Ecotoxicology and Environmental Safety 105 (2014) 80–89

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Bioabsorption of cadmium, copper and lead by the red macroalga Gelidium floridanum: Physiological responses and ultrastructure features Rodrigo W. dos Santos a,1, Éder C. Schmidt b,n,1, Marthiellen R. de L Felix a, Luz K. Polo a, Marianne Kreusch c, Debora T. Pereira c, Giulia B. Costa a, Carmen Simioni a, Fungyi Chow d, Fernanda Ramlov e, Marcelo Maraschin e, Zenilda L. Bouzon f a Plant Cell Biology Laboratory, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil b Postdoctoral Researcher of Postgraduate Program in Cell Biology and Development, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900, CP 476 Florianópolis, SC, Brazil c Scientific Initiation-PIBIC-CNPq, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil d Institute of Bioscience, Department of Botany, University of São Paulo, 05508-090 São Paulo, SP, Brazil e Plant Morphogenesis and Biochemistry Laboratory, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil f Central Laboratory of Electron Microscopy, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 21 October 2013 Received in revised form 20 February 2014 Accepted 24 February 2014

Heavy metals, such as lead, copper, cadmium, zinc, and nickel, are among the most common pollutants found in both industrial and urban effluents. High concentrations of these metals cause severe toxic effects, especially to organisms living in the aquatic ecosystem. Cadmium (Cd), lead (Pb) and copper (Cu) are the heavy metals most frequently implicated as environmental contaminants, and they have been shown to affect development, growth, photosynthesis and respiration, and morphological cell organization in seaweeds. This paper aimed to evaluate the effects of 50 μM and 100 μM of Cd, Pb and Cu on growth rates, photosynthetic pigments, biochemical parameters and ultrastructure in Gelidium floridanum. To accomplish this, apical segments of G. floridanum were individually exposed to the respective heavy metals over a period of 7 days. Plants exposed to Cd, Cu and Pb showed discoloration of thallus pigmentation, chloroplast alteration, especially degeneration of thylakoids, and decrease in photosynthetic pigments, such as chlorophyll a and phycobiliproteins, in samples treated with Cd and Cu. Moreover, cell wall thickness and the volume of plastoglobuli increased. X-ray microanalysis detected Cd, Cu and Pb absorption in the cell wall. The results indicate that Cd, Pb and Cu negatively affect metabolic performance and cell ultrastructure in G. floridanum and that Cu was more toxic than either Pb or Cd. & 2014 Elsevier Inc. All rights reserved.

Keywords: Gelidium floridanum Ultrastructure Chloroplasts Cadmium Lead Copper

1. Introduction Over the last few years, increasing human population and industrial development have led to an increase of contaminants in aquatic systems (Rocchetta et al., 2007). Accordingly, studies reporting the effects of heavy metals on aquatic organisms are currently attracting more attention, particularly those focusing on industrial and urban pollution. The contamination of coastal

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Corresponding author. E-mail address: [email protected] (É.C. Schmidt). 1 Rodrigo W. dos Santos and Eder C. Schmidt should be considered as first authors. http://dx.doi.org/10.1016/j.ecoenv.2014.02.021 0147-6513 & 2014 Elsevier Inc. All rights reserved.

waters with trace metals through sewage and other anthropogenic sources has become a severe problem (Mamboya et al., 1999). Heavy metals, such as lead (Pb), copper (Cu), cadmium (Cd), zinc (Zn), and nickel (Ni), are among the most common pollutants found in both industrial and urban effluents (Sheng et al., 2004). In low concentrations, some heavy metals (Cu, Zn, Ni, and Mn) are essential trace elements for photosynthetic organisms; however, in higher concentrations, these metals cause severe toxic effects (Hu et al., 1996). Heavy metals affect all organisms, especially those in the aquatic ecosystem, in many important ways. Several studies have shown such effects as decreasing macroalgal growth rates (Mamboya et al., 1999), changes in photosynthetic pigments (Bouzon et al., 2012a; Rocchetta et al., 2007), and photosynthetic

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efficiency (Bouzon et al., 2012a; Mamboya et al., 1999), as well as increasing total proteins and lipid contents (Rocchetta et al., 2007). Some reports have shown changes in the ultrastructure of the red algae Audouinella savina (F.S. Collins) Woelkerling (Talarico, 2002), Ceramium ciliatum (J. Ellis) Ducluzeau (Diannelidis and Delivopoulos, 1997), Hypnea musciformis (Wulfen) Lamouroux (Bouzon et al., 2012a), and Gracilaria domingensis (Kützing) Sonder ex Dickie (Santos et al., 2012, 2013); the green algae Dunaliella minuta Lerche (Visvik and Rachlin, 1992) and Enteromorpha flexuosa (Wulfen) J.Agardh (Andrade et al., 2004); the photosynthetic euglenoid Euglena gracilis Klebs (Rocchetta et al., 2007); and the brown alga Padina gymnospora (Kützing) Sonde (Andrade et al., 2002). Gradual increase in the discharge of heavy metals and other pollutants into the environment directly exposes marine organisms to different levels of toxicity, affecting development and decreasing both growth and biodiversity (Torres et al., 2008). Heavy metals in high concentrations are non-biodegradable pollutants (Mallick and Rai, 2001) and can be accumulated in macroalgae, thereby decreasing growth rates (Amado Filho et al., 1997). Cadmium is one of the heavy metals most frequently implicated in environmental contamination. This metal is utilized in the manufacture of various products, such as batteries, chipsets, pigments, television receivers, and semiconductors (Hashim and Chu, 2004; Hu et al., 1996). Cadmium can bind to sulfated groups, as well as metalloproteins and metalloenzymes, thereby neutralizing their functions (Pinto et al., 2003). However, Cd has no nutritional value for algae (Visvik and Rachlin, 1992). Cadmium is not part of organic molecules, and it has been associated with decreasing photosynthesis, Cd absorption, and growth rate of seaweeds (Diannelidis and Delivopoulos, 1997; Visvik and Rachlin, 1992). Lead is a major environmental contaminant that arises from human, agricultural and industrial activities, e.g., mining, burning of coal, effluents from storage battery manufacture, automobile exhaust, metal plating and finishing operations, fertilizers, pesticides, and additives in pigments and gasoline (Eick et al., 1999). This metal is not an essential element for biological processes, but it can be easily absorbed and accumulated in different parts of the organism (Sharma and Dubey, 2005). On the other hand, Cu is an essential micronutrient for plant growth and development. This metal is a structural element in regulatory proteins and participates in photosynthetic electron transport, mitochondrial respiration, oxidative stress, cell wall metabolism, transcription, protein trafficking, and hormone signaling (Yruela, 2005). However, in excess, it can inhibit growth and impair important cellular processes like photosynthetic electron transport, photosynthesis, and respiration. Membrane transport systems seem to be a target of this metal, playing a central role in toxicity processes (Yruela, 2005). Several sources of Cu, including industrial and domestic waste, agricultural practices, copper marine drainage, copper-based pesticides, and antifouling paints, have led to a clear increase in Cu concentrations in aquatic environments (Callow and Callow, 2002). