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Ecotoxicology and Environmental Safety 73 (2010) 762–770

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Ecotoxicology and Environmental Safety

0147-65

doi:10.1

n Corr

Ambien

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E-m

journal homepage: www.elsevier.com/locate/ecoenv

Effect of different microcystin profiles on toxin bioaccumulationin common carp (Cyprinus carpio) larvae via Artemia nauplii

El Ghazali Issama, Saqrane Sanaaa, Carvalho Antonio Paulob,c, Ouahid Younessd,Del Campo Francisca F.d, Oudra Brahima, Vasconcelos Vitorb,c,n

a 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, Moroccob Centro Interdisciplinar de Investigac- ~ao Marinha e Ambiental, CIIMAR/CIMAR-LA, Rua dos Bragas 289, Porto 4050-123, Portugalc Departamento de Zoologia e Antropologia, Faculdade de Ciencias, Universidade do Porto, 4169-007 Porto, Portugald Departamento de Biologia, Laboratorio de Fisiologia Vegetal, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain

a r t i c l e i n f o

Article history:

Received 11 September 2009

Received in revised form

6 December 2009

Accepted 7 December 2009Available online 31 December 2009

Keywords:

Cyanobacterial blooms

Microcystin variants

Accumulation

Transfer

Toxicity

Fish growth

13/$ - see front matter & 2009 Elsevier Inc. A

016/j.ecoenv.2009.12.015

esponding author at: Centro Interdisciplinar

tal, CIIMAR/CIMAR-LA, Rua dos Bragas 289

51 223380609.

ail addresses: vmvascon@fc.up.pt, vitor.vascon

a b s t r a c t

In this study, a 12-day growth trial was conducted to compare the effect of the variation in microcystin

(MC) composition in two Microcystis aeruginosa bloom samples on the growth performance and MC

accumulation/transfer in the common carp (Cyprinus carpio L.) larvae. Fish were fed Artemia salina

nauplii that had been preexposed to extracts from two M. aeruginosa natural blooms with different

microcystins (MCs) profiles. Bloom A had MC-LR as major toxin (74.05%) while bloom B had a diversity

of MC (MC-RR; MC-(H4)YR; MC-YR; MC-LR; MC-FR; MC-WR) with no dominance of MC-LR. Newly-

hatched Artemia nauplii were exposed separately to the two M. aeruginosa extracts A and B

(100 mg L�1 Eq MC-LR) for 2 h. The MC concentration in the nauplii was 73.6077.88 ng Eq MC-

LR g�1 FW (n=4, mean7SE) for bloom A and 87.04710.31 ng Eq MC-LR g�1 FW for bloom B. These

contaminated nauplii were given at the same ration to different groups (A and B) of fish larvae. Larval

weight and length from day 9 were significantly different between groups A and B, and in both cases

lower than that of a control group fed non-exposed nauplii. MCs accumulation by larvae, inversely

correlated with the growth performance, was also significantly different between groups A and B

(37.4372.61 and 54.5573.01 ng Eq MC-LR g�1 FW, respectively, at the end of the experimental

period). These results indicate that MC profile of a bloom may have differential effects on toxin

accumulation/transfer and toxicity.

& 2009 Elsevier Inc. All rights reserved.

1. Introduction

Cyanobacteria blooms represent a problem throughout theworld due to the eutrophication of the aquatic environment(Chorus and Bartram, 1999). Several cyanobacteria speciesproduce a variety of potent toxins, including a group ofhepatotoxins called microcystins (MCs) (Kaya, 1996). Thesemolecules are cyclic heptapeptides of which around 80 variantshave been identified (Dietrich and Hoeger, 2005), differing in thenature of the two L-amino acids and in the degree of methylsubstitution (Dittmann and Wiegand, 2006). The most frequentand studied variant is microcystin-LR (MC-LR) with the variableamino acids leucine (L) and arginine (R). Other variants that alsooccur more frequently are MC-RR, MC-YR and MC-LA (Figueiredo

ll rights reserved.

