Antioxidative response of the three macrophytes Ceratophyllum demersum, Egeria densa, and Hydrilla verticillata to a time dependent exposure of cell-free crude extracts containing

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Aquatic Toxicology 163 (2015) 130–139

Contents lists available at ScienceDirect

Aquatic Toxicology

j o ur na l ho me pag e: www.elsev ier .com/ locate /aquatox

ntioxidative response of the three macrophytes Ceratophyllumemersum, Egeria densa, and Hydrilla verticillata to a time dependentxposure of cell-free crude extracts containing three microcystinsrom cyanobacterial blooms of Lake Amatitlán, Guatemala

laudia Suseth Romero-Oliva, Valeska Contardo-Jara, Stephan Pflugmacher ∗

echnische Universität Berlin, Department of Ecotoxicological Impact Research and Ecotoxicology, Ernst-Reuter-Platz 1, 10587 Berlin, Germany

r t i c l e i n f o

rticle history:eceived 21 January 2015eceived in revised form 9 March 2015ccepted 1 April 2015vailable online 2 April 2015

eywords:icrocystin congenersxidative stressntioxidant defenseiotransformationacrophyte

a b s t r a c t

Microcystins (MCs) produced by cyanobacteria in natural environments are a potential risk to the integrityof ecosystems. In this study, the effects of cyanobacterial cell-free crude extracts from a Microcystisaeruginosa bloom containing three MC-congeners MC-LR, -RR, and -YR at environmental relevant con-centrations of 49.3 ± 2.9, 49.8 ± 5.9, and 6.9 ± 3.8 �g/L, respectively, were evaluated on Ceratophyllumdemersum (L.), Egeria densa (Planch.), and Hydrilla verticillata (L.f.). Effects on photosynthetic pigments(total chlorophyll (chl), chl a, chl b, and carotenoids), enzymatic defense led by catalase (CAT), perox-idase (POD) and glutathione reductase (GR), and biotransformation enzyme glutathione S-transferase(GST) were measured after 1, 4, and 8 h and after 1, 3, 7, and 14 days of exposure. Results show that inall exposed macrophytes, photosynthetic pigments were negatively affected. While chl a and total chldecreased with increasing exposure time, a parallel increase in chl b was observed after 8 h. Concomitantincrease of ∼5, 16, and 34% of antioxidant carotenoid concentration in exposed C. demersum, E. densa,and H. verticillata, respectively, was also displayed. Enzymatic antioxidant defense systems in all exposedmacrophytes were initiated within the first hour of exposure. In exposed E. densa, highest values of CATand GR activities were observed after 4 and 8 h, respectively, while in exposed H. verticillata highest value

of POD activity was observed after 8 h. An early induction with a significant increase of biotransforma-tion enzyme GST was observed in E. densa after 4 h and in C. demersum and H. verticillata after 8 h. Theseresults are the first to show rapid induction of stress and further possible MC biotransformation (basedon the activation of GST enzymatic activity included in MC metabolization during the biotransformationmechanism) in macrophytes exposed to crude extract containing a mixture of MCs.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

In natural environments, macrophytes play an important roles primary producers and producers of oxygen. They are alsonown to affect the structuralization of the littoral zone, as theyre actively involved in ecological processes (e.g., nutrient cycling)nd providers of refuge and food for juvenile fish (Magela and

ibeiro, 2010). Macrophytes are also involved in the maintenancef clear-water states in aquatic ecosystems (Cronin et al., 2006).ecently in both freshwater and marine ecosystems, eutrophi-

∗ Corresponding author. Tel.: +49 3031429023; fax: +49 3031429022.E-mail addresses: [email protected]

C.S. Romero-Oliva), [email protected] (V. Contardo-Jara),[email protected] (S. Pflugmacher).

ttp://dx.doi.org/10.1016/j.aquatox.2015.04.001166-445X/© 2015 Elsevier B.V. All rights reserved.

cation and climate change have provoked the proliferation andexpansion of harmful cyanobacterial blooms (O’neil et al., 2012).Since cyanobacteria have the ability to produce potent toxins,occurrence of blooms poses a threat for the ecological integrity ofaquatic ecosystems.

The most representative of all cyanotoxins, are the hepatotoxinsmicrocystins (MCs) of which more than 90 different congeners havebeen identified until today (Ufelmann et al., 2012). The cyanobac-terium Microcystis aeruginosa is known to produce several MCscongeners simultaneously, which are released by living cells andduring bloom senescence into the environment. So far, adverseeffects of most frequently occurring MC congener, MC-LR, have

been extensively studied in different aquatic organisms, such asin algae, macrophytes, zooplankton, bivalves, crayfish, and fish(Wiegand and Pflugmacher, 2005 and references therein). As spe-cific effects in chlorophytes and macrophytes growth inhibition

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nd fresh weight decrease (Babica et al., 2006 and referencesherein; Jiang et al., 2011; Mitrovic et al., 2005; Pflugmacher, 2002;omanowska-Duda and Tarczynska, 2002; Weiss et al., 2000),hanges in cytoskeletal organization (Szigeti et al., 2010) and evenlant death (Pflugmacher et al., 2001) have been observed. Fur-hermore, the activation of different adaptive/protective strategiesn aquatic organisms exposed to MCs, such as oxidative defense andiotransformation have also been reviewed (Babica et al., 2006).

These two strategies are the most potent defense mecha-isms in macrophytes against adverse environmental conditions,uch as exposure to MCs. On the one hand, oxidative defensegainst the overproduction of reactive oxygen species (ROS) isrocessed within a complex set-up of chemical reactions ledy several enzymes e.g., superoxide dismutase (SOD), peroxi-ase (POD), catalase (CAT), monodehydroascorbate peroxidaseMDHA), ascorbate peroxidase (APX), glutathione peroxidaseGPX), and dehydroascorbate reductase (DHA), etc., and non-nzymatic antioxidants e.g., ascorbate (ASH), glutathione (GSH),nd tocopherol (TCP) (Gill and Tuteja, 2010). Biotransformationechanism, on the other hand, deals with the metabolization

f toxic compounds, leading to their transformation to moreydrophilic substances and often less active derivatives. This ischieved by a series of reactions, which can be divided into threehases, including a wide array of enzymes. Phase I-functional trans-

ormation includes oxidative, reductive, and hydrolytic reactions,hich are responsible for the production of reactive groups. These

eactions are primarily managed by the cytochrome monooxyge-ase (CYP450) enzyme family. Phase II-conjugation is responsible

or the alignment either of the reactive groups produced inhase I or two compounds involving covalent attachments ofmall polar endogenous molecules i.e., glutathione. One of theix enzyme families dealing with conjugation is the glutathione-transferease (GST) family. This phase is also responsible ofhe formation of more polar, water-soluble compounds. Finally,hase III-transportation/compartmentalization, in non-excretoryrganisms like macrophytes, is responsible for the migration ofydrophilic molecules and subsequent storage of metabolites fromhe first two phases mostly in cell vacuoles and walls (Dixon et al.,010; Hoffmann et al., 2013; Sandermann, 1992).

