Journal of Fish Biology (2010) 76, 1415–1430

doi:10.1111/j.1095-8649.2010.02572.x, available online at www.interscience.wiley.com

Effects of the microcystin profile of a cyanobacterial bloomon growth and toxin accumulation in common carp

Cyprinus carpio larvae

I. El Ghazali*, S. Saqrane*, A. P. Carvalho†‡, Y. Ouahid§, F. F. DelCampo§, V. Vasconcelos†‡‖ and B. Oudra*

*Department of Biology, Laboratory of Biology and Biotechnology of Microorganisms,Microbiology and Environmental Toxicology Unit, Faculty of Sciences Semlalia Marrakech,

University Cadi Ayyad, P.O. Box 2390, Marrakech 40000, Morocco, †Centro Interdisciplinarde Investigacao Marinha e Ambiental – CIIMAR/CIMAR-LA, Rua dos Bragas 177, Porto4050-123, Portugal, ‡Departamento de Biologia, Faculdade de Ciencias, Universidade do

Porto, 4169-007 Porto, Portugal and §Departamento de Biologia, Laboratorio de FisiologiaVegetal, Universidad Autonoma de Madrid, Cantoblanco,

28049 Madrid, Spain

(Received 17 May 2009, Accepted 21 December 2009)

A 12 day growth trial was conducted to compare the effect of the variation in microcystins (MC)composition of two bloom samples of Microcystis aeruginosa on the growth performance andmicrocystin accumulation in common carp Cyprinus carpio larvae. Two M. aeruginosa naturalbloom samples with different MC profiles were collected and larvae were exposed to cyanobacterialcells through their diet. Three diets, a basal control diet and two diets prepared from the basal dietplus the same toxins content (60 ng MC g−1 diet) of each cyanobacterial bloom, were given at thesame ration level to three groups of larvae during the experimental period. Larval mass and standardlength from day 9 were significantly different between cyanobacterial treatments and in both caseslower than that of the control. The MC accumulation by larvae, inversely correlated with the growthperformance, was also significantly different between cyanobacterial treatments (26·96 v. 17·32 ngg−1 at the end of the experimental period). These results indicate that MC variants profile may haveeffects on the toxin uptake and toxicity. To date, this is the first laboratory study to show that fishaccumulate MC depending on the toxin profile of the cyanobacterial bloom. © 2010 The Authors

Journal compilation © 2010 The Fisheries Society of the British Isles

Key words: carp larvae; cyanobacterial blooms; fish growth; microcystins; microcystins accumulation.

INTRODUCTION

The occurrence of toxic cyanobacterial blooms in eutrophic lakes, reservoirs andrecreational waters has become a worldwide problem (Paerl et al., 2001). Severalcyanobacteria species, specifically Microcystis aeruginosa, may produce a variety ofpotent toxins, including a group of hepatotoxins called microcystins (MC), whichhave strong cytotoxic activity (Codd et al., 1997; de Figueiredo et al., 2004). These

‖Author to whom correspondence should be addressed. Tel.: +351 22 340 18 14; fax: +351 22 339 06 08;email: vmvascon@fc.up.pt

1415© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles

1416 I . E L G H A Z A L I E T A L .

(5) Adda

OCH3

CH3

CH2

CH3

CH3CH3

CH3

CH3NH2

NH

NNH

HN

HN

HN

C

O

N

H3CH3C

(6) D-GluCOOH

O

O

O

O

OO

NH

NH

H(4) L-Arg

Y

(7) N-methyldehydroAla

(1) D-Ala

(2) L-LeuX

(3) D-erythro-b-methylAsp

COOH

MC-LR leucin

leucintyrosin

tyrosin

tryptophanphenylanalin

argininargininarginin

argininargininarginin

MC-RRMC-LYMC-YRMC-WRMC-FR

X Y

Fig. 1. General structure of microcystin variants in this study. X and Y are variable L-amino acids.

molecules are cyclic heptapeptides, which present >76 variants generally differing inthe nature of the two l-amino acids and in the degree of methyl substitution (Fig. 1)(Dittmann & Wiegand, 2006). The toxicity of MC has been attributed to the highlyspecific inhibition of serine and threonine phosphatases (PP1–PP2A) (MacKintoshet al., 1990; Fischer & Dietrich, 2000a, b) and to the increased formation of reactiveoxygen species (ROS) (Ding et al., 1998, 2001; Li et al., 2003). It has been suggestedthat either of the above mechanisms could induce cytoskeletal damage leading to lossof cell morphology (Toivola & Eriksson, 1999; Ding & Ong, 2003).