de Investigac- ~ao Marinha e

, Porto 4050-123, Portugal.

celos@cimar.org (V. Vitor).

et al., 2004). The type of MC variants produced by a given strain iscontrolled by multienzymatic complexes (NRPS/PKS-I) involved inMC biosynthesis (Dittmann and Wiegand, 2006). These multi-enzymatic complexes are assembled into a modular structure,with each module responsible for the activation, thiolation,modification, and condensation of one amino acid substrate(Arment and Carmichael, 1996). Peptides produced by thismechanism are small (2–48 residues) with diverse structuresand a broad spectrum of biological activities. MCs are intracellularbut may rapidly and massively be released by cell lyses due tonatural senescence, herbicides or physical stress (Ross et al.,2006). The toxicity of microcystin (MC) has been attributed to thehighly specific inhibition of protein phosphatases (PP1, 2A, 4, 5)(MacKintosh et al., 1990; Yoshizawa et al., 1990). These secondarymetabolites (Wiegand and Pflugmacher, 2005) have been recog-nized as human and animal health hazards, since they have beenshown to cause adverse effects in mammals, fish, invertebrates aswell as plants (Wiegand and Pflugmacher, 2005; Malbrouck andKestemont, 2006). Toxicity has been reported at environmental

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exposure levels for several fish species, including catfish (Zimbaet al., 2001), carp (Xie et al., 2004), zebrafish (Oberemm et al.,1999; El Ghazali et al., 2009) and salmon (Anderson et al., 1993).MC affect a large number of fish organs, such as liver, intestine,kidney (Fischer and Dietrich, 2000), heart (Best et al., 2001) andgills (Carbis et al., 1997). Hematological disorder (Koop andHetesa, 2000), increased activity of some serum enzymes (Carbiset al., 1997), inhibition of protein phosphatases activity (Sahinet al., 1996) and death (Tencalla et al., 1994) have also beenreported. In addition to their effects on aquatic animals, MCs canalso be bioaccumulated (Ibelings and Chorus, 2007). MC accumu-lation has been shown to occur in zooplankton (Ferrao-Filho et al.,2002), mussels (Dionisio Pires et al., 2004), snails (Xie et al., 2007),crustaceans (Vasconcelos et al., 2001), fish (Xie et al., 2005) andrecently in turtle and water bird (Chen et al., 2009b). MCaccumulates in vertebrate cells due to active transport by ahighly expressed unspecific organic anion transporter (bile acidcarrier transport system) (Wiegand and Pflugmacher, 2005). It hasbeen suggested that accumulation and toxic effects of MC aregoverned by the presence/absence, type and expression level ofthis organic anion transport proteins (Dietrich and Hoeger, 2005).Moreover, it is reported that MC can be transferred along foodchains in the natural environment (Ibelings et al., 2005; Smith andHaney, 2006). Trophic transfer also has been demonstrated underlaboratory conditions in which hepatotoxins were transferredfrom zooplankton to fish (Karjalainen et al., 2005). Other potentialvectors include aquatic plants and macroalgae (Saqrane et al.,2007; Pflugmacher et al., 2007). Serious implications exist for thetransfer of MC to a higher trophic level through the food web(Ozawa et al., 2003). Chen et al. (2009a) recently present amilestone work in MC exposure research that MCs weretransferred along food chain from contaminated fisheries pro-ducts to fishermen (a chronically exposed human population atLake Chaohu in China) together with indication of hepatocellulardamage.

The aim of this study was to compare the effect of the MCprofile of two Microcystis aeruginosa natural blooms on the growthperformance and MC accumulation/transfer in common carp(Cyprinus carpio L.) larvae. Toxins are provided via Artemia salina

nauplii preexposed to the bloom extracts. With this approach, wewanted to study, under laboratory conditions, the relationshipbetween different MC profiles taken up by carp larvae viacontaminated Artemia nauplii and MC accumulation and effectsin fish.

