Biochemical and growth performance of the aquatic

Biochemical and growth performance of the aquatic macrophyte Azolla filiculoides to sub-chronic exposure to cylindrospermopsin Catarina Santos, Joana Azevedo, Alexandre Campos, Vitor Vasconcelos & Ana L. Pereira Ecotoxicology ISSN 0963-9292 Ecotoxicology DOI 10.1007/s10646-015-1521-x

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Author's personal copy Ecotoxicology DOI 10.1007/s10646-015-1521-x

Biochemical and growth performance of the aquatic macrophyte Azolla filiculoides to sub-chronic exposure to cylindrospermopsin Catarina Santos1 • Joana Azevedo1 • Alexandre Campos1,3 • Vitor Vasconcelos1,2 Ana L. Pereira1

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Accepted: 15 July 2015 Springer Science+Business Media New York 2015

Abstract Physiological and biochemical effects of cylindrospermopsin (CYN), a cyanobacterial toxin that inhibits protein synthesis and released during a harmful cyanobacterial bloom, has been overlooked in plants. Therefore, at the present research, the toxic effects (physiological and biochemical) of a crude extract containing CYN were assessed in the aquatic fern Azolla filiculoides exposed to three concentrations (0.05, 0.5 and 5 lg CYN mL-1). At 5 lg CYN mL-1, fern growth rate has showed a drastic decrease (0.001 g g-1 day-1) corresponding to a 99.8 % inhibition, but at the concentrations of 0.05 and 0.5 lg CYN mL-1 the growth rate was similar to the control plants. Growth rate also indicated a IC50 of 2.9 lg CYN mL-1. Those data point to the presence of other compounds in the crude extract may stimulate the fern growth and/or the fern is tolerant to CYN. Chlorophyll (a and b), carotenoids and protein content as well as the activities of glutathione reductase (GR) and glutathioneS-transferase (GST) has increased at 5 lg CYN mL-1 which may indicate that photosynthesis and protein

Catarina Santos and Ana L. Pereira researchers have contributed equally to the research. & Ana L. Pereira [email protected] 1

Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), BBE (Blue Biotechnology and Ecotoxicology), University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal

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Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, 4069-007 Porto, Portugal

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Present Address: Department of Clinical and Experimental Medicine, Cell Biology, Faculty of Health Science, Linko¨ping University, 581 83 Linko¨ping, Sweden

synthesis are not affected by CYN and the probable activation of defense and detoxifying mechanisms to overcome the effects induced by the presence of CYN. Low uptake of cylindrospermopsin (1.314 lg CYN g-1 FW) and low bioconcentration factor (0.401) point towards to a safe use of A. filiculoides as biofertilizer and as food source, but also indicate that the fern is not suitable for CYN phytoremediation. Keywords Azolla filiculoides Cylindrospermopsin Antioxidative enzymes Growth rate Photosynthetic pigments

Introduction Blooms of cyanobacteria are increasing worldwide due to eutrophication of water bodies. Some cyanobacteria species that forms those blooms can synthesise toxic compounds which contaminate water after their release (Apeldoorn et al. 2007; Cruz et al. 2013). Cylindrospermopsin (CYN) is an alkaloid with 415 Da synthesised through a polyketide gene cluster having a tricyclic guanidine moiety and a hydroxymethyluracil. Since the molecule is zwitterionic it is highly soluble in water (Banker et al. 2001). This cyanotoxin has been isolated from the cyanobacteria Cylindrospermopsis raciborskii, Aphanizomenon ovalisporum, Raphidiopsis curvata, Anabaena bergii, Anabaena flos-aquae, Umezakia natans and Lyngbya wollei mainly from tropical countries. However, CYN-producing cyanobacteria are invading regions with temperate climate including European countries such as France, Italy, Finland and German amongst others due probably to climate changes, increasing temperature and others. In nature, concentrations of CYN range from 0.002

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Author's personal copy C. Santos et al.

