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Toxicon 53 (2009) 786–796

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Toxicon

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Physiological changes in Triticum durum, Zea mays, Pisum sativum andLens esculenta cultivars, caused by irrigation with water contaminatedwith microcystins: A laboratory experimental approach

Sana Saqrane a, Youness Ouahid b, Issam El Ghazali a, Brahim Oudra a, Lahcen Bouarab a,Francisca F. del Campo b,*

a Department of Biology, Laboratory of Biology and Biotechnology of Microorganisms, Microbiology and Environmental Toxicology Unit, Faculty of SciencesSemlalia, B.P 2390, Marrakech, University Cadi Ayyad, Avenue Prince Moulay Abdallah, 40000-Marrakech, Moroccob Laboratorio de Fisiologıa Vegetal, Departamento de Biologıa, Universidad Autonoma de Madrid, Darwin 2, 28049-Madrid, Spain

a r t i c l e i n f o

Article history:Received 10 October 2008Received in revised form 26 January 2009Accepted 27 January 2009Available online 4 February 2009

Keywords:CyanobacteriaCyanotoxinMicrocystinWheatMaizePeaLensHPLCLC-MSPlant toxicityHealth risk

* Corresponding author. Tel.: þ34 91 4978189; faE-mail address: francisca.delcampo@uam.es (F.F.

0041-0101/$ – see front matter � 2009 Elsevier Ltddoi:10.1016/j.toxicon.2009.01.028

a b s t r a c t

The aim of the present study was to investigate the effect of exposure to a microcystin(MC)-containing extract from a cyanobacteria bloom on growth, development, mineralnutrient accumulation, and photosynthetic activity of Triticum durum, Zea mays, Pisumsativum and Lens esculenta cultivars. The MCs in the extract, identified by HPLC and/ormass spectrometry (MS) were: MC-RR, -LR, -YR, -(H4)YR, -WR, and -FR. Plant growth anddevelopment was tested along 30 exposure days. After this period, MC-extract causeda clear reduction in plant growth and productivity, as well as deleterious effects ondevelopment and Photosystem II activity, measured by Fv/Fm fluorescence. However, thechlorophyll (aþ b) content hardly varied, and the accumulation of Naþ, Kþ, Ca2þ, P and Nwas enhanced. All the effects observed were plant species, MC concentration, and expo-sure-time dependent. Relative accumulation of each MC variant greatly varied amongplant species and plant organ. The data obtained supports the idea that the use of surfacewater containing MCs for crop irrigation can affect both plant yield and quality, andsecondly, that MC accumulation in edible plants might pose a potential risk for human andanimal health, if the MC intake exceeded the recommended tolerable limits.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

It is widely recognized that toxins produced by cyano-bacteria blooms can be an important problem for fisheries,aquaculture, farming, and human health. Therefore,surveys of cyanobacteria and cyanotoxins have beencarried out in several countries, aimed at avoiding sanitaryrisks by a safe use of cyanotoxin contaminated water(Chorus and Bartram, 1999). However, cyanotoxins in waterutilized to irrigate food plant crops have not yet been

x: þ34 914978344.del Campo).

. All rights reserved.

considered within any official monitoring program onwater quality, in spite of edible plants constitutinga significant indirect route of human exposure to cyano-bacteria toxins.

Previous studies clearly indicate that irrigation withwater containing MC-cyanobacteria can be a threat for boththe quality and yield of crop plants. This fact highlights theneed to examine the MC threshold which may be detri-mental to crops (Pflugmacher et al., 2006; Crush et al.,2008). In 1995, Kos et al. reported for first time that bothMCs and crude extracts of MC-containing cyanobacteriainhibited the growth of mustard seedlings. Since then, theresearch interest on phytotoxic effects of cyanobacteriaon terrestrial plants has increased, demonstrating

S. Saqrane et al. / Toxicon 53 (2009) 786–796 787

morphological and physiological alterations by cyanotoxinsin a range of terrestrial plants, including Brassica napus L.,Lolium perenne, Oryza sativa L., Sinapis alba, Solanumtuberosum and Trifolium repens (Kos et al., 1995; McElhineyet al., 2001; Chen et al., 2004). In 2006, Pflugmacher et al.reported that germination of alfalfa seeds was inhibited byboth purified MCs and anatoxin-a from a toxic cyanobac-teria bloom, and by a cell-free crude extract from the samebloom. An increase in reactive oxygen detoxifying enzymeswas also observed, indicating a response to oxidative stresscaused by cyanotoxins (Pflugmacher et al., 2006).