Aquatic organisms can be exposed to MC via the consumption of toxic cyanobac-teria (Li et al., 2004; Xie et al., 2004) or aquatic organisms that had previouslyaccumulated MC in their tissues. Although negligible amounts of toxins enter thesystem through the gills or epithelium (Tencalla et al., 1994; Kent et al., 1996), itis generally believed that the oral route is the most important (Ernst et al., 2001).Several studies have been performed to assess the effect of MC on fishes (Buryet al., 1996; Kotak et al., 1996; Tencalla & Dietrich, 1997; Fischer & Dietrich,2000a; Fischer et al., 2000; Jacquet et al., 2004), although most of them focusedon the histopathological changes after the acute administration of MC by intraperi-toneal route. Nevertheless, others focused on the study of different effects on fishorally exposed to MC or to extracts of toxic cyanobacteria (Oberemm et al., 1999;Wiegand et al., 1999; Mohamed et al., 2003; Soares et al., 2004; Prieto et al., 2007).Toxic effects have been observed in various fish species, including Salmoniformes,Siluriformes, Cypriniformes as well as Perciformes, and clear differences in sus-ceptibility to toxic cyanobacteria in general (as cell suspensions or bloom material)and microcystins [primarily microcystin-LR (MC-LR)] have been found (Malbrouck& Kestemont, 2006). Microcystins affect a large number of fish organs, such asliver, intestine, kidney (Fischer & Dietrich, 2000a), heart (Best et al., 2001) andgills (Carbis et al., 1997; Bury et al., 1998a, b). Haematological disorder (Koop& Hetesa, 2000), increased activity of some serum enzymes (Carbis et al., 1997),

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

M I C RO C Y S T I N S AC C U M U L AT I O N I N C Y P R I N U S C A R P I O L A RVA E 1417

inhibition of protein phosphatases activity (Sahin et al., 1995) and death (Tencallaet al., 1994) have also been reported.

Aquatic animals not only can be damaged by cyanobacterial toxins but can bioac-cumulate them as well (Tencalla et al., 1994; Mohamed et al., 2003; Soares et al.,2004). Since the uptake of cyanobacterial toxins by fishes results primarily follow-ing oral ingestion of toxic cyanobacterial cells or contaminated tissues and manyfishes, such as those living in small eutrophicated lakes and aquaculture ponds, arenot able to avoid the ingestion of these toxic materials through food (Tencalla &Dietrich, 1997; Magalhaes et al., 2001), the accumulation of MC in fishes via thefood chain could be a threat to human food safety. In a recent study, MC were foundto be transferred mainly from contaminated aquatic animals to a chronically exposedhuman population (fishermen at a Lake Chaohu, China) together with indication ofhepatocellular damage (Chen et al., 2009).

In aquatic environments, surface aggregations of some cyanobacteria may accu-mulate to form massive scum with high cell density and toxin concentrations. Thisphenomenon often occurs in shallow littoral areas, which are the primary environ-ments for newly hatched fish larvae. As these larvae have limited ability for escapingexposure and their diet allow for the possible ingestion of toxin-containing cells,significant exposures could occur, leading to chronic effects or even death of theorganisms (Sivonen & Jones, 1999; Chorus, 2001). Studies on the development ofnewly hatched fishes are therefore critical, contributing to the overall evaluation ofthe ecological effects of MC toxicity in aquatic systems. Zhang et al. (2008) showedthat high MC concentrations retarded egg development (2–10 h delays) and larvalgrowth, reduced hatching rate (up to 45%) and caused high malformation rate (up to15%) and hepatocytes damage.

The purpose of the present study was to compare the effect of dietary intakeof two M. aeruginosa bloom samples on growth performance and MC accumu-lation in larvae of the common carp Cyprinus carpio L. Cyprinids are especiallyimportant fish species in freshwater ecosystems because of their role as direct con-sumers of phytoplankton and zooplankton, their potential for biological managementof cyanobacterial blooms and their value as food (Opuszynski & Shireman, 1995;Xie & Liu, 2001). These characteristics of cyprinids make them suitable to study theeffect of natural variations of MC composition (type and number of MC variants) inthe same toxic cyanobacterial strain. Indeed, the type of MC variants produced by agiven strain is controlled by multienzymatic complexes (NRPS/PKS-I) involved inMC biosynthesis (Dittmann & Wiegand, 2006). These multienzymatic complexes areassembled into a modular structure, with each module responsible for the activation,thiolation, modification and condensation of one amino acid substrate (Arment &Carmichael, 1996; Kleinkauf & von Dohren, 1996; Marahiel et al., 1997). Peptidesproduced by this mechanism are small (two to 48 residues) with diverse structuresand a broad spectrum of biological activities.

