresh weight during4 days and was undetectable after In this study at the end of the3-week depuration period L stagnalis tissues still contained up to21 mg g1 DW of total MCs (with 15 mg g1 DW of bound MCs)

45 Conclusion

The total MCs content (free and bound MCs) needs to beconsidered in order to assess the intoxication risk to the food webfrom gastropods The present study reveals for the first time animportant accumulation of total MCs (up to 70 mg g1 DW after 5weeks) by gastropods after cyanobacterial exposure with a highproportion (maximum of 67) of bound MCs We previously


reported that around 47 of ingested MCs were extracted bymethanol (free MCs) in adult L stagnalis 5-week exposed to Pagardhii containing MCs (5 mg MC-LReq L1) The remaining 53 ofingested MCs could have been eliminated in the gizzard or thedigestive gland fraction of the faeces or accumulated in thedigestive gland in a covalent form (Lance et al 2006) According tothe present study in which 37 of accumulated MCs were cova-lently bound MCs in the tissues of L stagnalis similarly exposed wecan hypothesize that at least 84 of ingested intracellular MCs areaccumulated in a free or covalent form by adult L stagnalis afterconsumption of toxic cyanobacteria Hence gastropods mayrepresent a high source of MC transfer to the food web However asquestioned by Williams et al (1997bc) and Ibelings and Chorus(2007) the covalent complex MCs-Ppases is probably not toxic asan intact entity or not bioavailable for the next trophic levelInvestigations are required to demonstrate the transfer patternsand the toxicity of bound MCs

Acknowledgements

Authors thank the Institut National de Recherche en Agronomie(Rennes France) for providing individuals L stagnalis and theMuseum National drsquoHistoire Naturelle (Paris France) for providingthe P agardhii PMC 75-02 strain Authors are grateful to the CIMO(Center for International Mobility) and to the French and FinnishMinistries of Foreign Affairs which partly supported this workthrough a 3-month fellowship to E Lance for a stay in Finland inthe laboratory of Dr J Meriluoto M-R Neffling acknowledges theNational graduate school in Informational and Structural Biology(ISB) and Academy of Finland decision number 108947 for funding

References

Carriker MR 1946 Observations on the functioning of the alimentary system ofthe snail Lymnaea stagnalis appressa say Biol Bull 91 88ndash111

Cazenave J Bistoni MA Pesce SF Wunderlin DA 2006 Differential detoxifi-cation and antioxidant response in diverse organs of Corydoras paleatusexperimentally exposed to microcystin-RR Aquat Toxicol 76 1ndash12

Chen J Xie P Guo L Zheng L Ni L 2005 Tissue distributions and seasonaldynamics of the hepatotoxic microcystins-LR and -RR in a freshwater snail(Bellamya aeruginosa) from a large shallow eutrophic lake of the subtropicalChina Environ Pollut 134 423ndash430

Chorus I Bartram J 1999 Toxic cyanobacteria in water In A Guide to PublicHealth Consequences Monitoring and Management E and FN Spon on behalf ofWHO London p 416

Dietrich DR Hoeger SJ 2005 Guidance values for microcystin in water andcyanobacterial supplement products (blue-green algae supplements)a reasonable or misguided approach Toxicol Appl Pharmacol 203 273ndash289

Dionisio Pires LM Karlsson KM Meriluoto JAO Kardinaal E Visser PMSiewertsen K Van Donk E Ibelings BW 2004 Assimilation and depurationof microcystin-LR by the zebra mussel Dreissena polymorpha Aquat Toxicol 69385ndash396

Ernst B Hoeger SJ OrsquoBrien E Dietrich DR 2007 Physiological stress andpathology in European whitefish (Coregonus lavaretus) induced by subchronicexposure to environmentally relevant densities of Planktothrix rubescens AquatToxicol 79 31ndash40

Fischer WJ Hitzfeld BC Tencalla F Eriksson JE Mikhailov A Dietrich DR2000 Microcystin-LR toxicodynamics induced pathology and immunohisto-chemical localization in livers of blue-green algae exposed rainbow trout(Oncorhynchus mykis s) Toxicol Sci 54 365ndash373

Gerard C Brient L Le Rouzic B 2005 Variation in the response of juvenile andadult gastropods (Lymnaea stagnalis) to cyanobacterial toxin (microcystin-LR)Environ Toxicol 20 592ndash596