Several authors have shown that terrestrial plants areable to accumulate MCs in sufficient concentration toinduce morphological and physiological changes(Gehringer et al., 2003; Chen et al., 2004; Pflugmacheret al., 2006). MCs can also affect seed germination, early-state development, and chlorophyll content (Abe et al.,1996; McElhiney et al., 2001; Pflugmacher et al., 2006).Recently, Peuthert et al. (2007) reported the uptake of MC-LR and MC-LF by seedling roots of 11 agricultural plants,and their translocation to shoots.

In Morocco, dense cyanobacteria blooms are frequentlyobserved in different water ecosystems, including naturallakes, rivers, and ponds (Oudra et al., 2002a,b), Microcystisgenerally being the dominant genus (Oudra et al., 1998,2001). Some of the water systems are partially utilized forirrigation, as it is the case of Lalla Takerkoust lake, whichused to irrigate 9800 ha of crops (Oudra et al., 2001).Nevertheless, until recently there has been no study inMorocco related to the effect of irrigation water contami-nated with toxic cyanobacteria, on the quality and yield ofedible crop plants. We just reported that germination andseedling growth of four edible plants (Lens esculenta, Pisumsativum, Triticum durum and Zea mays) were impaired after8-day exposure to an aqueous MC-containing bloomextract from Lalla Takerkoust reservoir (Saqrane et al.,2008). In the present work we study the toxic effectsproduced in four plant species by exposure to a MC-bloomextract, and analyze the ability of those plants to uptakeand translocate the different MCs present in the extract, byobserving how the exposure to the MC-extract affectedthe growth, development, productivity (fresh and drybiomass), photosynthetic activity and accumulation ofmineral nutrients. Besides, the MC variants present both inthe extract and in different plant organs after exposure tothe extract are analyzed.

2. Materials and methods

2.1. Cyanobacteria crude extract preparation

A cell-free extract from a dense MC-rich bloom was usedthroughout the work. The bloom was collected inSeptember 2005 from Lalla Takerkoust lake-reservoir(35 km from Marrakech, Morocco), where toxic Microcystisaeruginosa blooms frequently occur during summer andautumn. The bloom biomass was collected with a 27 mmphytoplankton net. The dominant species in the bloom wasidentified as M. aeruginosa by microscopy. The collectedsample was frozen and stored at �25 �C. After de-freezing,the sample was ultrasonicated on ice (3 kHz� 10 min), and

centrifuged at 4000 g for 15 min. The supernatant was theextract used for treating the plants.

2.2. Identification and quantification of microcystins in thecyanobacterial extract

Microcystins were analyzed by HPLC, and identified bytheir UV absorption spectrum and retention time (Lawtonet al., 1994), as well as by HPLC-Mass Spectrometry (LC-MS)when no standard was available. HPLC was performed ina Waters equipment (model 2695) provided with a photo-diode array detector (PDA, model 996). The column usedwas Chromolith C18 (250� 4.6 mm, 5 mm). The mobilephase system was a non-linear gradient of two solvents: (A)waterþ 0.05 % (v/v) trifluoroacetic acid (TFA), and (B)acetonitrile (MeCN)þ 0.05 % (v/v) TFA. The gradient profilewas as follows (t in min): t0, A¼ 60% t2, A¼ 60% t12, A¼ 55%t22, A¼ 55% t32, A¼ 52% t45, A¼ 0% t50, A¼ 0% t52, A¼ 60%t55, A¼ 60%. The injection volume was 50 mL, and the flowrate 1 mL min�1. Six purified MC standards were utilized toidentify the extract MCs; 4 of them commercially available,MC-LR, -YR, -RR, and -LF (Calbiochem, Germany), andanother two, MC-FR and -WR, obtained in our laboratory bysuccessive HPLC runs of the corresponding peak fractions.The purity of MC-FR and -WR was checked, and the identityof MC(H4)YR achieved by tandem LC-Mass spectrometry(LC-ESI-QTOF). LC-MS experiments were carried out in anAgilent equipment (1100 series HPLC MC, Agilent Tech-nologies, CA) that included a vacuum degasser, binarypump, autosampler and DAD detector, coupled to a hybridquadrupole time of flight (QTOF) instrument (QStar/Pulsari; Applied Biosystems, CA.) equipped with a turbospray ionsource interface. The LC column used was MED SEA18(Teknokroma), and the mobile phase the same gradientpreviously described, except that the TFA concentrationwas 0.1%. Full scan MS spectra were acquired in the positiveion mode, using a source potential of 5000 V, over the massrange of 50–1500 at 1 s. ESIMS/collision induced dissocia-tion (CID) mass spectra were measured using N2 as a colli-sion gas (collision energy, 3 kV) in the pressure range of65 bar. The N2 drying temperature was set a 300 �C, and thecone voltage was fixed at 70 V.