To the best of our knowledge, the available informationegarding effects of MCs in macrophytes is limited to the usef commercial MCs and single MC congeners obtained fromyanobacterial cultures. The effects of the mixture of several MCsepresenting more accurately the natural environment of a, e.g.,. aeruginosa, bloom have been scarcely evaluated until today.

his suggests a possible underestimation of MC effects under realnvironmental conditions. The only available studies in whichixtures of MCs were used, could demonstrate that e.g., a mix-

ure of 223, 8, and 2 �g/L of MC-LR, -RR, and -YR, respectively,ed to a significant stimulation of biotransformation enzyme GSTnd antioxidative enzyme glutathione reductase (GR) after 96 h ofxposure in the aquatic microalga Pseudokirchenerialla subcapitataBártová et al., 2010). Furthermore, in Triticum aestivum, expo-ure to a daily concentration of around 20 �g/L of MC-LR and -RR,espectively, provoked a significant enhancement in expression ofenes encoding SOD, CAT, APX, and DHA enzymes after 5 h of expo-ure, implying possible MC metabolization/accummulation. Thisas further corroborated as plants showed the accumulation ofC-LR in a time dependent manner with concomitant biotransfor-ation of MC-RR within the 91 days of exposure (Kettner, 2014).

In the light of the need to better understand processes occurringn natural ecosystems, the following study evaluated the induc-

ion of stress in three macrophytes coexisting with M. aeruginosalooms, from Lake Amatitlán, Guatemala. In order to recreateatural conditions, three local macrophytes found in the naturalssemblage of Lake Amatitlán’s flora; Ceratophyllum demersum (L.),

xicology 163 (2015) 130–139 131

Egeria densa (Planch.), and Hydrilla verticillata (L.f.) were exposedto cell-free crude extracts (CE) obtained from the lake. The cell-free CE contained a mixture of three MCs (MC-LR, -RR, and -YR)at a final concentration of 104.4 ± 7.6 �g/L; which represents pre-viously reported MCs concentrations at bloom events for LakeAmatitlán, Guatemala (Romero-Oliva et al., 2014).

2. Material and methods

2.1. Microcystin extraction from cyanobacterial bloom material

Cyanobacterial bloom material mainly consisting of M.aeruginosa (i.e., 99.991%) was collected in Lake Amatitlán,Guatemala. Sampling and method for the re-concentration ofbloom material were described in an earlier study by Romero-Olivaet al. (2014). From the re-concentrated bloom material obtainedin the field, cell-free CE was obtained. Extraction was conductedwith deionized water (DIW) and to provoke cell lysis, bloom mate-rial followed a freeze (−40 ◦C)–thaw (22 ◦C) cycle of 11 h and 13 h,respectively. This procedure was conducted from 7 to 11 consecu-tive times, depending on re-concentrated volume of bloom materialused (i.e., 600–1000 g) and integrity of M. aeruginosa cells (i.e., mon-itored by microscopy) during each CE preparation. In average, atotal of 728 ± 324 mL of water for 732 ± 294 g fresh weight (FW)of bloom material was required during the extractions. On everyoccasion, material was centrifuged at 2880 × g for 1 h at 4 ◦C. Initialsupernatants were separated and to the pellets DIW was added.The remaining pellets were finally extracted with MeOH (100%) toobtain a final solution of 23% MeOH. The solution was mixed for4 h and centrifuged for 1 h at 2880 × g. All resulting supernatantswere mixed to obtain a homogeneous cell-free CE and were storedat −20 ◦C until MCs quantification with LC–MS/MS.

2.2. Microcystin quantification in cell-free crude extracts

Determination and quantification of the MCs and congenersMC-LR, -RR, and -YR were performed by LC–MS/MS (Alliance 2695UHPLC combined with a Micromass Quattro microTM, Waters). Thechromatographic separation was carried out on a reverse phase col-umn using a KinetexTM C18 column (2.1 × 50 mm, 2.6 �m pore size,Phenomenex). The mobile phase consisted of solution A (Milli-Qwater containing 0.1% trifluoro acetic acid (TFA) and 5% acetoni-trile (ACN)) and solution B (ACN containing 0.1% TFA) at a flow rateof 0.2 mL/min. The linear gradient conditions were as follows: 0 min65% A; 3.75–7 min 35% A, and 7.8–12 min 100% A. The column oventemperature was set at 40 ◦C with an injection volume of 20 �L. Elu-tion peaks for each of the MC congeners were observed at 7.1 minfor MC-RR, 7.34 min for MC-YR, and 7.44 min for MC-LR. Desolva-tion gas N2 was set as trigger gas and Argon (Ar) as collision gas.Parent compound and its fragment ions, respectively, were scannedat the following mass-to-charge ratio (m/z): MC-LR 995.5 → 135.1;-RR 519.9 → 135.3, and -YR 1045 → 135. ESI+ conditions for all MCswere set as follows: capillary voltage of 3 kV, source temperature of120 ◦C, desolvation temperature of 500 ◦C, and cone gas flow-rateof 100 L/h. For MCs congeners MC-LR and MC-YR collision energywas 65, cone voltage was 60 V and for MC-RR collision energy was35, cone voltage was 20 V. Desolvation gas flow-rate was 1000 L/h.Limit of detection was set at 1 ng/mL (signal to noise S/N > 3) andlimit of quantification at 5 ng/mL (S/N > 5) for all MCs congeners.