to 800 lg L-1 with the highest record of 1.5 mg L-1. Due to this wide range of CYN concentrations, bioaccumulation studies must consider those environmental concentrations while in ecotoxicological research CYN concentrations can be higher. Cylindrospermopsin has hepatotoxic, cytotoxic and neurotoxic effects and act through the inhibition of glutathione and protein synthesis (Apeldoorn et al. 2007; Kinnear 2010; Cruz et al. 2013). Although contaminated water can be ingested by animals or used for irrigation of crop fields posing a health and environmental threat to animal and humans, there are no international guidelines for CYN in drinking and recreational water. Studies about phytoremediation potential, bioaccumulation, physiological and biochemical damages or induction of oxidative stress in plants (terrestrial or aquatic) due to CYN exposure are scarce. Pollen germination of Nicotiana tabacum was inhibited in a concentration of 5–1000 lg CYN mL-1 (Metcalf et al. 2004). However, CYN at 0.57, 5.7 and 57 lg L-1 did not inhibited seed germination of Lactuca sativa, Phaseolus vulgaris, Pisum sativum and Solanum lycopersicum (Silva and Vasconcelos 2010). Roots are one of the plants organs most affected by exposure to CYN. At a range of concentrations between 25–400 lg L-1 CYN has induced an increase in root growth in Hydrilla verticillata (Kinnear et al. 2008), at 0.5–40 lg CYN mL-1 induced a decrease in root elongation and necrosis in root cortex of Phragmites australis (Beyer et al. 2009) and at 0.01–20 lg CYN mL-1 has inhibited xylem differentiation and lateral roots in Sinapsis alba (Ma´the´ et al. 2013). On the other hand, at concentrations of 0.57, 5.7 and 57 lg L-1, root elongation in S. lycopersicum and P. sativum has decreased, but in L. sativa and P. vulgaris it increased (Silva and Vasconcelos 2010). Growth and biomass are also influenced with CYN: biomass of Oryza sativa has decreased after 9 days exposure to 2.5 lg CYN L-1 (Prieto et al. 2011) but biomass of the aquatic macrophyte Spirodela oligorrhiza (Kinnear et al. 2007) and growth of H. verticillata (Kinnear et al. 2008) increased or decreased depending on concentration and exposure time. Photosynthetic pigments content are influenced by CYN. Chlorophyll a and b content and ratio a/b in S. oligorrhiza has showed small changes for a range of CYN concentrations between 7.5 and 120 lg L-1 (Kinnear et al. 2007). On the other hand, chlorophylls a and b has decreased for all CYN concentrations (25–400 lg L-1) in H. verticillata, but the ratio a/b has increased (Kinnear et al. 2008). Concerning antioxidative enzymes, the information also is scarce. In O. sativa, activities of glutathioneS-transferase (GST) and glutathione peroxidase (GPx) has increased in roots and leaves pointing to an increase in oxidative stress (Prieto et al. 2011) probably due to the generation of reactive oxygen species (ROS). In the

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microalgae Chlorella vulgaris, the activity of GPx and GST has increased at 18.4 and 179 lg CYN L-1 (Campos et al. 2013). Those few studies could indicate plants defence mechanisms respond differently depending on CYN concentrations and also depend of the plant species. Uptake and bioconcentration of CYN in plant tissues (roots, stems or leaves) poses a potential health and environmental problem. CYN has been detected in the aquatic macrophytes H. verticillata (White et al. 2005) and S. oligorrhiza (Kinnear et al. 2007) and in the agronomic crops P. vulgaris, L. sativa and P. sativum (Silva and Vasconcelos 2010), O. sativa (Prieto et al. 2011) and B. juncea and B. oleracea var. sabellica (Kittler et al. 2012). However, given that the bioconcentration factor in all these plants was inferior to 1, those plants did not showed CYN bioconcentration. But the detection of CYN in roots and shoots point to cylindrospermopsin translocation from roots to shoots. Aquatic plants (also called macrophytes) such as Lemna sp., Myriophyllum sp., Eichhornia crassipes are of up most importance since those plants are in direct, permanent and continuous contact with water contaminants such as heavy metals, phosphorus, nitrogen, antibiotics, cyanotoxins such as CYN, among others. Phytoremediation can be achieved by aquatic macrophytes (submerged or floating) by biosorption or bioaccumulation of the contaminants through roots in the case of floating plants or through the whole plant organs in the case of submerged plants (Tel-Or and Forni 2011). Floating aquatic plants (meaning that their roots are hanging in the water column) are in continuous contact with CYN and given that CYN are continuously released by cyanobacteria to water, macrophytes are exposed to increasing content of CYN for days or weeks. Also, since CYN is very stable to a broad range of pH, temperature and light intensity (Chiswell et al. 1999) this molecule will persist in the water column even after the cyanobacterial blooms already disappeared. So, the use of aquatic macrophytes to evaluate the effects due by CYN is crucial. One of the most studied aquatic plants regarding phytoremediation of contaminated waters is Azolla. Azolla is a small freefloating aquatic pteridophyte with a worldwide distribution mainly in tropical and temperate regions covering large water areas (such as rivers or dams) forming dense blooms when environmental conditions are optimal (light, temperature, water flow, water nutrients). The sporophyte (Fig. 1) is formed by a main rhizome with lateral ramifications and covered by small deeply bilobed leaves (an aerial thick chlorophyllous dorsal lobe and a thin hyaline floating or partially submersed ventral lobe). The adventitious roots hanging into water protrude from the ventral side of the rhizome. This fern has a permanent symbiosis with the filamentous heterocystic nitrogen-fixing cyanobacterium Anabaena azollae inhabiting an ovoid cavity at the foliar dorsal

Author's personal copy Biochemical and growth performance of the aquatic macrophyte Azolla filiculoides…

Hoagland H-40 pH 6.1-6.2, in controlled conditions (Pereira and Carrapiço 2009). Cyanobacterium A. ovalisporum (LEGE X-001) obtained from the cyanobacteria culture collection of Laboratory of Ecotoxicology, Genomics and Evolution (LEGE) was grown in Z8 medium (Kotai 1972) in a growth chamber at 25 C, light intensity 10 lE m-2 s-1 and photoperiod (light/dark) 14/10 h. Extraction and quantification of cylindrospermopsin in a crude extract

Fig. 1 Sporophyte of the fern A. filiculoides

lobe. In this symbiosis, the fern provide carbohydrates to the cyanobiont and in return the cyanobiont provides all the nitrogen that the fern needs. Although this fern has sexual and asexual (also called vegetative) reproduction, the sexual reproduction is very rare in nature. So, the main way of dispersal is by fragmentation of the lateral ramifications of the main rhizome (Wagner 1997; Carrapiço 2010). This symbiosis has been investigated for use as biofertilizer especially in rice fields (Wagner 1997; Carrapiço et al. 2000), as animal food (Khatun et al. 1999) and for the phytoremediation of nitrogen, phosphorus and heavy metals of industrial and domestic waters (Costa et al. 2009; Tel-Or and Forni 2011; Sood et al. 2012). However, the environmental persistence of CYN can hinder the utilization of Azolla in several of those purposes. Therefore, it is important to understand the toxicological effects and the bioaccumulation of CYN in this aquatic macrophyte. The aims of the present study were to evaluate the effects of a CYN-containing crude extract from the cyanobacterium Aphanizomenon ovalisporum in three concentrations (0.05, 0.5 and 5 lg mL-1) in three physiological parameters (growth, photosynthetic pigments and antioxidant enzymes) in the aquatic maprophyte A. filiculoides, providing an indication for their potential as phytoremediator, biofertilizer, animal food and environmental stress marker.

Materials and methods

CYN was extracted following the methods of Welker et al. (2002) and Pinheiro et al. (2013) with modifications. Briefly, lyophilised biomass of A. ovalisporum was 5-times extracted with 0.1 % trifluoroacetic acid (TFA, v/v) in ultrapure water (Millipore, Madrid, Spain), homogenised and stirred for 30 min, followed by ultrasound at 35 Hz (RK100H, Bandelin Sonorex, Berlin, Germany) for 15 min in a water bath, probe sonication (VibraCell, Sonics & Materials Inc, Danbury, USA) at 20 Hz for 5 min in ice and centrifuged at 45429g, 4 C, 10 min. The supernatant fraction was collected and stored at -20 C. CYN was quantified following a modified method of Pinheiro et al. (2013) by analytical HPLC using a HPLC Waters Alliance e2695 (Waters, Sacave´m, Portugal) equipped with a photodiode array (PDA) detector 2998 and a reverse phase column (LiChrospher 100 RP-18 endcapped, 25 cm 9 4.6 mm, 5 lm) (Merck Chemicals, Lisbon, Portugal) kept at 40 C. An isocratic elution of 5 % MeOH acidified with 0.1 % TFA was the mobile phase with a flow rate was 0.9 mL min-1, injection volume of 10 lL and PDA range was 210–400 nm with a fixed wavelength at 262 nm. CYN spectrum and retention time of samples and standard (NRC, CRM-CYN, Lot #200505310387, Ottawa, Canada) were compared. For the calibration, a set of seven standard concentrations (0.5–25 lg mL-1) was used with a limit of detection (LOD) of 0.3 lg mL-1 and a signal to noise ratio higher than 3 (S/N C 3) and a limit of quantification (LOQ) of 0.8 lg mL-1 and S/N C 10. Concentration of CYN in samples were obtained by the linear regression equation y = 14341x - 237.1 (R2 = 1). All the solvents used were of HPLC grade, filtered in a 0.2 lm hydrophilic polypropylene membrane filters (Pall Life Sciences, Madrid, Spain) and degassed before use. Exposure of A. filiculoides to a crude extract containing cylindrospermospin

A. filiculoides and A. ovalisporum growth conditions Pteridophyte A. filiculoides (accession number FI1001) obtained from the germplasm collection at International Rice Research Institute (IRRI) grown in the medium

Sporophytes of A. filiculoides (approximately 1.3 g corresponding to a plant density of 167.21 ± 1.78 g m-2) were exposed to three concentrations of CYN (0.05, 0.5 and 5 lg mL-1) for 7 days (Fig. 2). A control (without crude

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extract) was included. For each concentration and control were used three replicates. After exposure time, biomass was harvested, washed in distilled water, water excess removed in absorbent paper for 5-10 min, weighted and stored at -80 C. Growth and biochemical parameters Relative growth rate (RGR) (Maejima et al. 2001), percent inhibition of relative growth rate (Ir) and inhibition concentration at 50 % (IC50) (OECD 2002) were estimated. To extract chlorophylls and carotenoids, sporophytes (100 mg) were sliced and immerse in 96 % EtOH, overnight, at 4 C, in darkness (Lichtenthaler 1987). The absorbance of the extracts was measured in a 96-well plate at 470, 648, 664 and 750 nm. Content of chlorophylls (a and b) and carotenoids were calculated according to Lichtenthaler (1987) equations. Extraction of phycobiliproteins (PBP) followed the method of Kaplan et al. (1986) with modifications. Sporophytes (100 mg) were ground to a fine powder in liquid nitrogen, extracted with 2.5 mM sodium phosphate buffer, pH 7, homogenized and centrifuged at 45429g, 20 min, 4 C. Absorbance was measured in a 96-well plate at 572, 612, 647 and 750 nm. Content of three phycobiliproteins, phycoerythrocyanin (PEC), phycocyanin (PC) and allophycocyanin (APC) were calculated according to Kaplan et al. (1986) equations. Protein extraction followed a modified method of Yin et al. (2005). Sporophytes (500 mg) were ground to fine

powder in liquid nitrogen and were extracted with 100 mM sodium phosphate buffer pH 7, on ice. Samples were homogenized, centrifuged at 45429g, for 30 min, at 4 C and the protein-containing fraction stored at -80 C until their use. Proteins were quantified with the Bradford method (Bradford 1976) using a commercial kit (SigmaAldrich, Sintra, Portugal) following the manufacturer instructions for 96-well plate. Protein extract was used to quantify three antioxidative enzymes. Glutathione-S-transferase (GST, EC 2.5.1.18) activity was quantified by measuring the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione (GSH) following the method of Habig et al. (1974). Glutathione peroxidase (GPx, EC 1.11.1.9) activity was measured indirectly through the NADPH decrease (Flohe´ and Gu¨nzler 1984). Glutathione reductase (GR, EC 1.6.4.2) activity was quantified by monitoring the NADPH decrease (Carlberg and Mannervik 1975). Absorbance was measured at 340 nm, 25 C in a kinetic assay with 21 reads at 15 s intervals for 5 min. All the absorbances were measured in a Synergy HT spectrophotometer (BioTek, Madrid, Spain) equipped with Gen5 2.00 software. Bioaccumulation of cylindrospermopsin in A. filiculoides About 250 mg of A. filiculoides sporophytes were exposed to 0.05, 0.5 and 5 lg CYN mL-1 for 7 days. Each assay had three replicates. After 7 days, the sporophytes were collected, washed in distilled water, excess of water removed in absorbent paper for 5–10 min, weighted and stored at -80 C. The medium of each assay also were collected, frozen at -20 C, lyophilised and resuspended in 0.1 % TFA in ultrapure water. Biomass of A. filiculoides was ground to fine powder with liquid nitrogen and CYN extracted as previously described for A. ovalisporum (see Extraction and quantification of cylindrospermopsin in a crude extract). All the samples were analysed by HPLC as formerly described (see Extraction and quantification of cylindrospermopsin in a crude extract). Cylindrospermopsin concentration in A. filiculoides was expressed as lg CYN g-1 FW. The bioconcentration factor (BCF) was calculated by dividing the cylindrospermopsin concentration in A. filiculoides (lg CYN mL-1) by their concentration in the growth medium (lg CYN mL-1) (BCF = [CYN]A. filiculoides/[CYN]medium) (Karjalainen et al. 2003). Data analysis