MCs were quantified using MC-LR as a reference. Allchemicals were of chromatographic grade (Scharlau ChimieBarcelona, Spain).

2.3. Plant material, culture and exposure experiments

The experiments were performed with four agriculturalplants: two cereals, T. durum and Z. mays, and two legumi-nosae, P. sativum and L. esculenta. In each case, 10 seeds weregerminated in pots containing sand. Seed germination andplant growth were carried out under green house condi-tions: temperature, 15/28 �C (night/day); relative humidity,ca. 38/65% (day/night); and approximately 1380 mmolm�2 s�1 midday photon fluence rate. During germination,and growth until appearance of the first leaves, plants wereirrigated at 3-day intervals with 100 mL of a nutrient solu-tion containing 0.3 g/L of an organic fertilizer EC-NPK(13.40.13) with microelements. When the first leavesappeared, the plants were irrigated at 3-day interval with

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200 mL of a solution containing both the fertilizer plus theconveniently diluted cyanobacteria extract to get various MCconcentrations: 0.5, 1.05, 2.1 and 4.2 mg equivalent MC-LR/mL. During 30 days of MC-extract exposure, samples werewithdrawn at different times, depending on the assay.

2.4. Assays of plant growth and development

The morphological parameters measured were: plantheight for the growth test, and leaf number for the devel-opment test. Measurements were carried out at 7-dayintervals along the 30-day exposure to the MC-extract. At theend of the treatment, fresh and dry biomass was determined,to evaluate the MC-extract effect on productivity.

2.5. Detection and characterization of microcystins inexposed plants

To evaluate the accumulation of MCs in plants, frozenmaterial (1–5 g) was extracted twice with 5 mL of aqueousmethanol 70% (v/v). Extracts were prepared from threeplant parts: root, stem and leaf. To obtain sufficient mate-rial, the different organs of 10 plants were pooled. Themethanolic extracts were vacuum dried (Gyrovap; GLHowe, UK) for 3 h at 40 �C, the residue treated with 300 mLof aqueous methanol 70% (v/v), and the suspension passedthrough a GF/C glass filter prior to HPLC analysis. MCcontent is expressed as MC-LR equivalents (MC-LR equiv).

2.6. Photosynthetic parameters

2.6.1. Chlorophyll analysisAfter 30 days of exposure to Microcystis aqueous extract,

chlorophyll (aþ b) content was determined to assess theimpact of cyanotoxins on photosynthetic pigments. Chlo-rophylls a and b were extracted in the dark, and analyzedaccording to Geider and Osborne (1992). Briefly, 1 g ofleaves was homogenized in 0.5 mL of aqueous acetone 90%(v/v), centrifuged (10000 g for 5 min), and the pellet wasextracted twice with 0.5 mL of 90% acetone. The concen-tration of chlorophyll a and b (mg/mg FW) was calculatedfrom the absorbance at 664 and 647 nm.

2.6.2. Photosynthetic activityPhotosynthetic activity was assayed by fluorescence in

dark-adapted leaves, using a portable fluorometer (HandyPlant Efficiency Analyser, Hansatech Instruments Ltd), afterillumination at a light intensity of 3500 mmol photonsm�2 s�1. Photosystem II was estimated by the ratio(Fm� F0)/Fm (also known as Fv/Fm) (Maxwell and John-son, 2000), where F0 is the minimum fluorescence, corre-sponding to the open state of PSII reaction centers, and Fm,the maximum fluorescence, corresponding to a close stateof PSII reaction centers. The fluorescence parameters werecalculated automatically using Handy PEA v 1.3 software.