2.3. Exposure scenario

Three local macrophytes from Lake Amatitlán, Guatemala; C.demersum (L.), E. densa (Planch.), and H. verticillata (L.f.) werechosen for the laboratory experiments because of their physiolog-ical plasticity and resistance to adverse environmental conditions

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32 C.S. Romero-Oliva et al. / Aqua

Bianchini et al., 2010; Champion and Tanner, 2000; Mony et al.,007), their xenobiotics and natural toxin uptake capacitiesNimptsch et al., 2008), and high abundance in Lake Amatitlán,uatemala (Romero-Oliva et al., 2014). Shoots from C. demer-

um and E. densa were purchased from Wakus (GmbH, Berlin,ermany) and H. verticillata from Extraplant (Extra-group GmbH,ünster, Germany). In short, macrophytes were cultured with

rovasoli’s medium according to Romero-Oliva, et al. (2014) inre-cultivated glass tanks (46 cm × 30 cm × 30 cm) at 25 ± 1 ◦C in

day:night cycle of 12:12 h, under cool white fluorescent light at8 �E/m2/s (lichtflux) for 7 days to assure their acclimation to lab-ratory conditions. A total biomass of 4.5 ± 0.5 g fresh weight (FW)rom each macrophyte was separately exposed to 500 mL cell-freeE containing a total MCs concentration of 104.4 ± 7.6 �g/L (MC-R 49.3 ± 2.9; -RR 49.8 ± 5.9, and -YR 6.9 ± 3.8 �g/L) for a periodf 2 weeks. These concentrations were used since they resemblehose found in Lake Amatitlán, Guatemala (Romero-Oliva et al.,014). Required MC concentrations were achieved by the dilu-ion of concentrated cell-free CE with culture medium. To evaluatehe effects of the experimental conditions, control (CTR) macro-hytes were exposed to culture medium free of MCs in parallel.ach exposure group, exposed and non-exposed macrophytes con-isted of five independent replicates. To prevent changes in theatio between macrophyte biomass and exposure medium (500 gW macrophyte: 500 mL exposure medium), culture medium notontaining MCs was added up to its original volume to exposednd non-exposed replicates when needed. Samples were collectedfter 1, 4, and 8 h and 1, 3, 7, and 14 days of exposure. At eachampling event plants were washed with 100 mL of DIW to removeurface-attached MCs and shock-frozen in liquid nitrogen. Mediumontaining cell-free CE from each replicate was stored at −40 ◦C;hereas, macrophyte material was stored at −80 ◦C until further

nalysis. Additionally, macrophyte material from each species wasollected from the original cultures representing the 0 h control.

.4. Photosynthetic pigment concentrations

Macrophyte material was ground to a fine powder in liquiditrogen using mortar and pestle. A total of 0.1 g for E. densa and.05 g for C. demersum and H. verticillata were homogenized in 5 mL,N-dimethylformamide (Sigma–Aldrich Co., LLC) and preserved

n dark for 3 days at 4 ◦C according to Inskeep and Bloom (1985).he solution was centrifuged at 3800 × g for 15 min at 4 ◦C and thebsorbance (O.D.) of the supernatant fraction was determined at47 nm and 664.5 nm, for chlorophyll a (chl a) and b (chl b), respec-ively, and at 480 nm for carotenoids in a Kontron (Uvikon) UV 941®

Munich, Germany). The five independent replicates of each expo-ure group were measured in triplicate. Concentrations of chl a, chl, and total chlorophyll (chl) were calculated according to Inskeepnd Bloom (1985) and carotenoid content according to Wellburn1994).

.5. Enzyme extraction and activity assays

Enzyme extraction was performed according to Pflugmacher2004) with minor modifications. The frozen macrophytes wereulverized in liquid nitrogen. Macrophyte material (2.5 ± 0.2 g

or C. demersum, 1.5 ± 0.2 g for E. densa and H. verticillata) wase-suspended in 4 mL ice-cold 20% glycerol, dithioerythritol-DTE1.4 mM) sodium phosphate buffer (0.1 M, pH 6.5). The homogenateas then centrifuged at 10,600 × g for 15 min at 4 ◦C (Eppendorf

entrifuge 5417R, Hamburg, Germany) and the supernatant was

ater concentrated through ammonium sulfate precipitation to

80% final concentration. Solutions were centrifuged for 1 h at0,800 × g and pellets were re-suspended in 1 mL for E. densa and. verticillata and in 0.5 mL for C. demersum of sodium phosphate

xicology 163 (2015) 130–139

buffer (20 mM, pH 7). Solutions were desalted by gel filtration usingSephadex G-25 (NAP- columns), shock-frozen in liquid nitrogenand stored at −80 ◦C until enzyme activity analysis.

GST (EC 2.5.1.18) activity was measured according to Habiget al., (1974) by employing 1-chloro-2,4-dinitrobenzene (CDNB)as substrate. The increase in conjugation product GSH-CDNB wasspectrophotometrically monitored at 340 nm. GR (EC 1.6.4.2) activ-ity was measured according to Carlberg and Mannervik (1985) bymonitoring the consumption of NADPH during the regeneration ofGSH from oxidized glutathione (GSSG) at an absorbance of 340 nm.POD (EC 1.11.1.7) activity was measured according to Pütter (1974)using guajacol as substrate and by measuring its oxidation to tetra-guajacol in a reaction catalyzed by POD at an absorbance of 436 nm.CAT (EC 1.11.1.6) activity was measured according to Aebi (1974)by monitoring the degradation of hydrogen peroxide (H2O2) at anabsorbance of 240 nm. Protein concentrations were determined at595 nm using the protein dye Bradford, according to the method ofBradford (1976). Enzymatic activities were expressed in either nkator �kat/mg protein, with [kat] being the conversion rate of 1 molof substrate per second. Protein concentration and all enzymaticmeasurements were performed in triplicate for each replicate.