2. Material and methods

2.1. Bloom sampling

Two cyanobacterial bloom materials (A and B) were collected respectively in

14 and 28 September 2005, from Lalla Takerkoust reservoir localized at 35 km

south-west of Marrakesh (311360N, 8120W), with a 27 mm Nitexs phytoplankton

net. Samples were collected close to shore from the surface layer. The main bloom-

forming species was identified as M. aeruginosa by both microscopy and PCR

detection. The collected samples were freeze-dried and stored at �26 1C until MC

quantification by HPLC-PDA analysis.

2.2. Microcystins detection and quantification

The toxin extraction and pre-purification were done according to Lawton et al.

(1994). Briefly, 1 g of lyophilized cyanobacterial cells was extracted three times

with 70% methanol (10 mg dry cells per mL of methanol). For each extraction, the

suspension was centrifuged at 4000g (10 min, 4 1C). Afterwards, the supernatant

was retained and the pellet was further extracted. The three methanolic extracts

were diluted with Milli-Q ultra-pure water to a final methanol concentration of

20% (v/v). For the MC pre-purification, the final extract was passed through

Octadecyl-silicagel ODS-C18 environmental (1 g) Sep-Pak cartridges (Waters,

Chromatography Division/Millipore Corp.). In this procedure, the ODS columns

were previously activated with 20 mL of methanol (100%) and washed with 20 mL

of 20% methanol. Then the methanolic extract was applied to the cartridges and

washed with water and with 10 mL of 20% methanol. The MCs were finally eluted

with 20 mL of 70% methanol. The last collected fraction containing the toxins was

completely evaporated at 40 1C and resuspended in 1 mL of methanol/Milli-Q

ultra-pure water (50:50, v/v) and filtered through a GF/C glass filter before being

subjected to HPLC analysis.

Chromatographic analysis was performed by a HPLC Waters equipment

(model 2695) with a photodiode array detector (model 996). The column used

was Chromolith C18 (250�4.6 mm2, 5 mm). The mobile phase was as follows:

(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 achieved using the solvent gradient from 70% to 0% (A) and from 30% to 100%

(B), respectively. The sample volume injected was 50 mL, and the mobile phase run

at 1 mL min�1. The UV spectrum for each separated fraction was checked and the

MCs variants were preliminarily identified by their characteristic UV spectrum

(max absorbency at 238 nm). Standard MC-LR, -YR and -RR were purchased from

Calbiochem (Germany). MC-FR and -WR were purified in the Laboratory of Plant

Physiology of the Autonomous University of Madrid. Other MCs 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 by Liquid

chromatography–mass spectrometry, the LC–MS experiments were carried out on

an Agilent 1100 series HPLC system (Agilent Technologies, CA), consisting of a

vacuum degasser, a binary pump, an autosampler and DAD detector, coupled to a

hybrid quadrupole time of flight (QTOF) instrument (QStar/Pulsar i; Applied

Biosystems, CA) equipped with a turbospray ion source interface. The column used

was Teknokroma, MED SEA18, and the mobile phase was a gradient of two

eluents: (A) H2O+0.1% TFA, and (B) MeCN+0.1% TFA. The mobile phase runs at

1 mL min�1. The chromatogram obtained in HPLC-MS, was identical to those

obtained using HPLC-DAD.

Full scan MS spectra were acquired in the positive ion mode, using a source

potential of 5000 V, over the mass range of 50–1500 at 1 s. ESIMS/collision

induced dissociation (CID). Mass spectra were measured using N2 as a collision gas

(collision energy, 3 kV) in the pressure range of 65 Bar. The N2 drying temperature

was set at 300 1C, and the cone voltage was fixed at 70 V. Mass signals of unknown

compounds with sufficient intensities (41000 counts in accumulated spectra)

were analyzed, and fragment patterns were compared with those from known or

partly characterized MCs. All chemicals were of chromatographic grade (Scharlau

Chimie Barcelona, Spain).