2.7. Mineral nutrient analysis

After 30 days of plant treatment, roots were harvested,rinsed and weighed for mineral nutrients analysis. Todetermine the inorganic ions Naþ, Kþ and Ca2þ, 1 g of dried

root was ashed in a muffle furnace for 6 h at 550 �C. Rootashes were dissolved in 20% (v/v) sulfuric acid, and thendiluted with distilled water. The cations were analyzed ina flame-photometer (Jen Woy. PFP7).

Total nitrogen (N) and phosphororus (P) were analysedessentially according to AFNOR (1983). For N determina-tion, 0.25 g dried roots were digested with 6 mL sulfuricacid (90%; v/v) at 420 �C for 90 min, after being mixed with3 g of a catalyst mixture (K2SO4: CuSO4.5H2O: Se;100:10:1). After digestion, the volume was adjusted to200 mL with distilled water, the solution transferred toKjeldhal bottles containing 40 mL of 10 M NaOH, and theresulting mixture subjected to distillation. The distillate(80 mL) was collected on boric acid, and titrated with0.05 M sulfuric acid.

For P determination, root digestion was carried out asfor total nitrogen. The sulfuric extract was filtered (What-man no. 5), the filtrate (10 mL) mixed with 10 mL ofa reactive mixture (ammonium molybdate and vanada-teþ nitric acid), and the volume adjusted to 100 mL withdistilled water. After 30 min, the absorbance at 430 nm wasread. P content was calculated using standards treated inthe same way as the samples.

2.8. Statistical analysis of data

Statistical analyses were performed using SPSS for windows10.0 setup. Data are presented as means� SD of three separatedeterminations (n¼ 3). Data were analyzed by one way anal-ysis of variance (p< 0.05) with Tukey’s post hoc test.

3. Results

3.1. Quantification and identification of microcystins incyanobacteria bloom extract

MC concentration in the cyanobacteria bloom extractwas 4.2 mg equiv MC-LR/mL. The HPLC chromatogramrevealed the presence of six components with a typical MCspectrum (Fig. 1). According to commercial standards, threeof them were MC-RR, -YR and -LR (Retention time, 6.78,13.51 and 15.48 min, respectively). Another two wereidentified as MC-FR and -WR (Rt, 18.32 and 19.86, respec-tively), after the standards obtained in the laboratory.Finally, the sixth MC was identified by LC-MS as MC-(H4)YR. The approximate abundance percentage of eachMC variant in the extract was: MC-RR, 31.6; MC-LR, 27; MC-YR, 16; MC-(H4)YR, 14.2; MC-WR, 6.6; and MC-FR, 4.6.

3.2. Effects of the cyanobacteria MC-extract on plant growth,development, and productivity

Exposure of plants to the cyanobacteria extract atvarious MC concentrations, caused a significant negativeeffect on growth and development. When compared to thecontrol untreated plants, a net reduction in plant height, aswell as in leaf number was observed (Fig. 2). Plant yield,determined by either fresh or dry biomass, was alsodecreased (Fig. 3).

All effects were plant species, dose and time dependent(Figs. 2 and 3). The parameter change caused by a given MC

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dosage and time exposure varied for each plant species. Ingeneral, T. durum appeared as the most sensitive plant, andL. esculenta the most resistant. In T. durum, plant height andleaf number (Fig. 2), as well as fresh weight (Fig. 3) weresignificantly reduced after 15 days of exposure to0.5 mg MC/mL, while in L. esculenta height was hardlyaffected (ca. 5%), similarly to leaf number (1% reduction),and fresh weight was reduced about 25%.

The highest MC concentration used, 4.2 mg MC/mL,caused after 30-day exposure similar growth impairmentin the 4 plants, about 30% height reduction. However, thelevel of impaired development varied among the plants,with L. esculenta exhibiting the smallest developmenteffects: 26% decrease in leaf number, as compared to a 50%decrease in T. durum (Fig. 2).

3.3. Effect of cyanobacteria MC-extract on physiologicalactivities

3.3.1. Photosynthetic activityThe extract caused a statistically significant decrease in

chlorophyll (aþ b) content in Z. mays and L. esculenta at thehigher MC concentrations (2.1 and 4.2 mg/mL), with thedecrease respect to the untreated plants being 22–25%(Fig. 4). However, no significant effect on T. durum andP. sativum was observed.