Fig. 2 Diagram of intoxication experimental design

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Prior to the analysis of variance, the ANOVA assumptions normality and homogeneity of variance were verified by Shapiro–Wilk and Levene tests, respectively. Data


fulfilling the ANOVA assumptions were analysed by oneway ANOVA and to compare means the post hoc Tukey test were performed. Data not following the ANOVA assumptions were analysed by the nonparametric test Kruskal–Wallis and to compare means the post hoc Mann– Whitney test were used (Zar 1999). The significance level (a) was 0.05. Statistical analyses were made using the IBM SPSS Statistics 21.0.

Results Growth of Azolla filiculoides Relative growth rate of A. filiculoides has shown an abrupt decrease (0.001 g g-1 days-1) at the highest CYN concentration (5 lg mL-1), which was statistically significant different from control and the other two CYN concentrations (0.05 and 0.5 lg mL-1) (Fig. 3). RGR also has indicated a growth stimulation of 8.42 ± 0.07 and 13.71 ± 0.05 % at the concentrations 0.05 and 0.5 lg CYN mL-1, respectively. Instead, a growth inhibition of 99.8 % was showed by the fern at the highest CYN concentration (5 lg mL-1). The IC50 value that inhibited A. filiculoides growth rate at 50 % was of 2.9 lg CYN mL-1.

Although the content of the three phycobiliproteins PEC, APC and PC (Fig. 4c) has increased with all the CYN concentrations used in the present study, those differences were not statistically significant. Proteins and antioxidative enzymes Protein content of A. filiculoides has showed a marked increase (1.66 lg mL-1) in plants exposed at the highest CYN concentration (5 lg mL-1). These increased was statistically significant different to control and the other two lower concentrations (Fig. 5). Concerning antioxidative enzymes of the glutathione system, differences in the GPx activity related to the fern exposure to a cyanobacterial crude extract containing CYN

Photosynthetic pigments Photosynthetic pigments chlorophyll a and b (Fig. 4a) and carotenoids (Fig. 4b) has increased with all the CYN concentrations. Statistically significant differences for the analysed pigments were observed in the ferns that grew at the highest concentration (5 lg CYN mL-1) (content of 162.0, 50.0 and 44.0 lg g-1 FW for chlorophyll a, chlorophyll b and carotenoids, respectively) compared with control and the other two CYN concentrations (0.05 and 0.5 lg mL-1).

Fig. 3 Relative growth rate of A. filiculoides after 7 days exposure to a crude extract with CYN. The values are mean ± se (n = 3). Different letters indicate statistically significant differences between treatments by the post hoc Tukey test (p B 0.05)

Fig. 4 Photosynthetic pigments of A. filiculoides-A. azollae after 7 days exposure to a crude extract with CYN. a Chlorophyll a and b content. b Carotenoids content. c Content of phycoerythrocyanin (PEC), phycocyanin (PC) and allophycocyanin (APC). The values are mean ± se (n = 3). Different letters indicate statistically significant differences between treatments for each pigment by the post hoc Tukey (p B 0.05) (chlorophylls), Mann–Whitney (p B 0.05) (carotenoids) and ANOVA (p [ 0.05) (phycobiliproteins) tests

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were not statistically significantly different (Fig. 6a). On the other hand, GR activity has increased with increasing CYN concentration having the highest activity (0.05 nmol min-1 mg-1 protein) at 5 lg CYN mL-1 being statistically significant different from control and the other two CYN concentrations (0.05 and 0.5 lg mL-1) (Fig. 6a). GST activity has showed a marked increase (1.1 nmol min-1 mg-1 protein) in the fern subject to the highest CYN concentration (Fig. 6b), and this value was statistically significantly different from the control and other two CYN concentrations (0.05 and 0.5 lg mL-1). Uptake of CYN and bioconcentration factor HPLC analysis of A. filiculoides extracts has showed no CYN in control plants and no uptake of CYN in plants growing at the concentrations 0.05 and 0.5 lg CYN mL-1 (Fig. 7a). However, extracts of A. filiculoides that grew at the highest CYN concentration (5 lg CYN mL-1) did showed CYN (Fig. 7a, b) in a concentration of 1.314 ± 0.11 lg CYN g-1 FW indicating that the fern uptake CYN. The bioconcentration factor (BCF) in the fern for the exposure at 5 lg CYN mL-1 was 0.401 ± 0.04.