MATERIALS AND METHODS

B L O O M S A M P L I N GThe cyanobacterial bloom material was collected on 14 (A) and 28 (B) September 2005,

from Lalla Takerkoust reservoir situated at 35 km south-west of Marrakesh (31◦ 36′ N;

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

1418 I . E L G H A Z A L I E T A L .

8◦ 2′ W), with a 27 μm phytoplankton net. Samples were collected close to shore fromthe surface layer. The bloom-forming species was mainly M. aeruginosa identified by bothmicroscopy and polymerase chain reaction (PCR) detection. The collected samples werefreeze-dried and stored at −26◦C until microcystins quantification by high performance liquidchromatography with photodiode-array detection (HPLC-PDA) analysis.

M I C RO C Y S T I N S D E T E C T I O N A N D Q UA N T I F I C AT I O N

The toxin extraction and pre-purification were done according to Lawton et al. (1994).Briefly, 250 mg of lyophilized cyanobacterial cells were extracted three times with 70%methanol (50–75 mg dry cells ml−1 of methanol). For each extraction, the suspension wascentrifuged at 4000 g (10 min, 4◦ C). Afterwards, the supernatant was retained and thepellet was further extracted. The three methanol extracts were diluted with Milli-Q ultra-purewater to a final methanol concentration of 20% (v:v). For the microcystin pre-purification,the final extract was passed through octadecyl silicagel ODS-C18 environmental (1 g) Sep-Pak cartridges (Waters, Chromatography Division, Millipore Corp.; www.waters.com). In thisprocedure, the ODS columns were previously activated with 20 ml of methanol (100%) andwashed with 20 ml 20% methanol. Then the diluted methanolic extract was applied to thecartridges and washed with 10 ml of 20% methanol. The MC were finally eluted with 10 ml of70% methanol. The last collected fraction containing the toxins was completely evaporated at40◦ C and resuspended in 1 ml of methanol–Milli-Q ultra-pure water (50:50, v:v) and filteredthrough a GF/C glass filter before being subjected to HPLC.

Chromatographic analysis was performed by HPLC Waters equipment (model 2695; www.waters.com) with a photodiode-array detector (model 996). The column used was Chro-molith C18 (250 mm × 4·6 mm, 5 μm; www.merck-chemicals.com). The mobile phase was:(A) water (H2O) + 0·05% (v:v) triflouroacetic acid (TFA) and (B) acetonitrile (MeCN) +0·05% (v:v) TFA. During the HPLC running time of 55 min, the separation was achievedusing the solvent gradient from 70 to 0% (A). The sample volume injected was 50 μl, and themobile phase run at 1 ml min−1. The UV spectrum for each separated fraction was checkedand the microcystins variants were preliminarily identified by their characteristic UV spectrum(maximum absorbency at 238 nm). Standard MC-LR, MC-YR and MC-RR were purchasedfrom Calbiochem (Merck Chemicals). MC-FR and MC-WR were purified in the Laboratoryof Plant Physiology of the Autonomous University of Madrid. Other MC different from theprevious ones were quantified using MC-LR as a standard. The results are presented as MC-LR equivalent by adding the mass of all the variants found. MC-(H4)YR was identified byliquid chromatography–mass spectrometry. The LC-MS experiments were carried out on anAgilent 1100 series HPLC system (Agilent Technologies; www.chem.agilent.com), consist-ing of a vacuum degasser, a binary pump, an autosampler and diode array detector (DAD),coupled to a hybrid quadrupole time of flight (QTOF) instrument (QStar/Pulsar i; AppliedBiosystems; www.appliedbiosystems.com) equipped with a turbospray ion source interface.The column used was Teknokroma, MED SEA18, and the mobile phase was a gradient oftwo eluents: (A) H2O + 0·1% TFA and (B) MeCN + 0·1% TFA. The mobile phase ran at1 ml min−1. The chromatogram obtained in HPLC-MS was identical to those obtained usingHPLC-DAD.