The photosynthesis performance was determined afterdark adaptation by analyzing Fv/Fm, which represents themaximum quantum yield of PSII (Maxwell and Johnson,2000). The control Fv/Fm value in the four type plants wasslightly inferior to that considered optimal, around 0.8(Johnson et al., 1993). Values lower than this will be seenwhen the plant has been exposed to stress, indicating inparticular the phenomenon of photo-inhibition.

In all the plants tested, and at the various MC concen-trations, cyanobacteria extract treatment produceda decrease in the Fv/Fm control (Fig. 5). The Fv/Fm declinewas dose and plant species-dependent, with the most

Fig. 1. HPLC chromatogram of the Microcystis bloom extract utilized thro

sensitive species being P. sativum, in which the Fv/Fmreduction attained at 1 mg/mL was around 70% (Fig. 5).Fv/Fm decrease clearly indicates that MCs impaired thephotosynthetic activity of all exposed plants, especially ofP. sativum, and that the impairment was at least partiallydue to a damage of PSII reaction centers.

3.4. Mineral nutrient accumulation

After 30-day exposure to the MC-extract, the content ofmineral nutrients in roots (Naþ, Kþ, Ca2þ, P and N)increased. The increase depended on the plant species, andwas generally proportional to the MC concentration (Fig. 6).The greatest effect was observed in the case of Ca2þ, whosecontent in the plants treated with the highest MCconcentration tripled that of the control. Thus, controllevels, ca.10 g/kg DW (L. esculenta and P. sativum), 12(Z. mays), and 14 (T. durum), changed respectively to ca.38, 32, 39, and 32 g/kg DW (Fig. 6).

As for Naþ and Kþ, the accumulation in all extract-treated plants was at least two fold than that of untreatedplants. With respect to P, the accumulation was much lessthan that for the other elements (inserts, Fig. 6). Thegreatest increase was observed in Z. mays, with theconcentration detected in control, 0.1 g/kg DW, increasingto 0.65 g/kg DW in plants exposed to 4.2 mg equiv MC-LR/mL. With regard to N, significant increases caused by theMC-extract were observed, with the highest change in allplants being more than 2 fold (Fig. 6). It should be high-lighted that in T. durum the highest increase was alreadyattained with the smallest MC concentration.

3.5. Microcystin accumulation in plants

The distinct effects of MC-extract on the 4 plants usedcould be due to different MC accumulation in various plantorgans. To confirm this idea, MC content in roots, stem andleaves were analyzed by HPLC (Table 1). When the plants

ughout the work. Insert, absorption spectrum of MC components.

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were exposed to the highest MC concentration, MCs weredetected in all organs, clearly indicating that they aretranslocated within the plant. MC accumulation was dosedependent, and the maximum level attained varied in eachplant species. However, for a given extract treatment, MCcontent in the various plant parts differed. Generally, thehighest MC content was found in roots, followed by leavesand stem. P. sativum appeared to be the best MC accumu-lator, followed by L. esculenta, taking into account both theaccumulation capacity (maximum level of accumulated

Fig. 2. Effect on plant growth and development of 30-day exposure to Microcystis bMC-LR/mL. Growth parameter: plant height. Development parameter: leaf number.(D). Data are the mean� SD (n¼ 3). Significant differences between control and tr

MCs) and the accumulation efficiency, when one considersthe minimum MC concentration at which accumulationwas detected. After exposure of P. sativum to the highest MCcontent (4.2 mg/mL), MC content was about 191 mg/g DW inroots, 79 in stem and 157 in leaves. Thus, roots and leaves ofP. sativum exhibited a similar overall MC accumulation. Incontrast, with the same treatment, T. durum accumulatedmuch less MCs: about 16 mg/g DW in root, 1 in stem, and 16in leaf. After treatment with the lowest MC concentration(0.5 mg/mL), no MC was detected in Z. mays, but

loom extract. MC concentration in irrigation solution: 0.5–4.2 mg equivalentPlants: Triticum durum (A), Zea mays (B), Pisum sativum (C) and Lens esculentaeated plants are indicated by: * p< 0.05, and ** p< 0.01.