2.6. Statistical analysis

All data were tested for normality and homogeneity of vari-ance using Shapiro–Wilks W and parametric and non-parametricLevene’s test, respectively. When a set of data was not normallydistributed or test of homogeneity of variances failed, a squareroot transformation was performed in order to fit Y into a linearregression model. When applicable, two-way analysis of variances(ANOVA) was performed comparing treatment (cell-free CE andCTR macrophytes), sampling time (1, 4, and 8 h and 1, 3, 7, and 14days) as well as the interaction between treatment and time. Post-hoc comparisons were conducted by Tukey’s HSD. For data sets notfiting normality, statistical comparison was performed using thenon-parametric Kruskal–Wallis test. For photosynthetic pigmentratios (chl a/chl b and carotens/total chl) Spearman’s rank corre-lations (rho) coefficients were determined. The level of statisticalsignificance was set at p < 0.05 (*) for ANOVA comparisons, p < 0.05(+) for Kruskal–Wallis, and p < 0.01for all correlations. All statisticalanalyses were performed with SPSS 21.0 (SPSS, 2012).

3. Results

3.1. Photosynthetic pigment contents

Photosynthetic pigment concentrations, such as chl a, chl b, andcarotenoids were significantly influenced by the exposure dura-tion (*p < 0.05) and treatment (*p < 0.05). In all three macrophytesexposed to cell-free CE containing MCs, lower concentrations ofchl a and total chl with concomitantly higher chl b concentrationswere observed toward the end of the experiments (i.e., after 7 and14 days) differing significantly from CTR macrophytes (+p < 0.05 inC. demersum and H. verticillata and *p < 0.05 in E. densa). ExposedC. demersum had similar chl a concentrations as CTR macrophyteswithin the first hours of exposure, but its decrease toward 1 dayevidenced significant difference (+p < 0.05) when compared withtheir respective CTR (Table 1).

In turn, an increase in chl b in CE exposed C. demersum could beobserved within the first hour of exposure continuing througout theentire experiment. Siginificant differences when compared to the

respective CTR macrophytes could be observed after 1 h, 3 and 14days (+p < 0.05). Changes in pigment pattern (i.e., based on the ratiobetween chl a/chl b) from CE exposed C. demersum were evidencedthrougout the entire experiment. A higher ratio in CE exposed C.

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Table 1Mean ± SD concentrations of chlorophyll a and b, total chlorophyll, and carotenoids after 1, 4, and 8 h as well as after 1, 3, 7, and 14 days in the three macrophytes C. demersum,E. densa, and H. verticillata exposed to cyanobacterial cell-free crude extract (CE) and toxin-free medium (CTR) expressed in �g/g fresh weight (FW). Bold and italic numbersindicate significant differences between CTR and CE by (a) parametric ANOVA (p < 0.05) and (b) non-parametric Kruskal–Wallis (p < 0.05). Higher (↑) and lower (↓) values inCE when compared to CTR.

Time in hours (h) and days (d) Chlorophyll a (�g/g FW) Chlorophyll b (�g/g FW) Total chlorophyll (�g/g FW) Carotenoids (�g/g FW)

CTR CE CTR CE CTR CE CTR CE

C. demersum0 h 465 ± 31 149 ± 14 1096 ± 66 1969 ± 2051 h 463 ± 45 478 ± 25 (b↑) 151 ± 11 160 ± 16 (b↑) 1040 ± 104 1082 ± 55 (b↑) 1986 ± 130 2103 ± 1484 h 477 ± 28 473 ± 38 148 ± 22 164 ± 10 982 ± 47 1086 ± 64 1974 ± 290 2081 ± 195 (b↑)8 h 470 ± 47 417 ± 34 155 ± 7 166 ± 11 946 ± 96 939 ± 74 (b↓) 1714 ± 207 1811 ± 141 (b↑)1 d 414 ± 36 395 ± 20 (b↓) 144 ± 7 163 ± 16 851 ± 82 866 ± 48 1741 ± 291 1979 ± 963 d 415 ± 28 340 ± 35 142 ± 7 163 ± 8 (b↑) 896 ± 67 807 ± 70 1723 ± 158 1882 ± 117 (b↑)7 d 418 ± 21 379 ± 47 138 ± 11 143 ± 8 950 ± 45 933 ± 19 1798 ± 138 1911 ± 82 (b↑)14 d 422 ± 32 407 ± 23 147 ± 9 155 ± 2 (b↑) 952 ± 33 939 ± 55 1980 ± 116 2074 ± 88 (b↑)

E. densa0 h 300 ± 9 101 ± 10 676 ± 63 749 ± 151 h 315 ± 13 374 ± 6 107 ± 5 112 ± 2 752 ± 54 841 ± 126 (a↑) 756 ± 17 939 ± 134 h 347 ± 5 395 ± 7 117 ± 7 126 ± 7 780 ± 100 898 ± 81 (a↑) 804 ± 49 1004 ± 188 h 374 ± 13 426 ± 17 119 ± 5 142 ± 8 861 ± 34 942 ± 20 (b↑) 858 ± 55 1028 ± 301 d 402 ± 2 370 ± 5 (a↑) 122 ± 5 124 ± 7 832 ± 103 831 ± 58 839 ± 46 842 ± 503 d 372 ± 9 344 ± 9 122 ± 8 123 ± 9 820 ± 21 813 ± 70 807 ± 56 865 ± 657 d 359 ± 8 327 ± 8 118 ± 2 129 ± 11 808 ± 114 734 ± 155 796 ± 27 824 ± 1914 d 326 ± 10 274 ± 10 109 ± 1 118 ± 6 729 ± 72 702 ± 65 (b↑) 743 ± 68 864 ± 40

H. verticillata0 h 641 ± 37 211 ± 15 1465 ± 82 2786 ± 491 h 618 ± 68 616 ± 33 197 ± 34 205 ± 23 (b↑) 1387 ± 102 1382 ± 42 2501 ± 127 2772 ± 2884 h 613 ± 75 633 ± 81 214 ± 16 238 ± 17 1410 ± 29 1334 ± 177 2718 ± 215 2841 ± 276 (b↑)8 h 602 ± 68 599 ± 48 197 ± 29 214 ± 23 1492 ± 155 1350 ± 126 2587 ± 410 2805 ± 3491 d 624 ± 60 551 ± 11 215 ± 15 218 ± 18 1491 ± 132 1234 ± 122 2766 ± 285 2704 ± 339 (b↓)

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3 d 592 ± 52 426 ± 70 (b↓) 197 ± 30 17 d 575 ± 40 541 ± 60 (b↓) 189 ± 14 214 d 574 ± 74 579 ± 78 (b↑) 180 ± 10 2

emersum was evidenced from 8 h and toward the end of the exper-ment. Significant differences when compared to their respectiveTR group were exhibited after 3 and 14 days (+p < 0.05) (Fig. 1A).urthermore, in CE exposed C. demersum an increase in total chl wasbserved within the first exposure hour until 1 day. Significant dif-erences when compared to the respective CTR macrophytes coulde observed after 1, 8 h and 1 day (+p < 0.05). Thereafter, decrease

n chl b concentration from CE exposed C. demersum was displayednd remained until the end of the experiment.