Discussion Investigations about phytotoxicity, bioconcentration or phytoremediation of cylindrospermopsin in plants (agronomic and non-agronomic, terrestrial or aquatic) are very sparse. Given that aquatic plants are in permanent contact with water contaminants, they must be in the forefront of the CYN research. One of such aquatic plants should be the fern Azolla. Although the potential of the Azolla-A. azollae symbiosis to remove heavy metals, nitrogen and phosphorus from water has been thoroughly studied (Tel-Or and

Fig. 5 Protein content in of A. filiculoides-A. azollae after 7 days exposure to a crude extract with CYN. The values are mean ± se (n = 3). Different letters indicate statistically significant differences between treatments by the post hoc Tukey test (p B 0.05)

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Fig. 6 Antioxidative enzymes activities of A. filiculoides-A. azollae determined after 7 days exposure to a crude extract with CYN (a) Glutathione peroxidase (GPx) and glutathione reductase (GR) activity. b Glutathione-S-transferase (GST) activity. The values are mean ± se (n = 3). Different letters indicate statistically significant differences between treatments for each antioxidative enzyme by the post hoc Tukey (p B 0.05) (GR and GST) and ANOVA (GPx) (p [ 0.05) tests

Forni 2011; Sood et al. 2012), there are no research about the effect of cylindrospermopsin on this fern. Bioconcentration and phytoremediation of CYN by A. filiculoides Bioaccumulation of CYN in plant tissues is an environmental and health problem due to their potential transfer to higher trophic levels, but also can indicate the potential to phytoremediate water contaminated with CYN. Plants accumulate xenobiotics and toxins when the bioconcentration factor (BCF) is higher than one (Karjalainen et al. 2003). Macrophytes H. verticillata (White et al. 2005) and S. oligorrhiza (Kinnear et al. 2007) as well as the edible plants L. sativa, P. vulgaris, P. sativum, S. tuberosum (Silva and Vasconcelos 2010), O. sativa (Prieto et al. 2011), B. oleraceae var. sabellica and B. juncea (Kittler et al. 2012) has showed a bioconcentration factor for CYN lower than 1. The same occurred with the macrophyte A. filiculoides in the present research with a BCF of 0.401 at the highest CYN concentration (5 lg mL-1) meaning the non-bioaccumulation of CYN. However, at the present study, CYN concentration in which occurred CYN uptake was much higher than that ones used for H. verticillata


Fig. 7 HPLC of A. filiculoides extracts after 7 days exposure to a cyanobacterial crude extract containing CYN. a Chromatogram A. filiculoides samples (curve 1 control; curve 2 exposure to 0.05 lg CYN mL-1; curve 3 exposure to 0.5 lg CYN mL-1; curve 4

exposure to 5 lg CYN mL-1) and CYN standard (curve 5). b Absorbance spectrum of CYN peak in A. filiculoides tissues exposed to 5 lg CYN mL-1

(25-400 lg CYN L-1) (White et al. 2005), S. oligorrhiza (7.5–120 lg CYN L-1) (Kinnear et al. 2007), O. sativa (2.5 lg CYN L-1) (Prieto et al. 2011) and B. juncea (22–444 lg CYN L-1) (Kittler et al. 2012) pointing to a very low uptake of CYN by A. filiculoides. Another explanation to the low detection of CYN in plant tissues would be biosorption of CYN instead of uptake. In fact, Kinnear et al. (2007) point out that necrotic fronds of S. oligorrhiza could facilitate the sorption of CYN to plant tissues rather than their uptake. This could be the case of the present research, since at the highest CYN concentration the brownish zones in the leaves and roots of A. filiculoides rhizome may facilitate the biosorption of CYN. Regardless of the explanation (uptake or biosorption) to the detection of CYN in A. filiculoides, the present results could indicate A. filiculoides might not be an efficient CYN phytoremediator, but could be used as biofertilizer and as animal feed since it does not bioaccumulate CYN. However, the presence of an environmental cyanotoxin in water bodies where A. filiculoides grows could induce changes in the fern that can be useful as stress markers.