2.3. Experimental set up

2.3.1. Brine shrimp

Cysts of the brine shrimp A. salina were incubated in seawater at 25 1C for 24 h

with vigorous continuous aeration at a constant irradiance (1.9�10�6 mmol m�2

s�1) provided by white fluorescent lights. Newly-hatched A. salina nauplii were

transferred to fresh seawater and exposed to M. aeruginosa extracts A and B

(100 mg L�1 Eq MC-LR) separately in aerated 1-L glass bottles for 2 h at 25 1C. After

exposure the nauplii were collected in a 100-mm-mesh-sized net, rinsed with

200 mL of seawater and given to the fish larvae from the same group (A and B). The

control nauplii were treated similarly, but without exposure to MCs. Brine shrimp

nauplii were observed under a binocular stereomicroscope after the first

incubations to check if M. aeruginosa extracts A and B increased mortality. No

differences between the treatments were observed, and we concluded that MCs

concentration used did not affect nauplii survival.

2.3.2. Fish

First-feeding 7 days-old larvae (2.670.4 mg wet weight and 7.6470.19 mm

standard length) of common carp (C. carpio L.) were used in the present

experiment. Larvae were issued from eggs obtained through induced spawning

of a broodstock with pituitary extract and incubated at 25 1C. At the beginning of

the experiment, larvae were randomly assigned to 9 rearing units filled with

dechlorinated tap water, in order to have triplicates of two treatments (A and B)

and a control. Each rearing unit consisted of a set of two plastic tanks, an internal

5 L-tank with lateral screened windows (mesh size of 500 mm), inside of which

larvae (100 per tank) were placed, and an external 8 L-tank, as described by

Charlon and Bergot (1984). The water temperature was kept at 25.371.2 1C and

the photoperiod at 16 h light (PAR of 1.9�10�6 mmol m�2 s�1) throughout the

experimental period. Every 24 h larvae from each rearing unit were transferred

(as in Charlon and Bergot, 1984) to a clean unit with renewed water, to avoid the

accumulation of excreted ammonia and the formation of a bacterial film at the

bottom, as well to ensure acceptable levels of oxygen (Z5.6 mg L�1) and pH

(ranged between 7 and 8). Larvae were exposed to the MC via Artemia because

they prefer live food and it is a more coherent way to contaminate the larvae via

zooplankton. Once a day fish from groups A and B were fed preexposed nauplii

correspondent to each group. Control groups were fed non-preexposed nauplii. In

all cases, during the 12-day experiment, a total of 3200 nauplii per day were

distributed in each tank, which were rapidly consumed by carp larvae.

Observations on fish development were made under a Zeiss Stemi DV4

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stereomicroscope and images of larvae were taken using an attached Canon

PowerShot A620 digital camera and analyzed using Adobes Photoshop CS3. Larval

standard length was measured from these images, using the UTHSCSA Image-Tool

v3.00 program (developed at the University of Texas Health Science Center at San

Antonio, TX, USA). At the start of the experiment mean wet weight and standard

body length of carp larvae was 2.670.4 and 7.6470.19 mm (mean7SE),

respectively. On days 3, 6, 9 and 12, ten larvae per tank were randomly sampled,

anaesthetized, weighed and measured for standard body length. These larvae were

not sacrificed and were put back to the tanks after the measurements.

For MCs analysis in larvae, ten larvae per tank were randomly sampled on days

6 and 12, starved for 1 day, anaesthetized, weighed and frozen at �26 1C until MCs

extraction. To avoid the sacrifice of many fish we did not aim to study the

accumulation dynamics but rather the result of a 12 day accumulation

experiment. Fish were handled in accordance with European Union regulations

concerning the protection of experimental animals.

2.4. Determination of total MCs content in fish tissue and A. salina nauplii by ELISA

MCs extraction procedure was performed as described by Smith and Haney

(2006) with minor modifications. Due to the small size of the larvae it was not

possible to isolate the different organs, so fish tissue was homogenized with an

Ultraturrax homogenizer for 5 min. The suspension was then sonicated with an

ultrasonic processor for 2 min at ca. 80 amplitude (Sonics Materials, Vibra Cell 50).