Gerard C Poullain V 2005 Variation in the response of the invasive speciesPotamopyrgus antipodarum (Smith) to natural (cyanobacterial toxin) andanthropogenic (herbicide atrazine) stressors Environ Pollut 138 28ndash33

Gerard C Carpentier A Paillisson JM 2008 Long-term dynamics and commu-nity structure of freshwater gastropods exposed to parasitism and other envi-ronmental stressors Freshwat Biol 53 470ndash484

Gerard C Poullain V Lance E Acou A Brient L Carpentier A 2009 Influenceof toxic cyanobacteria on community structure and microcystin accumulationof freshwater molluscs Environ Pollut 157 609ndash617

Gilroy DJ Kauffman KW Hall RA Huang X Chu FS 2000 Assessing potentialhealth risks from microcystin toxins in blue-green algae dietary supplementsEnviron Health Perspect 108 435ndash439



ARTICLE IN PRESS

Goldberg J Huang H Kwon Y Greengard P Nairn AC Kuriyan J 1995 Three-dimensional structure of the catalytic subunit of protein serinethreoninephosphatase-1 Nature 376 745ndash752

Harada KI Murata H Qiang Z Suzuki M Kondo F 1996 Mass spectrometricscreening method for microcystins in cyanobacteria Toxicon 34 701ndash710

Hastie CJ Borthwick EB Morrison LF Codd GA Cohen PTW 2005 Inhibitionof several protein phosphatases by a non-covalently interacting microcystinand a novel cyanobacterial peptide nostocyclin Biochim Biophys Acta (G)1726 187ndash193

Ibelings BW Chorus I 2007 Accumulation of cyanobacterial toxins in freshwaterlsquolsquoseafoodrsquorsquo and its consequences for public health a review Environ Pollut 150177ndash192

Jurczak T Tarczynska M Izydorczyk K Mankiewicz J Zalewski M Meriluoto J2005 Elimination of microcystins by water treatment processes-examples fromSulejow reservoir Poland Water Res 9 2394ndash2406

Kotak BG Zurawell RW Prepas EE Holmes CFB 1996 Microcystin-LRconcentration in aquatic food web compartments from lakes of varying trophicstatus Can J Fish Aquat Sci 53 1974ndash1985

Lance E Brient L Bormans M Gerard C 2006 Interactions between cyano-bacteria and Gastropods I Ingestion of toxic Planktothrix agardhii by Lymnaeastagnalis and the kinetics of microcystin bioaccumulation and detoxificationAquat Toxicol 79 140ndash148

Lance E Paty C Bormans M Brient L Gerard C 2007 Interactions betweencyanobacteria and Gastropods II Impact of toxic Planktothrix agardhii on thelife-history traits of Lymnaea stagnalis Aquat Toxicol 81 389ndash396

Lance E Bugajny E Bormans M Gerard C 2008 Consumption of toxic cyanobacteriaby Potamopyrgus antipodarum (Gastropoda Prosobranchia) and consequences onlife traits and microcystin bioaccumulation Harmful Algae 7 464ndash472

Lance E 2008 Impact of toxic cyanobacteria on freshwater gastropods and ontheir role as vector in food web microcystin transfer PhD thesis UniversiteRennes 1 France pp 289

Malbrouck C Kestemont P 2006 Effects of microcystins on fish Environ ToxicolChem 25 72ndash86

Martins JC Vasconcelos VM 2009 Microcystin dynamics in aquatic organisms JToxicol Environ Health Part B 12 65ndash82

Maynes JT Luu HA Cherney MM Andersen RJ Williams D Holmes CFBJames MNG 2006 Crystal structures of protein phosphatase-1 bound tomotopurin and dihydromicrocystin-LA elucidation of the mechanism ofenzyme inhibition by cyanobacterial toxins J Mol Biol 356 111ndash120

Meriluoto JAO Lawton L Harada K-I 2000 Isolation and detection of micro-cystins and nodularins cyanobacterial peptide hepatotoxins In Holst O (Ed)Methods Mol Biol Bacterial Toxins Methods and Protocols vol 145 HumanaPress Totowa NJ pp 65ndash87

Meriluoto JAO Spoof LEM 2005 Analysis of microcystins by high-performanceliquid chromatography with photodiode-array detection In Meriluoto JAOCodd GA (Eds) Toxic Cyanobacterial Monitoring and Cyanotoxin AnalysisAringbo Akademi University Press Turku Finland pp 77ndash84

Metcalf JS Beattie KA Pflugmacher S Codd GA 2000 Immuno-crossreactivityand toxicity assessment of conjugation products of the cyanobacterial toxinmicrocystin-LR FEMS Microbiol Lett 189 155ndash158