toxic compounds. In fact, environmental pollutants such as heavy metals also induce a growth increase of A. filiculoides exposed at low concentrations (Sańchez-Viveros et al. 2011). Moreover, the presence of other water- soluble metabolites in the crude extract (Pietsch et al. 2001; Kinnear et al. 2008) especially at lower concentrations may interfere with the CYN toxic effects in A. filiculoides and thus the fern grow at a rate superior to the control. Pinheiro et al. (2013) point that growth stimulation of microalgae (Chlamydomonas reinhardtii, C. vulgaris, Nannochloropsis sp.) at low CYN concentrations (0.25–0.5 mg L-1) could be due to other compounds present in CYN crude extract regarded as extra nutrients. Growth drastic decrease (99.8 % of inhibition) has only occurred in A. filiculoides at very high CYN concentrations (5 lg mL-1) thus indicating that the aquatic fern A. filiculoides may be tolerant or resilient to CYN. The IC50 of 2.9 lg CYN mL-1 for A. filiculoides was much lower than IC50 of S. alba (800 and 18.2 lg CYN mL-1 for crude extract and pure CYN, respectively) (Vasas et al. 2002) and N. tabacum (300 CYN mL-1 for crude extract) (Metcalf et al. 2004). The growth and IC50 indicates that at environmental CYN concentrations (0.002–800 lg mL-1) A. filiculoides will growth and will be tolerant but at high CYN concentrations deleterious effects will occur.

Growth performance of A. filiculoides exposed to a CYN-containing crude extract CYN at very low concentrations (2.5 lg L-1) have no effect on biomass of O. sativa after 6 days exposure, but after 9 days exposure the biomass of O. sativa has decreased (Prieto et al. 2011). On the other hand, the aquatic plants S. oligorrhiza (Kinnear et al. 2007) and H. verticillata (Kinnear et al. 2008) has showed an increase in the relative growth rate even at a concentration of 113 and 400 lg L-1 respectively. In A. filiculoides also has occurred a growth increase with the two lower CYN concentrations (0.05 and 0.5 lg mL-1). The increase in biomass production can be an approach of plants (aquatic or terrestrial) to deal with a cyanobacterial bloom and release of

Biochemical response of A. filiculoides to an A. ovalisporum crude extract containing CYN Chlorophylls and carotenoids are photosynthetic pigments that can be used as plant fitness biomarkers as changes in their content can point to plant response to stress. In H. verticillata, content of chlorophyll a and b has decreased with CYN concentrations (25–400 lg L-1) and exposure time (7 and 14 days) meaning CYN affects chlorophyll synthesis or increase the pigment degradation (Kinnear et al. 2008). On the other hand, chlorophylls (a and b) content in

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S. oligorrhiza were similar for CYN concentrations ranging 8–110 lg L-1 (Kinnear et al. 2007). In the present study, both chlorophylls (a and b) has increased with the highest CYN concentration (5 lg mL-1) probably indicating that chlorophyll synthesis is not inhibited or the degradation rate is not affected. Nothing is known about the effects of CYN in carotenoids in aquatic or terrestrial plants but in the present research carotenoids content follows the same pattern of chlorophylls meaning their highest content was obtained at 5 lg CYN mL-1. A. filiculoides intoxicated with arsenate (another water contaminant) induced a decline in chlorophylls and an increase in carotenoids (Sańchez-Viveros et al. 2011). Increase in chlorophylls and carotenoids when A. filiculoides has showed almost 100 % growth inhibition seems contradictory, but may reveal that this aquatic fern handle with the deleterious effects of CYN or other compounds present in the crude extract though the increase of those photosynthetic pigments. Phycobiliproteins of the cyanobiont A. azollae are fully characterized (Kaplan et al. 1986) but they are not analysed to ascertain the cyanobiont fitness. These pigments are accessory antenna proteins associated with the photosystem II and also function as antioxidant and ROS scavenging. In this study, the content of three phycobiliproteins (PEC, PC and APC) did not showed significant changes with the three CYN concentrations used. This may indicate that the cyanobiont metabolism especially bilin pathway was not affected by the decrease of A. filiculoides growth at the highest CYN concentration. Also, these data indicate that probably A. filiculoides did not interrupt the carbohydrate flux to the cyanobiont A. azollae that is essential for the maintenance of the cyanobiont metabolism. Maybe the supply of nitrogen by the cyanobiont to the fern was not interrupted and contributing to cope with the environmental stressor CYN and the maintenance of A. filiculoides metabolism. While cylindrospermopsin is an inhibitor of protein synthesis, glutathione and cytochorme P450 (Apeldoorn et al. 2007; Kinnear 2010; Pearson et al. 2010) their impact in the inhibition of plant protein synthesis only was studied in N. tabacum pollen germination (Metcalf et al. 2004). In the present study, CYN at concentrations of 0.05 and 0.5 lg mL-1 did not affect protein synthesis, but at the highest concentration (5 lg CYN mL-1) protein content showed a drastic increase. This could indicate that protein synthesis was not inhibited even when occurred a severe decrease of A. filiculoides growth but in fact the fern metabolism was bootsed at very high CYN concentrations to overcome possible deleterious effects. Detoxifying mechanisms of ROS or xenobiotics include enzymes (superoxide dismutase, catalase and others), enzymes of the glutathione system and antioxidative compounds. Those mechanisms are important for plant tolerance to biotic and abiotic stress (Gill and Tuteja 2010).