Samples were extracted for 24 h in 80% methanol at 4 1C. Extracted samples were

then clarified by centrifugation at 3000g for 10 min and filtered through a 0.2 mm

filter (Acrodisc, Polyethersulfone, VWR International). Fish extract was evaporated

to dryness and resuspended in ultra-pure water (Milli-Qs, Millipore).

For A. salina, the nauplii were collected in a 100-mm-mesh-sized net after

toxins exposure, rinsed with 200 mL of seawater and dried prior to freezing at

�26 1C. MCs extraction procedure was performed as described above.

ELISA was performed as described by the Envirogards Microcystins Plate Kit

(Strategic Diagnostic, Newark, DE, USA). The absorbency was determined in a

DENLEY We-Scan ELISA lector at a wavelength of 450 nm. Results are reported as

MC-LR equivalents.

2.5. Calculations and statistics

Accumulation rate is estimated based on the amount of toxins accumulated in

carp larvae at the end of the experiment (X) compared to the total amount of toxin

available to fish (Y):

Accumulation rate %ð Þ ¼ X=Y� �

� 100

Means, standard deviations and standard errors for all experimental

parameters were calculated using the Microsofts Excel 2007 software. Statistical

analysis of data was performed by one-way analysis of variance (ANOVA) at a

probability level of 0.05 and means were compared by the Tukey’s test using the

SPSS 11.5 software.

3. Results

In spite of a comparable MCs content in both M. aeruginosa

blooms A and B (968 mg g�1 d.w. in bloom A and 976 mg g�1 d.w.in bloom B), differences were found respecting the type, numberand percentage of MC variants, as revealed by HPLC-PDA analysis(Fig. 1). In bloom A, MC-LR was the predominant variantrepresenting 74.05% of the total, while no dominance of aspecific MC in bloom B. MC-(H4)YR was identified by LiquidChromatography–Mass Spectrometry (LC–MS) analysis (Fig. 2).

The nauplii exposed to bloom A extract contained 73.6077.88 ng Eq MC-LR g�1 FW (n=4, mean7SE) measured by theELISA. While, MC concentration reached 87.04710.31 ng Eq MC-LR g�1 FW (n=4, mean7SE) in nauplii exposed to bloom Bextract. This difference was statistically non-significant (ANOVA,p=0.084). Based on these concentrations and on the amount ofnauplii provided as food, an intake of 0.097 ng Eq MC-LR larva�1

day�1 totaling 1.16 ng Eq MC-LR larva�1 at the end of the ex-periment in group A and of 0.102 ng Eq MC-LR larva�1 day�1

totaling 1.22 ng Eq MC-LR larva�1 in group B could be estimated.MCs were detected in all fish samples from groups A and B.In both groups, MCs rapidly accumulated in the fish tissueof carp larvae fed toxins-containing nauplii (Fig. 3). MCsaccumulation in groups A and B was, respectively, 22.0974.26

and 33.6073.48 ng Eq MC-LR g�1 FW (n=4, mean7SE) inday 6, and was significantly different (ANOVA, p=0.006). At theend of the experiment this concentration increased in bothgroups to 37.4372.61 and 54.5573.01 ng Eq MC-LR g�1 FW(n=4, mean7SE), respectively, the difference remaining statis-tically significant (ANOVA, p=0.001).

During the 12 days of the experiment, difference in mortalitywas not statistically significant among the three groups (data notshown) and no differences in fish behavior could be distinguishedamong the treatments and the control. Fish continued to consumeA. salina nauplii at the same rate throughout the experimentalperiod.

Larvae from groups A and B showed significant reductions(po0.05) in both body weight and length (Figs. 4 and 5). Fromday 3, standard length in fish fed toxins-containing nauplii wassignificantly lower (po0.05) than in fish fed the control nauplii,and from day 6 marked reductions (po0.05) in larvae bodyweight was also observed. Table 1 shows that specific growth rate(SGR) of the fish fed MCs-containing nauplii (A and B) wassignificantly lower (Tukey’s test, po0.05) than that fed thecontrol nauplii. Moreover, between toxins fed groups, group Bwas significantly more affected (ANOVA, po0.05) (Figs. 4 and 5and Table 1), and this difference increased during theexperimental period (Figs. 4 and 5).