Michelson EH 1957 Studies on the biological control of schistosome-bearingsnails predators and parasites of freshwater mollusca a review of the litera-ture Parasitology 47 413ndash426


Msagati TAM Siame BA Shushu DD 2006 Evaluation of methods for theisolation detection and quantification of cyanobacterial hepatotoxins AquatToxicol 78 382ndash397

Neffling M-R Lance E Meriluoto J Detection of free and covalently boundmicrocystins in animal tissues by liquid chromatographyndashtandem mass spec-trometry Environ Pollut in this issue doi101016jenvpol200910023

Ott JL Carmichael WW 2006 LCESIMS method development for the analysis ofhepatotoxic cyclic peptide microcystins in animal tissues Toxicon 47 734ndash741

Ozawa K Yokoyama A Ishikawa K Kumagai M Watanabe MF Park HD2003 Accumulation and depuration of microcystins produced by the cyano-bacterium Microcystis in a freshwater snail Limnology 4 131ndash138

Pflugmacher S Wiegand C Oberemm A Beattie KA Krause E Codd GASteinberg C 1998 Indentification of an enzymatically-formed glutathioneconjugate of the cyanobacterial hepatotoxin microcystin-LR The first step ofdetoxification Biochem Biophys Acta 1425 527ndash533

Sano T Nohara K Shiraishi F Kaya K 1992 A method for micro-determinationof total microcystin content in waterblooms of cyanobacteria (blue green algae)Int J Environ Anal Chem 49 163ndash170

Wiegand C Pflugmacher S 2005 Ecotoxicological effects of selected cyanobacterialsecondary metabolites a short review Toxicol Appl Pharmacol 203 201ndash218

Williams DE Craig M Dawe SC Kent ML Andersen RJ Holmes CFB 1997a14C-labeled microcystin-LR administered to Atlantic salmon via intraperitonealinjection provides in vivo evidence for covalent binding of microcystin-LR insalmon livers Toxicon 35 985ndash989

Williams DE Craig M Dawe SC Kent ML Holmes CFB Andersen RJ 1997bBioaccumulation and clearance of microcystins from salt water mussels Mytilusedulis and in vivo evidence for covalently bound microcystins in mussel tissuesToxicon 35 1617ndash1625

Williams DE Dawe SC Kent ML Andersen RJ Craig M Holmes CFB 1997cEvidence for covalently bound microcystins in mussel tissues Chem ResToxicol 10 463ndash469

Xie L Yokoyama A Nakamura K Park H 2007 Accumulation of microcystins invarious organs of the freshwater snail Sinotaia histrica and three fishes ina temperate lake the eutrophic lake Suwa Japan Toxicon 49 646ndash652

Yepremian C Gugger MF Briand E Catherine A Berger C Quiblier CBernard C 2007 Microcystin ecotypes in a perennial Planktothrix agardhiibloom Water Res 41 4446ndash4456

Zhang D Xie P Liu Y Chen J Liang G 2007 Bioaccumulation of the hepatotoxicmicrocystins in various organs of a freshwater snail from a subtropical Chineselake Taihu lake with dense toxic Microcystis blooms Environ Toxicol Chem26 171ndash176

Zurawell RW Kotak BG Prepas EE 1999 Influence of lake trophic status on theoccurrence of microcystin-LR in the tissue of pulmonate snails Freshwat Biol42 707ndash718

Zurawell RW Chen H Burke JM Prepas EE 2005 Hepatotoxic cyanobacteriaa review of the biological importance of microcystins in freshwater environ-ments J Toxicol Environ Health 8 1ndash37

Zurawell RW Holmes CFB Prepas EE 2006 Elimination of the cyanobacterialhepatotoxin microcystin from the freshwater pulmonate snail Lymnaea stag-nalis juguralis (say) J Toxicol Environ Health 69 303ndash318

Zurawell RW Goldberg JI Holmes CFB Prepas EE 2007 Tissue distributionand oral dose effects of microcystin in the freshwater pulmonate snail Lymnaeastagnalis jugularis (say) J Toxicol Environ Health 70 620ndash626



ARTICLE IN PRESS

et al 2004)] and may be important for MCs transfer through thefood web To evaluate bound MCs in gastropods is essential sincethey are consumed by numerous invertebrates and vertebrates(eg crayfish leeches insects fish waterfowl) (for reviewMichelson 1957) which in turn are consumed by aquatic andterrestrial predators