Full scan mass spectrometry (MS) spectra were acquired in the positive ion mode, usinga source potential of 5000 V, over the mass range of 50–1500 at 1 s. Electrospray ioniza-tion (ESI)–MS and collision induced dissociation (CID)–MS were measured using N2 as acollision gas (collision energy, 3 kV) in the pressure range of 65 bar. The N2 drying temper-ature was set at 300◦ C, and the cone voltage was fixed at 70 V. Mass signals of unknowncompounds with sufficient intensities (>1000 counts in accumulated spectra) were analysed,and fragment patterns were compared with those from known or partly characterized MC.All chemicals were of chromatographic grade (Scharlau Chimie; www.scharlau.com).

D I E T P R E PA R AT I O N

A control diet (Table I) was prepared by mixing the solid powdered ingredients (previouslyground to <100 μm) followed by the addition of the lipids emulsified in water, in order to

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

M I C RO C Y S T I N S AC C U M U L AT I O N I N C Y P R I N U S C A R P I O L A RVA E 1419

Table I. Composition and chemical analysis of cyanobacterial diets fed to Cyprinus carpiolarvae

Ingredients (g kg−1)Lyophilized beef liver 370·0Brewers yeast 550·0Cod liver oil 10·0Soybean lecithin (50%) 40·0Vitamin mix* 10·0Choline chloride (50%) 10·0Mineral mix† 10·0Lyophilized cyanobacteria‡ 0·1Chemical analysisDry matter (% MD) 94·0Crude protein (% MD) 51·1Crude lipid (% MD) 6·1Ash (% MD) 7·2Gross energy (kJ g−1 MD) 19·3Microcystins (ng g−1 MD)‡ 60·0*Per kg of vitamin mix: retinol, 1 800 000 IU; calciferol, 200 000 IU; α-tocopherol, 3·5 g; ascorbic acid,5 g; thiamin-HCl, 1·5 g; riboflavin, 2·5 g; Ca pantothenate, 5 g; nicotinic acid, 20 g; pyridoxine-HCl,0·5 g; folic acid, 1 g; cyanocobalamin, 2 g; menadion sodium bis., 1 g; biotin, 0·15 g; inositol, 40 g.†Per kg of mineral mix: cobalt sulphate–7H2O, 0·04 g; copper sulphate–7H2O, 0·5 g; iron carbonate,4 g; potassium iodide, 0·06 g; magnesium oxide, 50 g; manganese oxide, 2 g; sodium selenite, 0·03 g.‡Only for diets A and B.MD, dry mass.

form a moist blend. Diets A and B were obtained from this basal diet through the addition of0·1 g kg−1 of freeze-dried material of M. aeruginosa bloom A and B, respectively, includingthe same MC content (60 ng g−1 dry matter). The blend was pelletized using a grinder andthe resulting pellets were dried at c. 40◦ C. Dry pellets were crushed and sieved to obtainparticles with graded diameter (100–200, 200–400 and 400–600 μm).

Chemical analyses of diets (Table I) were performed according to the following procedures:dry matter after drying in an oven at 105◦ C until constant mass; ash by incineration in amuffle furnace at 450◦ C for 16 h; protein (N × 6·25) by the Kjeldahl method after aciddigestion; lipid by petroleum ether extraction in a Soxtec system (Tecator; www.tecator.se)apparatus; energy by direct combustion in an adiabatic bomb calorimeter.

F I S H A N D E X P E R I M E N TA L S E T U P

First-feeding 7 days-old larvae [mean ± s.e. 2·36 ± 0·15 mg wet mass (MW) and7·64 ± 0·19 mm standard length (LS)] of C. carpio were used in the present experiment.Larvae were from eggs obtained through induced spawning of a broodstock with pituitaryextract and incubated at 25◦ C. At the beginning of the experiment, larvae were randomlyassigned to nine rearing units filled with dechlorinated tap water. Each rearing unit con-sisted of a set of two plastic tanks, an internal 5 l tank with lateral screened windows (meshsize of 500 μm), inside of which larvae (100 per tank) were placed, and an external 8 ltank, as described by Charlon & Bergot (1984). The water temperature was kept at mean± s.e. 25·3 ± 1·2◦ C and the photoperiod at 16 L (PAR of 1·9 × 10−6 μmol m−2 s−1):8Dthroughout the experimental period. Every 24 h, larvae from each rearing unit were transferred(as in Charlon & Bergot, 1984) to a clean unit with renewed water, to avoid the accumulationof excreted ammonia and the formation of a bacterial film at the bottom, as well as to ensureacceptable levels of oxygen (≥5·6 mg l−1) and pH (7–8). The experiment lasted 12 days.