4. Discussion

In this study, a 12-day growth trial was conducted to comparethe effect of the variation in MC composition between twoM. aeruginosa bloom materials on the growth performance andMC accumulation/transfer in the common carp (C. carpio) larvae.Fish were exposed to cyanobacterial toxins through A. salina

nauplii that had been preexposed to extracts from twoM. aeruginosa blooms (A and B) with different MC profiles. Thisexposure takes place without direct contact between the fish andthe cyanobacteria; the zooplankton was used as the toxin vector.

The brine shrimp A. salina, a widely-used ecotoxicologybioassay organism, had been shown to be sensitive to MC(Campbell et al., 1994), but in this study the survival rate is notsignificantly different (Tukey’s test, p40.05) between control andexposed nauplii (result not shown), probably due to the shortexposure time (2 h). On the other hand, we found that this time isenough for MC accumulation in nauplii exposed to cyanobacterialextract from bloom A and B (73.6077.88 and 87.04710.31 ngEq MC-LR g�1 FW, respectively). MC have been found to accumu-late in several species of zooplankton (Watanabe et al., 1992;Thostrup and Christoffersen, 1999), and in natural zooplanktoncommunity (Ferrao-Filho et al., 2002). Contamination of theseorganisms can occur by exposure to soluble toxins or directconsumption of cyanobacterial cells.

In our work, the ingestion of contaminated nauplii induced amarked growth inhibition (obvious reductions in body length andweight) in fish larvae. There are several studies describing the oraltoxicity of MC in fish (Fischer and Dietrich, 2000; Li et al., 2004;Zhao et al., 2005), generally the toxic effect being attributed to themain MC congener in cyanobacterial cells. Following intestinalabsorption, the toxin is taken up into hepatocytes via a carrier-mediated transport system. When MC have reached the intracel-lular compartment they inhibit the activity of serine/threonineprotein phosphatases 1, 2A, 4 and 5 (Zurawell et al., 2005) by atwo-step mechanism (Craig et al., 1996), after an initial rapidnoncovalent binding, MCs with N-methyldehydroalanine canform a covalent bond with the Cys273 in PP1 or Cys266 in PP2A(MacKintosh et al., 1995; Runnegar et al., 1995). This inhibitioncould disturb the cellular phosphorylation balance and cause

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Fig. 1. HPLC-PDA chromatogram showing the microcystin variants and their percentages in the freeze-dried material of Microcystis aeruginosa natural bloom A and B.

Microcystin variants were determined according to the standard samples.

E.G. Issam et al. / Ecotoxicology and Environmental Safety 73 (2010) 762–770 765

hyperphosphorylation of a variety of functional proteins, whichleads to apoptosis and/or necrosis of hepatocytes (Tencalla et al.,1994; Fischer and Dietrich, 2000). In natural environment, fishcan be exposed to MC via the consumption of toxic cyanobacteria

(Li et al., 2004; Xie et al., 2004) or aquatic organisms that hadpreviously accumulated MCs in their tissues (Lance et al., 2007);however, negligible amounts enter the system through the gills orepithelium (Tencalla et al., 1994). Route of exposure has been

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Fig. 2. Full scan mass spectra of MC(H4)-YR.