Fig. 3. Effect on plant productivity of 30-day exposure to Microcystis bloom extract. MC concentration in irrigation solution: 0.5–4.2 mg equivalent MC-LR/mL.Productivity assayed as fresh and dry weight. Plants: Triticum durum (A), Zea mays (B), Pisum sativum (C) and Lens esculenta (D). Data are mean� SD (n¼ 3).Significant differences between control and treated plants are indicated by: * p< 0.05, and ** p< 0.01.

S. Saqrane et al. / Toxicon 53 (2009) 786–796 791

a significant level was appreciated in the other 3 plantspecies. Nevertheless, relative MC accumulation in thevarious organs differed among the plants and within eachplant.

To study the ability of each of the 4 plant species touptake, translocate and accumulate different MC variants,

qualitative MC analysis was performed with MC-treatedplants. Data from plants exposed to the lowest MC dose(Fig. 7A1–D1) should allow us to compare MC uptakeability, data from intermediate MC dose exposure shouldallow us to compare uptake and translocation (Fig. 7A2–D2), and data from the highest MC exposure should allow

Fig. 4. Chlorophyll (aþ b) content after 30 days of plant exposure to Microcystis bloom extract. MC concentration in irrigation solution: 0.5–4.2 mg equivalent MC-LR/mL. Triticum durum (A), Zea mays (B), Pisum sativum (C) and Lens esculenta (D). Data are mean� SD (n¼ 3). Significant differences between control and treatedplants are indicated by: * p< 0.05, and ** p< 0.01.

S. Saqrane et al. / Toxicon 53 (2009) 786–796792

us to compare accumulation capacity (Fig. 7A3–D3). In eachplant type, the easiest uptaken MC differed. Taking intoaccount the relative abundance of each MC in the cyano-bacteria extract and the amount of MC initially accumu-lated in root, the MCs most easily taken up were: MC-YR inT. durum (Fig. 7A1), MC-LR and MC-(H4)YR in Z. Mays(Fig. 7B2), MC-FR and MC-RR in P. sativum (Fig. 7C1), andMC-RR in L. esculenta (Fig. 7D1). In Z. mays no MC wasdetected with the lowest MC dose. Translocation and finalpartitioning of each MC also differed in the four cultivars(Fig. 7A2–D2 and A3–D3). It seemed that the degree of finalpartitioning in pea and lens was directly related to the MChydrosolubility, the highest level of MCs observed at all MCconcentrations tested corresponding to MC-RR and MC-LR.In general, the accumulation of the most lipophylic MCs,MC-FR and MC-WR was very low, except in Z. mays, whereMC-WR accumulation was considerable (Fig. 7B3), takinginto account the relative abundance of this MC in theextract. It is also remarkable in Z. mays that no RR wasdetected after any MC-extract exposure (Fig. 7B1–B3).

Fig. 5. Effect on Photosystem II activity of 30-day exposure to Microcystisbloom extract. MC concentration in irrigation solution: 0.5–4.2 mgequivalent MC-LR/mL. Photosynthetic activity was assayed as Fv/Fmfluorescence. Data are mean� SD (n¼ 5). Significant differences betweencontrol and treated plants are indicated by: * p< 0.05, and ** p< 0.01.

4. Discussion

The experiments show that growth, development,photosynthetic activity, productivity and mineral nutritionof T. durum, Z. mays, P. sativum and L. esculenta cultivarswere affected, although to a different degree, by a cyano-bacteria extract containing 0.5–4.2 mg equivalent MC-LR/mL. The data on growth inhibition agree with thoseobtained by other authors in other plant species differentfrom those in this study, utilizing either MCs and/or

cyanobacterial MC-extracts (Kos et al., 1995; Kurki-Helasmo and Meriluoto, 1998; Pflugmacher et al., 2001;Gehringer et al., 2003; Chen et al., 2004). Gehringer et al.(2003) observed a significant decrease in leaf and rootlengths, and in productivity of Lepidium sativum seedlingsby a cyanobacteria MC-extract. Pure MC-LR caused a similardecrease, indicating that the effect of the cyanobacteriaextract was due to MCs. Later, it was reported that differentspinach variants were all morphologically affected after 6-week exposure to a cyanobacteria crude extract containing0.5 mg/L MC-LR (Pflugmacher et al., 2007).