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

1420 I . E L G H A Z A L I E T A L .

Diets (control diet, diet A and diet B) were tested in triplicate. Larvae were fed by handtwice daily (0900 and 2000 hours) at a ratio of 1 mg diet larva−1 day−1, corresponding to0·06 ng MC day−1 or a total of 0·72 ng MC in the 12 days period per larva. All the dietsgiven to fish were consumed in c. 2 h.

For MC analysis in larvae, 10 larvae were randomly sampled from each tank on days3 and 12, starved for 1 day and frozen at −26◦ C until MC extraction. Observations onfish development were made under a Zeiss Stemi DV4 stereomicroscope (www.zeiss.com).Photographs were taken using a Canon PowerShot A620 digital camera (www.canon.com) andanalysed using Adobe Photoshop CS3 (www.adobe.com). Larval standard length (LS) wasmeasured from these images, using the UTHSCSA Image-Tool v3.00 programme (developedat the University of Texas Health Science Center at San Antonio; www.utsi.utexas.edu). Eachday, 10 larvae were used after anaesthesia for measurement of LS and MW. Fish were handledin accordance with E.U. regulations concerning the protection of experimental animals.

D E T E R M I NAT I O N O F TOTA L M C C O N T E N T I N F I S H T I S S U EB Y E L I S A

For MC analysis in fish tissue, larvae were anaesthetized, euthanized, weighed and mea-sured before freezing. MC extraction procedure was performed as described by Smith & Haney(2006) with minor modification. Fish tissue was homogenized with an Ultraturrax homoge-nizer (www.ika.net) for 5 min. The suspension was then sonicated with an ultrasonic processorfor 2 min at c. 80 amplitude (Sonics Materials, Vibra Cell 50; www.sonic.com). Samples wereextracted for 24 h in 80% methanol at 4◦ C. Extracted samples were then clarified by centrifu-gation at 3000 g for 10 min and filtered through a 0·2 μm filter (Acrodisc, Polyethersulphone,VWR International; www.vwr.com). Fish extract was evaporated to dryness and resuspendedin ultra-pure water (Milli-Qs, Millipore; www.millipore.com). Enzyme-linked immunosor-bent serologic assay (ELISA) was performed as described by the Envirogard MicrocystinsPlate Kit (Strategic Diagnostic; www.sdix.com). The absorbency was determined in a DEN-LEY We-Scan ELISA reader at a wavelength of 450 nm. Results are reported as MC-LRequivalents.

C A L C U L AT I O N S A N D S TAT I S T I C S

Means, s.d. and s.e. for all experimental variables were calculated using the MicrosoftExcel 2007. Statistical analysis of data was performed by one-way ANOVA at a probabilitylevel of 0·05 and means were compared by the Tukey test using the SPSS 11.5 software(www.spss.com).

RESULTS

Despite comparable content of MC in both M. aeruginosa blooms [968 μg g−1

dry mass (MD) in bloom A and 976 μg g−1MD in bloom B], differences were foundregarding the type, number and percentage of MC variants, as revealed by HPLC-PDA analysis (Fig. 2 and Table II). MC-LR clearly dominated in bloom A (74·05%),while no dominance of a specific MC was detected in bloom B. MC-(H4)YR wasidentified by liquid chromatography–mass spectrometry (LC-MS) analysis (Fig. 3).

No fish died during the 12 days of the experiment and no differences in fishbehaviour could be distinguished among the treatments and the control. Fish contin-ued to consume diets A and B at the same rate throughout the experimental period.

Significant differences in larval growth were observed among all groups. Fromday 9, MW and LS in fish fed toxins-containing diets were significantly lower thanin fish fed the control diet (Figs 4 and 5), which was reflected in the specific growth

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

M I C RO C Y S T I N S AC C U M U L AT I O N I N C Y P R I N U S C A R P I O L A RVA E 1421

(a) Spectrum index plot

210·00 210·00 210·00 210·00 210·007575nm nm nm

14 810 15 687

290·00 290·00 290·00 290·00 290·00nm nm

38 017 50 577

50·57239·238·02233·315·69239·214·80239·27·57239·2

Auto-scaled chromatogram

Auto-scaled chromatogram

Microcystins variants

Percentage

Microcystins variants

Percentage

MC-LR

MC-LR MC-YR MC-(H4)YRMC-RR

MC-FR MC-LY MC-(H4)YR DMC-LR

74·05 10·02 8·59 4·45 4·22

MC-FRMC-LY

MC-LR

MC-(H4)YR DMC-LR

50·5

77

38·0

17

15·6

8714

·810

7·57

5

0·14

0·12

0·10

0·08

0·06

0·04

0·02

0·00

5·00 10·00 15·00 20·00 25·00 30·00 35·00 40·00 45·00 50·00Time (min)