E.G. Issam et al. / Ecotoxicology and Environmental Safety 73 (2010) 762–770766

shown to be of great importance as exposure via medium causedfar less effects and no mortalities compared with the same lethaldosage applied orally (Tencalla et al., 1994). In addition, Oberemm(2001) reported that the young life stages of fish were moresensitive to MC hepatotoxic effects than adults or juveniles. In astudy using the loach (Misguruns mizolepis), embryos and larvae ofthis small freshwater fish, were shown to be affected by toxicity ofMC-LR which targets their liver and heart (Liu et al., 2002). Ingibel carp (Carassius auratus gibelio), high mortality was observedwhen fish were fed low MC content feed (1.02–10.76 mg kg�1

body weight) (Zhao et al., 2005).In the present study, the intake of MC did not affect the

survival of C. carpio larvae, but we found evidence for the directtransfer of MC from A. salina nauplii to fish and the subsequent

accumulation of toxin in the larvae tissue. Some studies havefound that MC can be transferred along the food web. Ibelingset al. (2005) studied the distribution of MC in the food web of LakeIJsselmeer and found that transfer of MC within the food webtakes place, despite no evidence for biomagnification. In anotherfield study, Xie et al. (2005) reported that MCs showed a generaltendency to accumulate up the food chain in Lake Chaohu: MC-LRwas relatively low in tissues and organs of phytoplanktivoroussilver carp, despite the direct feeding on toxic cyanobacteria, andhigher in predatory and omnivorous fish. Smith and Haney (2006)examined MC concentrations in three levels of an aquatic foodweb (phytoplankton, zooplankton, and sunfish) and foundevidence for the direct transfer of MC from zooplankton tosunfish and the subsequent accumulation of toxin in the liver

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tissue. Ibelings and Chorus (2007) indicated that toxin content ateach trophic level is dependent on biodilution and on thebioaccumulation vs. biotransformation capacity of the variousorgans. Moreover, MC appears to be absorbed through the GI tractof fish in greater proportion when the toxin is administeredthrough a vector (such as the zooplankton) rather than throughtoxic cyanobacteria directly (Smith and Haney, 2006). Due to theirstructure and size, MC does not readily penetrate the cellmembrane via simple diffusion but rather require the presenceof multi-specific organic anion transporting polypeptides foractive uptake (Monks et al., 2007). The transported toxin is alsosubject to detoxication and excretion. Detoxication means thatthe organism enzymatically can form a conjugate of the

Fig. 3. Microcystin accumulation in tissues of carp larvae fed the preexposed

nauplii from groups A and B. Significant differences were detected between

treatments (ANOVA, po0.05) in day 6 and 12 of the feeding experiment. Values

are mean7SE.

Fig. 4. Wet weight of common carp larvae during the experimental period. Significant d

among all groups as from day 9 (Tukey’s post hoc, po0.05). Values are mean7SE of 1

hepatotoxins, which helps the organism to survive undercyanobacterial stress (Pflugmacher et al., 1998). For the excretionof free toxins, animals have an ability to produce conjugates of MCin order to make the toxins more hydrophilic and less toxic(Metcalf et al., 2000).

In the present study, MC concentration in nauplii and fishtissues refers only to free MC, since a part of MC in the tissues ofaquatic organisms are irreversibly bound in a complex withprotein phosphatases (Williams et al., 1997a, b). In fact, thecovalent microcystin–PPase complex could not be extracted anddetected in this study as in similar studies that have measuredmethanol-extracted MC using ELISA (Zimba et al., 2001; Smithand Haney, 2006). This means that the MC concentrations foundin nauplii and fish tissues were probably underestimated in thepresent experiment. The question is to know if the covalentmicrocystin–PPase complex is still as toxic as unbound MC and ifthe covalently bound MC will be readily bioavailable for the nexttrophic level. Ibelings et al. (2005) reported that the toxicity of themicrocystin–PPase complex may be in the order of the micro-cystin–glutathione conjugates, but few studies have addressedthe issue of covalently bound MC in freshwater organisms andmore research needs to be conducted to determine if covalentlybound MC is transferable up the food web.