This was not the case in E. densa, as CE-exposed macrophytesisplayed an increase in chl a concentration during the first 8 hf exposure (Table 1 and Fig. 1B). Thereafter, a strong decrease inhl a was exhibited in CE-exposed E. densa significantly varyingrom their CTR group after 1 day (*p < 0.05) and remaining towardhe end of the experiment. In parallel, chl b concentration in CE-xposed E. densa was increased in a time-dependent manner andeached its highest value after 8 h (142 �g/g FW). At all sampingvents chl b concentration in CE-exposed macrophytes was higherhan in the respective CTR group. Changes in pigment pattern wereot evident and when compared to the respective CTR macrophyteso significant differences could be observed (Fig. 1B). Total chl in CExposed E. densa was higher than in the CTR group and statisticallyignificant differences between them could be observed after 1, 4 h*p < 0.05) and 8 h (+p < 0.05). This was later followed by a decreasen chl b in CE exposed E. densa which varied significantly to theespective CTR group after 14 days (+p < 0.05).

In CE exposed H. verticillata, lower concentrations of chl a werexhibited throughout the entire experiment. After 1 day CE exposed. verticillata evidenced a strong decrease in chl a (i.e., ∼20% of itsoncentration in earlier sampling point) concentration and when

ompared to the respective CTR group significant differences coulde observed after 3, 7, and 14 days (+p < 0.05). Chl b concentration

n CE-exposed H. verticillata was higher than in CTR macrophyteshrougout the entire experiment. In CE-exposed H. verticillata, the

1331 ± 139 1005 ± 117 (b↓) 2495 ± 271 2933 ± 47 (b↑)7 1269 ± 89 1158 ± 129 (b↓) 2361 ± 302 2483 ± 2073 (b↑) 1194 ± 168 1046 ± 173 1999 ± 453 2679 ± 272 (b↑)

decrease in chl a was positively correlated to the increase in chl bconcentrations (p < 0.01). Pigment pattern ratio (i.e., chl a/chl b) wassignificantly higher in CE-exposed H. verticillata than in the respec-tive CTR group after 1 day (+p < 0.05) and remained higher towardthe end of the experiment differing significantly again from therespective CTR group after 14 days (+p < 0.05) (Fig. 1C). Concomi-tantly, total chl concentration in CE-exposed H. verticillata was alsolower than in the respective CTR group througout the entire exper-iment and significant differences could be observed after 3 and 7days (+p < 0.05).

3.2. Carotenoid concentrations

In the production of antioxidant carotenoid, time (*p < 0.05), andtreatment with cell-free CE (*p < 0.05) had a significant effect onthe exposed plants. 2.5-fold higher carotenoid concentrations wereobserved in H. verticillata compared to C. demersum and E. densa(Table 1). Significant differences compared to the respective CTRgroup were observed in all macrophytes during the intial period,in the case of E. densa after 1 h (*p < 0.05) and in the case of C.demersum and H. verticillata after 4 h (+p < 0.05). Throughout theentire experiment all CE-exposed macrophytes displayed a higherconcentration of carotenoid than the respective CTR. CE-exposedC. demersum showed highest carotenoid concentrations at 4, 8 hand 3, 7, and 14 days; which differed significantly when comparedto their respective CTR (+p < 0.05). Ratios between carotenoid andtotal chl concentrations in CE-exposed C. demersum remained sim-ilar until 8 h (Fig. 1D), thereafter, the increase of carotenoids led tohigher difference observed after 3 days (+p > 0.05) which remaineduntil the end of the experiment. A similar though accentuated pat-

tern was also observed for CE-exposed H. verticillata after 3 days(Fig. 1F). This was not the case for CE-exposed E. densa, as valuesremained similar to their CTR group throughout the entire experi-ment (Fig. 1E).

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ig. 1. Chlorophyll a/b and total chlorophyll/carotene ratios (mean ± SD) in C. demefter 1, 3, 7, and 14 days exposure to cyanobacterial cell-free crude extract (CE, dotignificant differences (p < 0.05) between CE and CTR calculated with ANOVA or Kru

.3. Enzymatic defense system

In C. demersum, CAT activity was increased after 1 and 4 h inE-exposed macrophytes compared to the respective CTR groupFig. 2A). In turn, a subsequent decrease was observed after 8 hhich continued until 3 days. CE-exposed macrophytes evidenced

ignificant differences when compared to CTR macrophytes after and 3 days (*p > 0.05). After 7 and 14 days, no differences

n the activity of CAT between the two treatments could bebserved. In E. densa, a similar pattern of an immediate increase

n CAT activity and a significant decrease with longer exposureuration was observed (Fig. 2B). The activity increase in CE-xposed macrophytes was significant after 8 h (+p > 0.05) and itsecrease turned to be significantly different from the respec-ive CTR toward the end of the experiment (i.e., after 7 days*p > 0.05)). In CE-exposed H. verticillata, an increase in CAT activ-ty could be observed after 4 and 8 h of exposure (Fig. 2C), which

as significantly different compared to respective CTR after 8 h

*p > 0.05). A subsequent decrease in activity was observed after

h and the activity remained at a level similar to CTR group until 7ays.

A and D), E. densa (B and E), and H. verticillata (D and F) after 1, 4, and 8 h as well ases) and toxin-free medium (CTR, black lines). Asterisks (*) and crosses (+) indicate

Wallis test, respectively.