Here a chemical method has been adapted to detect total (boundplus free) MCs content in snail tissues through the formation of2-methyl-3-methoxy-4-phenylbutiric acid (MMPB) as an oxidationproduct of MCs (Sano et al 1992 Harada et al1996 Williams et al1997bc Ott and Carmichael 2006) and its detection by liquidchromatography electrospray ionisation tandem mass spectrometry(LCndashESI-MSMS) Free extractable MCs in snail tissues were alsoassessed using both enzyme-linked immunosorbent assay (ELISA)and LCndashESI-MSMS We examine the accumulation of bound andfree MCs in adult L stagnalis exposed during 5 weeks to cyanobac-teria (Planktothrix agardhii PMC 75-02) producing several variantsof MCs (Yepremian et al 2007) [5 and 33 mg MC-LReq L1] or todissolved pure MC-LR (33 and 100 mg L1) These concentrations canbe observed in eutrophic waters (for review Chorus and Bartram1999) We assessed the percentage of various MCs variants in Pagardhii and snail tissues as well as the proportion of free and boundMCs in snail tissues For all experiments the intoxication period wasfollowed by a 3-week depuration in order to determine the potentialdecrease of both free and bound MCs in gastropods

The discussion focuses on

the comparison between bound and free MCs accumulationand elimination in snails according to intoxication pathways(ingestion of MCs producing cyanobacteria vs dissolved MC-LRexposure) the change in MCs accumulation in snails depending on the

proportion of different MCs variants produced bycyanobacteria and the consequences in terms of potential MCs transfer in the

food web

2 Material and methods

21 Biological and toxic material

Adult L stagnalis were obtained from a laboratory population in the Experi-mental Unit of the Institut National de Recherche en Agronomie (U3E INRA Rennes)Prior to the experiment L stagnalis (25 3 mm shell length) were isolated in glasscontainers of 35 mL (1 snailcontainer) acclimated to the experimental conditions(1212 LD 20 1 C) and fed on organic lettuce for 7 days

P agardhii (strain PMC 75-02) originating from the recreational watersport siteof Viry (Essone France) was cultured as described in Lance et al (2006) This strainhas been shown to produce various MCs variants (ie dmMC-LR and MC-YR)(Yepremian et al 2007) The P agardhii suspension was provided twice a week to thegastropods and contained a total concentration of 5 or 33 mg MC-LReq L1 measuredby high pressure liquid chromatography with UV diode array detection (HPLC-DAD)using the method described in Lance et al (2006)

The natural extract of cyanobacterial bloom used to spike controls was preparedas in Jurczak et al (2005) and Neffling et al (in this issue) Purified MC-LR (AlexisCorporation USA) was solubilised with MeOH (1 mL L1) in dechlorinated water forfinal dissolved MC-LR concentrations of 33 and 100 mg L1

22 Experimental set up

After acclimation snails were divided into various treatment groups accordingto diet and medium and were held in

dechlorinated water with lettuce ad libitum (CONTR) dechlorinated water containing 33 mg of dissolved MC-LR L1 (D33LT) or

100 mg MC-LR L1 (D100LT) with lettuce ad libitum two P agardhii suspensions P1 and P2 (strain PMC 75-02 cultured in same

conditions but in two independent cultures at 8-month interval) both dilutedin order to obtain two MCs concentrations


- 5 mg MC-LReq L1 without additional food (CYAN5) and with lettuce adlibitum (CYAN5LT) both with the P1 and P2 suspensions

- 33 mg MC-LReq L1 without additional food (CYAN33) with P1 and P2 andwith lettuce ad libitum (CYAN33LT) only with P2

Each group consisted of 20 isolated individuals Cyanobacterial suspensions aswell as the medium of starved control and MC-LR exposed snails were renewedtwice a week Each treatment was maintained for 5 weeks after which all gastro-pods were maintained in dechlorinated water and fed solely on lettuce ad libitumfor a 3-week depuration period