In our study, MC accumulation by fish fed toxins-containingnauplii appeared to increase along the experimental period. Fromthe total administered toxin, 49.6% was accumulated in fish fromgroup A, this rate increasing to 62.16% in fish from group B,indicating that MC structural differences may have effects ontoxins transfer, uptake and/or accumulation. To date, this is theonly laboratory study to show that MCs transfer and accumula-tion depends on toxins profile of cyanobacterial blooms. Dietrichand Hoeger (2005) agree, on basis of experiments by Meriluotoet al. (1990), that minor structural changes, characteristic of thedifferent MC congeners, may have major effects on uptake, organdistribution and excretion of these toxins. Indeed, minor changesin the MC structure may alter the biophysical properties likelipophilicity (De Maagd et al., 1999; Vesterkvist and Meriluoto,

ifferences were detected between the control and groups A and B as from day 6 and

0 animals per treatment.

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Fig. 5. Standard larval length of common carp larvae during the experimental period. Significant differences were detected between the control and groups A and B as from

day 3 and among all groups as from day 9 (Tukey’s post hoc, po0.05). Values are mean7SE of 10 animals per treatment.

Table 1Mean initial and final wet weight and specific growth rate of carp larvae fed the

normal and preexposed nauplii from groups A and B.

Initial body

weight (mg)

Final body

weight (mg)

Specific growth

rate (%)

Control 2.670.4a 19.871.5a 143.7711.7a

A 2.670.3a 15.571.1b 107.779.1b

B 2.670.4a 12.071.3c 78.8712.4c

Specific growth rate (%)=[(final body length� initial body length)/time]�100.

Values are means7standard error.

Means in the same column with different superscripts are statistically different

(po0.05).

E.G. Issam et al. / Ecotoxicology and Environmental Safety 73 (2010) 762–770768

2003). The more hydrophobic MC-LF and MC-LW showed highersurface activity on artificial bilayers compared to the -LR variant,and thus might interact more easily with biological membranes(Vesterkvist and Meriluoto, 2003), and are believed to be morecell-permeable than the more hydrophilic MCs (Kuiper-Goodmanet al., 1999). Moreover, these MC variants might be lessdependent on the bile acid transporter system to penetrate lipidmembranes of animal cells (Sivonen and Jones, 1999). Thedifferences between hydrophilic and hydrophobic MC could resultin changes in organotropism, toxicokinetics and bioaccumulation(Vesterkvist and Meriluoto, 2003). Ward and Codd (1999) found apositive correlation between toxicity and hydrophobicity of MC inTetrahymena pyriformis cells. The cells were more sensitive to thetyrosine-containing MC-LY compared to the hydrophilic MC-LR.Lipophilicity of a chemical (as determined by the octanol-waterpartition coefficient, log Kow) is also a strong determinant of therisk for biotransfer (Ibelings et al., 2005); for example MC-LR has avery low log Kow (De Maagd et al., 1999) and its depuration frombiota is relatively fast (Yokoyama and Park, 2003). On the basis oftheir observations on the ratio of MC-LR:MC-RR in differenttissues and organs of silver carp, Xie et al. (2004) suggested thatMC-LR may be actively degraded during digestion, and its uptakebe selectively inhibited, whereas MC-RR is transported across the

intestines and embedded into body tissues. The intestinal tractcan also selectively inhibit the transportation of MC-LR (Tencallaand Dietrich, 1997). Additional research should be conducted toprovide a deeper understanding of the processes that drive theuptake and transfer of particular MCs variants in the food web.

5. Conclusion

Toxicity, measured as larvae length and weight reductioncompared to control and a MC-LR dominated sample, and MCaccumulation seems to be higher when fish larvae were exposedto a bloom containing a high diversity of equally abundantmicrocystins. Although most of the studies with pure MC useMC-LR, because it has been shown to be the most toxic tomammals, it seems that fish have a stronger reaction to a mixtureof MC variants. So data using pure MC-LR in experimental worksmay underestimate the real impact of a cyanobacteria bloomcontaining a mixture of MC variants.

Acknowledgments

This work is carried out within the framework of thecooperation Morocco–Portuguese (convention of cooperationCNRST-Morocco/FCT-Portugal; Prof. Brahim Oudra/Prof. V.M.Vasconcelos). And Morocco–Spanish (AECI project A/017389/08and bilateral agreement between UCAM and UAM; Prof. BrahimOudra/Prof. F. F. del Campo).

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