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In leaves of O. sativa after 6 days exposure with a crude extract of 2.5 lg CYN L-1, GPx activity did not showed significant differences, but GST activity increased (Prieto et al. 2011). On the other hand, in the microalgae C. vulgaris activity of the enzymes GST and GPx has increased at 18.4 lg CYN L-1 and decreased at 179 lg CYN L-1 (Campos et al. 2013). Those data indicate that CYN can induce oxidative stress and GST detoxifies it. In A. filiculoides the maintenance of GPx levels with all CYN concentrations probably indicate CYN or other water soluble compounds present in the crude extract does not induced the generation of ROS. But the increase of GST and GR levels at the highest CYN concentration may indicate an activation of the detoxifying mechanism through GST and important for plant tolerance under stress. Also, the increase in GR activity only at the highest CYN may point to the maintenance of high cellular levels of GSH/GSSG ratio and oxidation of NADPH. Given that the fern does not bioaccumulated CYN, the increase of GR and GST activities may be due to other stress conditions such as the presence of other compounds in the crude extract.

Conclusions In the present research, it was showed that A. filiculoides are not suitable to phytoremediate CYN from water bodies due to the low uptake of CYN (1.314 lg CYN g-1 FW). However, the very low BCF (0.401), makes this fern suitable to be used as biofertilizer and animal food. Data also point to possible tolerance of A. filiculoides to environmental concentrations of CYN given that this aquatic plant grown very well at 0.05 and 0.5 lg CYN mL-1. An IC50 of 2.9 lg CYN mL-1 at 5 lg CYN mL-1 may possibly indicate tolerance to CYN. Photosynthetic pigments (chlorophyll a and b), carotenoids and protein content has increased with 5 lg CYN mL-1 point towards a response of the fern to the presence of the toxin and thus maintaining the plant metabolism. Activities of antioxidative enzymes GST and GR has increased at 5 lg CYN mL-1 which may point to the activation of detoxifying mechanisms through the glutathione. The maintenance of GPx levels indicate probably the non generation of ROS. All of these data point that other water soluble compounds besides CYN may influence the response of the fern. While growth has almost stopped at the high CYN concentration, fern and cyanobiont still maintain their metabolism to try to overcome the deleterious effects of CYN and other compounds. Acknowledgments This research was partially supported by 1) the European Regional Development Fund (ERDF) through the COMPETE (Operational Competitiveness Programme) and national funds through FCT (Foundation for Science and Technology) under the project PEst-C/MAR/LA0015/2013 and 2) Porto University under the

Author's personal copy Biochemical and growth performance of the aquatic macrophyte Azolla filiculoides… project IJUP2011_3. The European Social Funding (FSE) under the Human Potential Operational Program (POPH) of National Strategic Reference Board (QREN) supports the fellowship SFRH/BPD/44459/ 2008 to Ana L. Pereira. Thanks to Stephan Haefele and Agnes Padre of IRRI for sending A. filiculoides (FI1001). Compliance with ethical standards Conflict of interest of interest.

The authors declare that they have no conflict

Ethical standard This article does not contain any studies with human participants or animals performed by any of the authors.

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