The impairment of photosynthesis by cyanobacterialtoxins in macroalgae, macrophytes (Pflugmacher, 2002)

Fig. 6. Mineral nutrient content in roots after 30 days of plant exposure to Microcystis bloom extract. MC concentration in irrigation solution: 0.5–4.2 mgequivalent MC-LR/mL. Insert illustrates phosphorus content. Triticum durum (A), Zea mays (B), Pisum sativum (C) and Lens esculenta (D). Data are mean� SD(n¼ 5). Significant differences between control and treated plants are indicated by: * p< 0.05, and ** p< 0.01.

Table 1Microcystin content in plant organs (roots, stem and leaves) of Triticumdurum, Zea mays, Pisum sativum and Lens esculenta after 30 days of irri-gation with Microcystis bloom extract, at various MC concentrations 0.5–4.2 mg equiv MC-LR/mL.

Plant Extract MCconcentration(mg/mL)

MC concentrationin plant organ(mg/g FW)

Roots Stem Leaves

Triticum durum 0.50 0.18 ND ND1.05 7.60 ND ND4.20 16.66 1.17 15.71

Zea mays 0.50 ND ND ND1.05 7.55 1.29 1.014.20 18.71 7.65 5.82

Pisum sativum 0.50 0.24 ND 0.411.05 6.23 0.88 5.524.20 190.85 79.19 156.80

Lens esculenta 0.50 16.00 ND 0.331.05 24.52 2.33 7.004.20 162.79 36.61 98.37

ND, not detected.

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and plants (Kos et al., 1995; Romanowska-Duda and Tarc-zynska, 2002; M-Hamvas et al., 2003; Weib et al., 2005) hasalso been previously reported. In Ceratophyllum Demersumthis impairment was accompanied by clear changes in theChl a/Chl b ratio (Pflugmacher, 2002). Nevertheless, amongthe studies performed so far with plants (both aquatic andterrestrial) to explore MC effects on photosynthetic activity,variable modulated fluorescence was used only in one ofthem (Jarvenpaa et al., 2007). No net decrease in Fm/Fv wasobserved upon irrigation of broccoli (Brassica oleracea var.italica) and mustard (S. alba) with water or mineralmedium supplemented with a purified MC-cyanobacteriaextract (Jarvenpaa et al., 2007). MC concentrations used byJarvenpaa et al. (2007) were much smaller (0.1–10 mg equiv.MC-LR/L) than those utilized here; therefore, one cannotdiscard the possibility that photosynthesis also decreasedat doses higher than 10 mg equiv MC-LR/L. However, oneshould take into account that plant sensitivity to MCsdiffers among species, among other reasons due to distinctability to uptake, translocate and/or accumulate the diverseMC variants.

In contrast with the decrease in photosynthetic activityobserved upon MC-extract exposure, overall mineralnutrient accumulation in plant roots clearly increased. Atpresent, there is no experimental evidence to explain suchfinding. The most obvious explanation would be a generalincrement of root membrane permeability, which in turncould be due either to a direct or indirect effect. Another

possible explanation relates to the observed impairment ofgrowth and development, since this would probably implydefects in nutrient allocation and utilization. Furtherexperiments should be carried out to understand the basisof the MC influence on mineral nutrient accumulation.

In general, the severity of any of the observed effectswas related to the amount of MCs accumulated in the plant.

Fig. 7. Percentage of microcystin variants present in roots, stem and leaves of Triticum durum (A), Zea mays (B), Pisum sativum (C) and Lens esculenta (D) exposedto Microcystis bloom extract at respectively 0.5 (A1,B1,C1,D1); 1.05 (A2,B2,C2,D2) and 4.2 mg equiv MC-LR/mL (A3,B3,C3,D3).

S. Saqrane et al. / Toxicon 53 (2009) 786–796794

The MC variants detected in the various organs of plantsexposed to the intermediate (Fig. 7A2–D2) and highest MCdose (Fig. 7A3–D3) were the same, indicating that afteruptake, MCs are translocated throughout the plant. Themajor MCs accumulated after plant exposure to the highestMC concentration (Fig. 7A3, B3, C3, D3) in general wereMC-LR and MC-RR, as expected, considering the MCcomposition of the extract. Yet, the relative abundance ofeach MC differed in the various organs of each plant, andfor a given organ in the 4 plant species.