Time (min)

(b)Spectrum index plot

210·00 210·00290·00 290·00 290·00 290·00 290·00 290·00

210·00 210·00 210·00 210·006773 11 011nm nm nm nm nm nm

13 567 15 560 18·381 19·896

239·2 239·2 232·1 239·2 239·2 222·7

292·2 281·6

13·57 15·57 18·38 19·906·77 11·02

MC-WR MC-FR3·013·7414·5519·8325·6233·26

0·14

0·12

0·10

0·08

0·06

0·04

0·02

0·00

5·00 10·00 15·00 20·00 25·00 30·00 35·00 40·00 45·00 50·00

MC-RR

MC-YRMC-(H4)YR

MC-LR

MC-FRMC-WR

19·8

96

18·3

81

15·5

60

13·5

67

11·0

11

6·77

3

Abs

orba

nce

units

Abs

orba

nce

units

Fig. 2. High performance liquid chromatography with photodiode-array detection chromatogram showing themicrocystins variants (see Fig. 1) and their percentages in the freeze-dried material of Microcystis aerug-inosa natural bloom (a) A and (b) B. Microcystins variants were determined according to the availablestandard samples.

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

1422 I . E L G H A Z A L I E T A L .

Table II. Amount of total microcystin (MC) and MC profile (see Fig. 1) in the two bloomsamples (A and B) collected in Lalla Takerskoust reservoir used in the assay

Bloom

Total MCμg g−1 dry

mass

MC-LR(%)

DMC-LR(%)

MC-FR(%)

MC-LY(%)

MC-RR(%)

MC-YR(%)

MC-WR(%)

MC-(H4)YR

(%)

A 976 74·05 4·22 10·02 8·59 4·45B 968 33·26 25·62 19·83 3·74 14·55

rate (G) (Table III). Moreover, between toxin fed groups, group fed diet B wassignificantly more affected than that fed diet A (Figs 4 and 5 and Table III).

The MC accumulation by fish fed diets A and B appeared to increase with exper-imental time, and was always significantly higher in fish fed diet B (Fig. 6). After3 days of feeding, the MC content was 10·87 ng g−1 and 16·21 ng g−1 in groupsA and B, respectively. In both groups, MC were rapidly accumulated in fish tis-sues after 12 days of continuous supply of toxins-containing diet (Fig. 6), but at theend the accumulation became much more important in fish nourished with diet B(26·96 ng g−1) than with diet A (17·32 ng g−1).

DISCUSSION

The intake of low and repeated doses of MC from cyanobacterial cells can inhibitgrowth, which may be due to toxic effects on C. carpio larvae. There are severalstudies describing the oral toxicity of MC in fishes after feeding them toxic algae(Fischer & Dietrich, 2000a; Li et al., 2004; Zhao et al., 2005), generally the toxiceffect being attributed to the main MC congener in cyanobacterial cells. These cellsare thought to be lysed in the fish gut after ingestion, and digestion releases inter-cellular MC into the intestinal lumen of fishes (Falconer, 1993). Following intestinalabsorption, the toxin is taken up into hepatocytes via a carrier-mediated transportsystem, and then inhibits the activity of serine and threonine protein phosphatases 1and 2A (Runnegar et al., 1993). This inhibition could disturb the cellular phospho-rylation balance and cause hyperphosphorylation of a variety of functional proteins,which leads to apoptosis and necrosis of hepatocytes (Tencalla et al., 1994; Carbiset al., 1996; Dawson, 1998; Fischer et al., 2000).