23 Quantitative analysis of free MCs in exposed snails using ELISA

Four snails were randomly chosen every week in the groups CYAN33 andCYAN33LT exposed to P2 and at the end of the intoxication and depuration periodsin all other groups Snails were starved for ca 24 h to empty their gut contents(Carriker 1946) MCs extraction from tissues and analysis by immuno-assay wereperformed as described in Lance et al (2006) with an ELISA Microcystin Plate Kit(Envirologix Portland ME USA) which detects all of the 6 purified hepatotoxins ofcommon bloom-forming cyanobacteria especially MC-LR and MC-RR (Gilroy et al2000) MCs were expressed in mg MCs g1 dry weight (DW) of snail tissue from0020 mg MCs g1 DW threshold and to the nearest 0005 mg MCs g1 DW The valueswere calculated by taking into account extraction recovery and possible signal over-or underestimation due to matrix interference with the ELISA test because ofunspecific binding to the antibodies The recovery for the extraction and the matrixeffect (ie effect of snail tissue) were assessed as described in Lance et al (2006) Theaverage recovery was 680 39 and matrix effect was negligible (from 03 to 75of differences between matrix and methanol results with an average of 29 04)Similar average recovery and matrix effect were already observed for L stagnalis(Lance et al 2006)

24 Quantitative analysis of free MCs in exposed snail tissues by LCndashESI-MSMS

241 Sample preparation MCs extraction from snail tissuesFor extraction of free MCs 10 mg of freeze-dried tissue material sample was

extracted with a mixture of 5 butanol (Merck Germany) 20 methanol (HPLCgrade Rathburn UK) and 75 water (purified to 182 U cm with Milli Q Synthesispurification system Molsheim France) and sonicated (15 min of bath and 1 min ofprobe sonication) The extract was cleaned-up and concentrated by solid-phaseextraction Oasis HLB 30 mg cartridges (Waters Milford MA USA)

242 LCndashESI-MSMS analysisThe chromatographic separation was achieved with Agilent 1100 LC Purospher

Star C-18e 4 mm 30 mm column with 3 mm particles The gradient mobile phaseconsisted of A) 01 formic acid (analytical grade Merck Germany) in water and B)acetonitrile (HPLC S grade Rathburn UK) The gradient was from 25 B to 90 B over8 min with flow rate 05 mL min1 and the column re-equilibrated to 25 B for 4 minwith flow rate 1 mL min1 The compounds were detected with a Micromass QuattroMicro triple-quadrupole instrument with the single ion recording (SIR) and multiplereactant monitoring (MRM) on positive ion mode The relevant ion recordings (inspiked controls see Section 243 and snail samples) were mz [dmMC-RRthorn2H]2thorn

5128 [MC-RRthorn2H]2thorn 5198 [dmMC-LRthornH]thorn 9815 [MC-LRthornH]thorn 9955 [MC-YRthornH]thorn 10455 and on the MRM mode the transitions from these ions to fragmentmz 1351 [MthornH]thorn

243 MCs quantification and signal response calculationsThe MCs quantification was based on control tissues of snails spiked with

a natural bloom extract (Jurczak et al 2005 Neffling et al in this issue) andextracted in the same way as the tissues of treated snails The concentrations of MCspresent in the spiking mixture had been determined by HPLC-DAD analysis (Mer-iluoto et al 2000 Meriluoto and Spoof 2005) The matrix from the snail tissuescaused severe signal suppression and the signal response fluctuated during the snailsample series The effects were monitored and corrected for with external standardsa mixture of MCs in the natural bloom extract also used for the spiking experimentsto normalise the signal response The amount of free MCs was expressed inmg MCs g1 DW with a detection threshold of 01 mg MCs g1 DW for the variantsMC-RR and dmMC-RR and 02 mg MCs g1 DW for the variants MC-LR dmMC-LRand MC-YR

25 Quantitative analysis of total MCs in exposed snails using the MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) method and quality control of MCsmeasurement

The method shortly described in this paper is described more in details ina related publication (Neffling et al in this issue)

251 Sample preparation Lemieux oxidation of snail tissuesFor MMPB analysis the tissue sample was trypsinated (trypsin 10 solution

25 g L1 SigmandashAldrich Chemie Germany) for two hours in pH 75 at 37 C and



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dmMC-LR dmMC-RR






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_ 2190 1160 6370 1082 1430 3649620 plusmn 458 2860 1570 6780 1210 360 1609390 plusmn 379 2110 548 5220 736 2660 481_ 3970 156 4690 1254 1330 939





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MCs in snailtissues

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_ 2190 1160 6370 1082 1430 3649620 plusmn 458 2860 1570 6780 1210 360 1609390 plusmn 379 2110 548 5220 736 2660 481_ 3970 156 4690 1254 1330 939





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Documents

Accumulation of free and covalently bound microcystins in tissues of Lymnaea stagnalis (Gastropoda) following toxic cyanobacteria or dissolved microcystin-LR exposure