T. durum appeared to be exceptional with respect to theother 3 plants tested. MC-YR seemed to be the MC uptakenbest (Fig. 7A1), and became a minor component in plantsexposed to intermediate MC concentration, but was absentin plants exposed to the highest MC concentration. Another

unexpected fact was that MC-(H4)YR, a minor MC in thecyanobacteria extract, became the major MC accumulatedin T. durum exposed to the highest MC concentration. Wehave no evidence to explain those findings; however, itcould be that once uptaken by the roots, each MC wouldface different resistance to move through the plant, and/orcould undergo different metabolic changes. These twopossible ideas could also explain the distinct relative MCabundance in the various organs of a given plant species. Inrelation to MC metabolism, previous reports have shownthat MC can form glutathione conjugates in liver (Kondoet al., 1992) and in plants (Pflugmacher et al., 2007).

Evidence of MC translocation within the plant is scarcelydocumented, the available reports being very recent, onseedlings of other agricultural plants, e.g. Medicago sativa

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L., Triticum aestivum L., Cicer arietinum L. (Peuthert et al.,2007) and broccoli and mustard (Jarvenpaa et al., 2007). Innone of those reports, a continuous assay of the differentMC variants present in the various plant parts was carriedout.

Comparison of the major MC accumulated in thedifferent plant organs and of physiological changes allowmany suggestions and/or speculations. For example, thefact that in all plants a generalized increase of nutrientaccumulation takes place, in spite of different MC variantsappearing in their roots (and even in other plant organs),suggests that all the MCs present in the cyanobacteriaextract are effective to induce the increase in nutrientaccumulation. Also, the 6 extract MCs appear to be effectivein impairing photosynthetic activity, since any major MC orMC mixture accumulated in leaves caused Fv/Fm decrease.No comparative MC effectiveness could be established withthe available data, either with respect to different MCs fora given plant or with respect to the same MC in differentplants.

Taking together the data and those from otherauthors, it seems clear that MC-containing extracts arephytotoxic; but the severity of each effect variesaccording to the plant species and to the cyanobacteriaextract used. In this regard, one should consider that thephytotoxicity of purified MCs, on an equivalent MC-LRconcentration basis, differs from that of MC-containingextract, implying that the action of MCs could bemodulated by other cyanobacteria components. Pietschet al. (2001) observed that a MC-cyanobacteria extractcaused a bigger impairment of photosynthetic activity inmicroalgae and macrophytes than purified MCs whenthese were used at an equivalent concentration.Although more comparative experiments of this sortshould be carried out, it appears that the use of cyano-bacteria extracts to assay cyanobacterial phytotoxicity ismore adequate than that of purified cyanobacteriatoxins, because it reflects better the natural situation ofplants irrigated by cyanobacteria-contaminated water.Several authors have already used cyanobacteria crudeextracts, instead of purified extracts to study phytotox-icity (Chen et al., 2004; Peuthert et al., 2007).

There exist few studies on the underlying mechanismsfor the phytotoxic effects of MCs, a major point whichrequires further research with horticultural and agricul-tural plants to gain further insight into key aspects of MCuptake, partitioning, degradation and transformation. Fromthe health risk point of view, special attention should bepaid to important topics as MC allocation in the plant,degree of accumulation and toxicity of the diverse MCs, aswell as possible relation among different cyanobacteriacomponents, which may lead to synergistic and antago-nistic effects.

To conclude, the conspicuous phytotoxic effects hereobserved in this study reinforces the idea that irrigationwith water contaminated by MC-containing bacteria couldbe accompanied by important undesired consequences, asreduction in plant yield; and most important, it might bea potential threat for human and animal health, if the MCintake via plant material ingest exceeded the recom-mended tolerable limits.

Acknowledgments

This work was funded by the Moroccan government, theSpanish AECI (bilateral Spain-Morocco Project A/011422/07accorded to Drs. del Campo and Oudra), and the Auto-nomous Community of Madrid (CCG07-UAM/AMB-1883).S. Saqrane was recipient of an excellence grant from theCNRST- Minister of National Education, Higher Education,Staff Training and Scientific Research. We acknowledge thecritical comments of the referees and the help of Dr. LeeRobertson in reading the manuscript.

Conflict of interest

The authors declare that there are no conflicts ofinterest.

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