Cell-free CE provoked an increase in POD activity in all testedmacrophytes. Increased POD activity in CE-exposed C. demersumwas observed throughout the entire exposure time, differing sig-nificantly from CTR macrophytes after 7 and 14 days of exposure(+p > 0.05) (Fig. 2D). In E. densa, a slight increase in POD activitycould be observed in CE-exposed macrophytes after 4 and 8 h; withsignificant differences when compared to CTR macrophytes after4 h (*p < 0.05) (Fig. 2E). Thereafter, POD activity was similar in bothCE-exposed and CTR macrophytes. In CE-exposed H. verticillata, arapid activation of POD activity could be observed after 4 and 8 hto 1 day of exposure (Fig. 2F), which was significantly differentwhen compared with the respective CTR group after 8 h (*p > 0.05).Thereafter, POD activity in CE-exposed H. verticillata decreased andremained similar to POD activity displayed by CTR macrophytes.

GST activity in all evaluated macrophytes was enhanced by cell-free CE (Fig. 3A–C). While in CE-exposed C. demersum increasesin GST activity followed a positive time dependency with high-est activity toward the end of the experiment. In E. densa and H.

verticillata, a rapid GST activity enhancement was observed withinthe first 1 to 4 h of exposure followed by a GST activity decreasetoward the end of the experiments. In C. demersum, a statistically

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C.S. Romero-Oliva et al. / Aquatic Toxicology 163 (2015) 130–139 135

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ignificant ∼5-fold increase in GST activity was observed in CE-xposed macrophytes after only 8 h. GST activity in CE-exposed C.emersum remained increased thereafter, until the end of the expo-ure. The differences were statistically significant when comparedo the respective CTR group after 8 h, 1 day (+p > 0.05) 3 and 14 days*p > 0.05). In contrast, in E. densa significantly increased GST activ-ty in CE exposed-macrophytes was observed within the first hours

f exposure (after 4 (+p > 0.05) and 8 h (*p > 0.05)) (Fig. 3B). GSTctivity remained increased in CE-exposed E. densa compared toTR macrophytes thereafter, although not statistically significantly.ST activities in CE-exposed H. verticillata were also immediately

C. demersum (A and D), E. densa (B and E), and H. verticillata (C and F) after 1, 4, and(CE, patterned columns) and toxin-free medium (CTR, black columns). Asterisks (*)

ith ANOVA or Kruskal–Wallis test, respectively.

increased after 1 and 4 h of exposure (Fig. 3C) and were statisti-cally different compared to respective CTR macrophytes after 4 h(*p < 0.05).

GR enzyme activity was influenced by cell-free CE in E. densaand H. verticillata. In CE-exposed C. demersum, no differences in GRactivity were found when compared to the CTR during the 14 daysof exposure (Fig. 3D). In contrast, significantly increased GR activity

was found after 3 days in CE-exposed E. densa when compared toCTR macrophytes (+p > 0.05) (Fig. 3E). In turn, CE-exposed H. ver-ticillata had significanlty decreased GR activitiy compared to therespective CTR group after 7 days (+p > 0.05) (Fig. 3F).

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136 C.S. Romero-Oliva et al. / Aquatic Toxicology 163 (2015) 130–139

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ig. 3. Activity (mean ± SD in kat/mg protein) of glutathione S-transferase (GST) andC and F) after 1, 4, and 8 h as well as after 1, 3, 7, and 14 days exposure to cyanobacolumns). Asterisks (*) and crosses (+) indicate significant differences (p < 0.05) betw

. Discussion

In this study, adverse effects provoked by cell-free CE obtainedrom M. aeruginosa blooms in Lake Amatitlán, Guatemala withn situ MCs concentrations of 104.4 ± 7.6 �g/L (MC-LR 49.3 ± 2.9;RR 49.8 ± 5.9, and -YR 6.9 ± 3.8 �g/L) were studied in three locallyccurring, widely distributed macrophytes (C. demersum (L.), E.ensa (Planch.), and H. verticillata (L.f.)). CE containing three MCongeners led to changes in the photosynthetic pigments of the

hree evaluated macrophytes. Shifts in photosynthetic pigmentattern (i.e., chl a/chl b) has earlier been documented in thehlorophyte Chladophora sp. and macrophytes C. demersum, M. spi-atum, and Typha australis exposed to concentrations of MC-LR of

thione reductase (GR) in C. demersum (A and D), E. densa (B and E), and H. verticillata cell-free crude extract (CE, patterned columns) and toxin-free medium (CTR, blackCE and CTR calculated with ANOVA or Kruskal–Wallis test, respectively.

0.1–0.5 �g/L, Vallisneria natans exposed to 0.1–25 �g/L and Lemnaminor to MC-RR of 5 mg/L. Moreover in macrophyte L. minor theformer was in parallel observed with a decrease in total chl (Jianget al., 2011; Pflugmacher, 2002; Weiss et al., 2000). Furthermore,macrophytes exposed to MC-LR, showed this same shift after 24 h(Pflugmacher, 2002), 14 days (Jiang et al., 2011) and those exposedto MC-RR after 6 days (Weiss et al., 2000). In this study, the generaldecrease in chl a with concomitant increase in chl b was observed inall macrophytes much earlier, after only 4 h in C. demersum and H.

verticillata and after 8 h in E. densa. This was further accompanied bya decrease in total chl (i.e., C. demersum −1.4%, E. densa −3.7%, andH. verticillata −12.4% less than in respective CTR group) toward theend of the experiments. The results of the present study suggest that

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C.S. Romero-Oliva et al. / Aqua

ell-free CE containing various congeners of MCs affects photosyn-hetic activity of macrophytes (based on photosynthetic pigmentontent) more (i.e., 20% higher) and earlier (i.e., after 8 h instead offter 1 day) than in C. demersum exposed to single congeners, asbserved in earlier reports (Pflugmacher, 2002, 2004).

Insights into the specific effects of MCs on photosynthesisave been recently documented (Campos et al., 2013; Phelan andowning, 2014). These insights highlight that MCs can inhibit thectivity of the photosystem-PS II by binding into the thylakoidembranes thus, affecting energy transport to active reaction

enters (Jüttner and Lüthi, 2008; Phelan and Downing, 2014). Addi-ionally, more pronounced effects in photosynthetic efficiency,ecrease in photosynthetic quantum yield in the PS II and electronransport were highly accentuated in chlorophytes and other algaehen treated with crude extracts obtained from M. aeruginosa cul-

ures at concentrations of 0.01 and 41.5 �g/L (Campos et al., 2013;erron et al., 2012).