The analysis performed in this work revealed an important difference in MCcongeners present in two different M. aeruginosa blooms, with almost the same totalMC content. The variation in MC composition between the two blooms seems toaffect significantly the uptake by C. carpio, and hence MC toxicity. This might be dueto the molecular affinity of the organic ion transporters responsible for carrying MCacross cell membranes, which would differ for each type of MC. Dietrich & Hoeger(2005) agree, on the basis of experiments by Meriluoto et al. (1990) using differentepimers of 3H-dihydro-MC-LR, that minimal structural changes in the MC moleculecan have major implications for the uptake, organ distribution and excretion kineticsof these toxins. In this study, evidence for different uptake of total MC based on theirdifferent accumulation in tissues of C. carpio fed bloom materials with diverse MCcomposition was found. It could be that more hydrophilic variants are better taken

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

M I C RO C Y S T I N S AC C U M U L AT I O N I N C Y P R I N U S C A R P I O L A RVA E 1423

240

230220

210

200

190

180170

160150

140

130120

110

100

90

80

70

60

50

40

30

20

100

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 95 1000 1050 1100 1150 1200

1031·60

1049·61 [M+H]+

122·10135·09 213·10 283·18303·18 375·22 435·26 446·26 506·29 599·37 624·38174·15

70·07

115·09

107·09

112·10 127·10

155·09 163·12

174·15213·10

200·13

195·10 218·16258·19 265·18

285·18

303·18 347·22

375·22

418·23

435·26

446·26

489·27

506·29599·37

607·35130·06

138·06 167·10

122·10

[Mdh

a-A

la-(

4H)T

yr-M

eAsp

-Arg

+H

]+

[Arg

-Add

a-G

lu +

H]+ o

r [M

eAsp

-Arg

-Add

a]+

[(C

11H

14O

)-(G

lu-C

O)-

Mdh

a−A

la+]

+

[(C

11H

14O

)-(G

lu-C

O)-

Mdh

a−A

la +

H]+

[(C

11H

14O

)-(G

lu-C

O)-

Mdh

a +H

]+

[(C

11H

14O

)-(G

lu-C

O)-

Mdh

a +H

]+

[(A

rg+N

H2)-

MeA

sp +

H]+

[C11H14O +H]+

[(Arg+NH) + 2H]+

[Glu−Mdha + H]+

[Mdh

a−A

la +

H]+

135·09 [PhCH2CH(OMe)]+

Inte

nsity

(co

unts

)

0·80

0·75

0·70

0·65

0·60

0·55

0·50

0·45

0·40

0·35

0·30

0·25

0·20

0·15

0·10

0·05

0·0050 100 150 200 250 300 350 400 450 500 550 600 650 750

Fig. 3. Full scan mass spectra of MC-(H4)YR (see Fig. 1).

up and produce more drastic effects than more lipophylic ones. This is not expectedsince MC-LR is more toxic to mammals than the more hydrophilic MC-RR. Fishesdo not respond the same way as mammals. Xie et al. (2004) showed that whensilver carp Hypophthalmichthys molitrix (Valenciennes) fingerlings were exposed toa bloom containing MC-LR and MC-RR, no MC-LR was detectable in the muscleand blood samples of the fish in spite of the abundant presence of this toxin in the

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

1424 I . E L G H A Z A L I E T A L .

00

2

4

6

8

Mw

(m

g)

10

12

14

16

18

20

3 6

Time (days)

9 12

*

**

*

Fig. 4. Wet mass (MW) of Cyprinus carpio larvae during the experimental period. Significant differences weredetected among all treatments as from day 9 (Tukey’s post hoc, P< 0·05). Values are mean ± s.e. of 10animals per treatment. , statistically significant difference between all groups at each sampling time(P< 0·05). Control diet ( ): no microcystins (MC); diet A ( ): MC-LR, DMC-LR, MC-FR, MC-LY(see Fig. 1); diet B ( ): MC-LR, MC-RR, MC-YR, MC-WR (see Fig. 1).

intestines. On the other hand, the maximum MC-RR in the blood, liver and muscleof the fish was 49·7, 17·8 and 1·77 mg g−1 MD, respectively. They concluded thatH. molitrix has a mechanism to degrade MC-LR actively and to inhibit MC-LRtransportation across the intestines and that the depuration of MC-RR concentrationsoccurred slowly than uptakes in blood, liver and muscle, and the depuration rate wasin the order of blood liver muscle (Xie et al., 2004).