The observed changes in chl pattern and overall decrease of totalhl in this study might also be correlated with oxidative impair-ent of chloroplast ultrastructure. Since chloroplasts are centers

f oxidation due to respiration, increases in ROS provoked by unfa-orable environmental conditions (i.e., interaction with cell-freeE-containing mixture of MCs), might intensify the negative effectsnd lead to the deterioration of photosynthetic apparatus of macro-hytes. Even if biosynthesis of photosynthetic pigments (i.e., basedn chl a, chl b, carotenoid, and total chl concentrations) was notompletely inhibited in all the macrophytes evaluated in this study,ecrease in their concentrations was observed in a time-dependentanner. These findings suggest that at longer exposure durations,

hloroplasts of macrophytes might be damaged and thus, photo-ynthetic fitness might be severely hindered.

In contrast to the detected decrease in total chl, higher produc-ion of antioxidant carotenoid and activity increase of antioxidantnzymes CAT and POD were also observed in all CE-exposed macro-hytes, suggesting the disturbance of cellular homeostasis and

ncreased oxidative stress. As earlier documented, MCs can stim-late the formation of ROS (Jiang et al., 2011; Pflugmacher, 2004;assilakaki and Pflugmacher, 2008) with concomitant activationf antioxidative defense systems of macrophytes (Pflugmacher,002). The protective function of carotenoids in excess of light, lowemperatures, and desiccation has been documented for the modellant Arabidiopsis. This protective function is strongly related withhe role of carotenoids in the inactivation of ROS and thus, protec-ion of the PS II structure, thylakoid membranes acting as structuraltabilizers of some photosynthetic pigment–protein complexesDomonkos et al., 2013). In earlier findings with C. demersumxposed to the cyanobacterial neurotoxin, anatoxin-a at a concen-ration of 15 �g/L, production of high levels of carotenoids wasisplayed after 12 h of exposure reaching their maximum valuesfter 48 h (Ha and Pflugmacher, 2013b). The results presented herehow the same pattern, though earlier in terms of exposure dura-ion and with a higher concentration increase of carotenoids. InE-exposed C. demersum and E. densa, highest carotenoid concen-rations were observed after only 4 and 8 h, respectively, and in. verticillata slightly later after 8 h to 3 days. These results show

hat all CE-exposed macrophytes used in the present study seemo rely greatly on their capacity to raise antioxidant carotenoidoncentration in order to cope with oxidative stress at differentimes of exposure. Interestingly, CE-exposed C. demersum and E.ensa continued to produce more carotenoids in comparison to CTRacrophytes toward the end of the experiments by almost 34 and

6%, respectively.

Oxidative stress is generally initiated by the overproduction of

OS due to adverse environmental conditions. The most represen-ative and potent ROS is H2O2 (Gill and Tuteja, 2010). The enzymesAT and POD catalyze the detoxification of H2O2 into H2O and

xicology 163 (2015) 130–139 137

O2. In combination, the ascorbate–glutathione cycle also scavengesH2O2 via a series of interdependent redox reactions involving theantioxidants ASH and GSH as well as enzymes APX, MDHA, andGR. Within this cycle, GR plays an important role as it sustains thereduced status of GSH by catalyzing NADPH-dependent reaction ofdisulphide bond of GSSG (Gill and Tuteja, 2010). In this study, detox-ification of H2O2 by CAT, POD, and partly GR in the three evaluatedmacrophytes exposed to cell-free CE were observed within theinitial period of exposure to CE. In C. demersum possible scaveng-ing of H2O2 occurred within the first hours of exposure, indicatedby the activation of CAT. Thereafter, a significant time-dependentincrease of POD activity after 8 h until the end of the experimentwas observed. GR did not seem to play a determining role in theantioxidant defense of this macrophyte as its activity was similarto that of CTR macrophytes throughout the exposure. This suggeststhat C. demersum counteracts oxidative stress produced by cell-free CE containing MCs by first moderately activating enzymaticdefense via production of CAT, followed by a second strong enzy-matic activity increase of POD (i.e., after 8 h) in conjunction with anincrease in antioxidant carotenoid content. In CE-exposed E. densa,enhanced concentrations of carotenoids and increased CAT activ-ity within the first 4 h of exposure were followed by an increase inGR activity within 3 days, suggesting that E. densa when in contactto cell-free CE containing MCs uses both antioxidant carotenoidand CAT activity increase as a first line of antioxidant defense fol-lowed by a putative activation of the ascorbate–glutathione cycle,evidenced by the increase in GR activity. Finally CE-exposed H.verticillata showed the enzymatic activation via an initial activityincrease of CAT followed by POD until 1 day as antioxidant defense.

The results presented here show that the studied macrophytesdisplay an individual strategy against oxidative stress by either rais-ing the production of antioxidants, enzymatic antioxidant defense,or both. While the majority of former studies describe growth inhi-bition and photosynthetic pigment pattern changes in macrophytesexposed to single MCs (Campos et al., 2013; Perron et al., 2012;Pflugmacher, 2007; Pflugmacher et al., 2007a,b), this study sug-gests that not only photosynthethic pigments are affected, but thata strong activation of plant species-dependent antioxidant defensesystems occur when exposed to cell-free CE containing MCs.

In general, representation of real environmental conditions,such as the senescence of Lake Amatitlán’s cyanobacterial bloomscould imply a plant species-specific antioxidant defense strategy,suggesting the adaptive potential of these plants to such adverseconditions. Appearance of M. aeruginosa bloom formations havebeen reported for the last decades in Lake Amatitlán (Perez et al.,2011), as well as the presence of submerged, floating, and emergedmacrophytes (Basterrechea, 1984). The former as well as the resultsobtained in this study suggest that the three studied macrophytes C.demersum, E. densa, and H. verticillata possess well-adapted antioxi-dant defense mechanisms to cope with the recurring cyanobacterialblooms. To the best of our knowledge, this is the first study thatmimics a real cyanobacterial bloom condition of cell-free CE con-taining a mixture of three MCs, which periodically coexist with themacrophytes from Lake Amatitlán, Guatemala.