It is important to highlight that MC detected in fish tissues only refer to free toxins,since it was not possible to detect MC bound to protein phosphatases or glutathioneusing the standard MeOH extraction applied in this work (Amorim & Vasconcelos,1999; Thostrup & Christoffersen, 1999; Zimba et al., 2001; Magalhaes et al., 2003).A large number of MC congeners, those that contain methyldehydroalanine, formcovalent bonds to protein phosphatases 1 and 2A in animal cells, and these cova-lently bound MC are not extracted using standard MeOH extraction (Williams et al.,1997a, b; Pires et al., 2004). Consequently, total MC concentration detected in fisheswas underestimated. Williams et al. (1997a, b) reported that only c. 13% of the totalMC load in fish liver was extractable via MeOH method, 24 h after exposure. Theremaining toxin was thought to be irreversibly, covalently bound to protein phos-phatases 1 and 2A, inhibiting protein phosphatases activity. In this study, fish fromgroup A accumulated 36·81% of the administered toxin after 12 days of feeding,this rate increasing to 50·15% for fish from group B, indicating that MC structuralchanges may affect toxin transfer and uptake. Yet, this remains an important pointto be confirmed in further experimental studies.

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

M I C RO C Y S T I N S AC C U M U L AT I O N I N C Y P R I N U S C A R P I O L A RVA E 1425

7

8

9

10

11

12

13

14

30 5 9 12Time (days)

Ls

(mm

)

*

**

*

Fig. 5. Standard length (LS) of Cyprinus carpio larvae during the experimental period. Significant differenceswere detected among all treatments as from day 9 (Tukey’s post hoc, P< 0·05). Values are mean ± s.e. of10 animals per treatment. , statistically significant difference between all groups at each sampling time(P< 0·05). Control diet ( ): no microcystins (MC); diet A ( ): MC-LR, DMC-LR, MC-FR, MC-LY(see Fig 1); diet B ( ): MC-LR, MC-RR, MC-YR, MC-WR (see Fig. 1).

According to Fischer et al. (2005), MC is taken up through the multispecifictransport system for organic anions (OATP). Some fishes could depurate MC accu-mulated in the liver through biliary excretion (Sahin et al., 1996) and some otherscan degrade MC (Xie et al., 2004). On the basis of their observations on the ratioof MC-LR:MC-RR in different tissues and organs of H. molitrix, Xie et al. (2004)suggested that MC-LR may be actively degraded during digestion, and its uptakebe selectively inhibited, whereas MC-RR is transported across the intestines andembedded into body tissues. The other reason for the different accumulation ratemight be due to the fact that bioaccumulation is commonly found for lipophilic

Table III. Mean ± s.e. initial (MWI) and final wet mass (MWF) and specific growth rate (G)of Cyprinus carpio larvae fed the experimental diets control A and B

MWI (mg) MWF (mg) G* (%)

Control diet 2·44 ± 0·07a 18·42 ± 0·39a 17·77 ± 0·25a

Diet A 2·21 ± 0·16a 15·33 ± 0·40b 15·94 ± 0·48b

Diet B 2·42 ± 0·07a 13·39 ± 0·26c 14·25 ± 0·26c

*G = 100(ln MWF − ln MWI)t−1, where t = time (days).

Means in the same column sharing different superscript lowercase letters are statistically different(P < 0·05).

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1415–1430

1426 I . E L G H A Z A L I E T A L .

3 12

Time (days)

0

5

10

15

MC

acc

umul

atio

n (n

g g

−1)

20

25

30

35

Fig. 6. Microcystin (MC) accumulation in tissues of Cyprinus carpio larvae fed diets A ( ) and B ( ).Significant differences were detected between fish treatments (ANOVA, P< 0·05) in days 3 and 12 ofthe feeding experiment. Values are mean ± s.e. Control diet: no MC; diet A: MC-LR, DMC-LR, MC-FR,MC-LY (see Fig. 1); diet B: MC-LR, MC-RR, MC-YR, MC-WR (see Fig. 1).

toxicants and less important for hydrophilic compounds (De Maagd et al., 1999).Vesterkvist & Meriluoto (2003) also demonstrated that certain MC congeners maybe more lipophilic than the hydrophilic MC-LR. These MC variants are believedto be more cell-permeable than the more hydrophilic MC (Kuiper-Goodman et al.,1999) and may be less dependent on the bile acid transporter system to pass a cellmembrane (Sivonen & Jones, 1999). Additional research should be focused on thesecongener specificities, for a better understanding of MC uptake kinetics. This under-standing may be crucial for proving risk assessment from MC exposure in naturalconditions.

This work was carried out within the framework of the Morocco–Portuguese coopera-tion (convention of cooperation CNRST-Morocco–GRICES Portugal; B. Oudra and V. M.Vasconcelos), and Morocco–Spanish cooperation (AECI project A/17389-08 and bilateralagreement between UCAM and UAM; B. Oudra and F. F. del Campo).

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