Levels of stress that led to the previously described results,might also be in related with the effort of the plant to biotransformMCs. Biotransformation of MCs is mediated by the conjugation ofGSH to MC-conjugates via soluble GSTs (Pflugmacher et al., 1998).While biotransformation of MC-LR has been documented in macro-phytes (Mitrovic et al., 2005; Pflugmacher et al., 1998, 1999, 2001),currently there are no available studies of biotransformation ofother MC congeners. In the present study, a mixture of cell-free

CE containing three MCs at an environmental relevant concentra-tion, provoked an enhancement in the biotransformation enzymeGST in the three exposed macrophytes. In CE-exposed E. densa andH. verticillata a rapid activation was observed within the first hours

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38 C.S. Romero-Oliva et al. / Aqua

f exposure; in CE-exposed C. demersum it occurred slightly laterfter 8 h. This was followed by an activity decrease in E. densa and. verticillata after 8 h, but not in C. demersum in which GST activ-

ty continued to increase in a time-dependent manner reachingts highest activity toward the end of the experiment. Assuminghat GST activity increase was in proportion with MC biotrans-ormation in the CE-exposed macrophytes, its early activation in. densa and H. verticillata might have provoked additional stress,eading to a higher ROS formation and consequently to the observedarly activation of the antioxidant defense systems of the plant (i.e.,ntioxidant–carotenoid and enzymatic response). On the one hand,hese two macrophytes, when in contact with MCs, might imme-iately take MCs up from the surrounding medium as a protectivetrategy with a consequent activation of the antioxidant defenseystems. On the other hand and based on the results obtainedor photosynthetic pigment content and both antioxidant defensend biotransformation enzymes, possible MC uptake might haverovoked an overproduction of ROS, strongly activating the antiox-

dant defense of macrophytes. In the case of exposed C. demersum,ven if activity increase of antioxidant enzymes and carotenoidsas observed, increased activation of GST was higher than in the

ther studied macrophytes. Based on the increase of GST activitys a possible indicator of MC uptake, uptake and further biotrans-ormation for C. demersum can be suggested. Immediate uptake of

Cs (i.e., after 30 min) in C. demersum has been previously observedPflugmacher et al., 1999), supporting the former proposed.

Assuming that macrophyte capacity to take up and biotransformCs would lead to higher ROS level and thus, activation of different

ntioxidant defense mechanisms in the macrophytes evaluated inhis study, it can be suggested that all three tested macrophytesave the ability to cope with the applied environmentally-realisticC concentrations. The high tolerance of C. demersum to MCs asell as its uptake capacity has been earlier documented and when

xposed to a much higher concentration (compared to the presenttudy) of 14C-MC-LR (2.5 mg/L), exposed macrophytes were stillble to take up 1.11% of this MC congener after 7 days (Pflugmachert al., 1998). The results obtained in this study highlight the higherolerance to CE mixture of MCs by C. demersum in comparison tohe other evaluated macrophytes (i.e., E. densa and H. verticillata).evertheless, assessment of the capacity of the macrophyte to takep, biotransform, and accumulate MCs from CE should be furthertudied in order to clarify the correlation between the antioxi-ant defense of the plant and biotransformation mechanisms whenxposed the plant is exposed to real environmental MC concentra-ion and mixtures.

In general, the results of the present study corroborate earlierndings, in which exposure to MCs from cell-free CE obtained from

iving cyanobacteria (laboratory cultures) (Bártová et al., 2010;innear et al., 2008; Pflugmacher, 2002) or from natural environ-ents led to higher activity of enzymatic defense systems thanhen exposed to commercial MCs (Campos et al., 2013; Perron

t al., 2012; Pflugmacher, 2007; Pflugmacher et al., 2007a,b). Fur-hermore, it was evidenced that C. demersum, E. densa, and H.erticillata can coexist at relatively high concentrations of MCs104.4 ± 7.6 �g/L) via the activation of their antioxidant defense

echanisms (i.e., antioxidant carotenoid production and enzy-atic antioxidant defense). Moreover, the tested macrophytesight play an active role in the elimination of MCs from natural

nvironments viatoxin biotransformation or bioaccumulation.

. Conclusions

Exposure of C. demersum, E. densa, and H. verticillata to cell-freeE containing three MC congeners at an environmental rele-ant concentration (MC-LR 49.3 ± 2.8, -RR 49.8 ± 5.9, and -YR

xicology 163 (2015) 130–139

6.9 ± 3.8 �g/L) affected photosynthetic and enzymatic antioxi-dant defense systems of these macrophytes. The general shift inphotosynthetic pigment pattern observed in all three exposedmacrophytes might imply that cell-free CE containing MCs firstaffects photosynthesis, leading further on to increased oxidativestress. Plant species-dependent antioxidant defense mechanismsindicated that for CE-exposed C. demersum an early enhancementof antioxidant carotenoid was followed by a general enzymaticantioxidant defense which displayed a positive time-dependenttrend. A similar trend for carotenoid content was observed in CE-exposed E. densa; however, significant and early activity increasewas observed for CAT. Finally, CE-exposed H. verticillata evidencedan early stimulation (i.e., after 1 h) of both, CAT and POD activities,which were later followed by the increase in antioxidant carotenoidcontent after 1 day. Furthermore, a similar time-dependent increas-ing activity trend for biotransformation enzyme GST was observedin all three macrophytes, suggesting the potential of these macro-phytes to metabolize MCs. The results show that environmentallyrelevant mixtures and concentrations of MCs might have a strongernegative effect in the antioxidant and biotransformation systems ofmacrophytes. To the best of our knowledge, interactions betweencoexisting macrophytes and cyanobacterial blooms, have not beenaddressed in former studies, making this study the first of its char-acter. Insights into the strategies of macrophytes to cope withcyanobacterial blooms, like antioxidant defense and biotransfor-mation might help to better understand the fate of MCs in aquaticecosystems.

Acknowledgments

We would like to thank Sandra Kühn for her assistance in the lab-oratory and the Deutscher Akademischer Austausch Dienst-DAADfor granting Claudia Suseth Romero-Oliva financial support to con-duct her Doctoral studies.

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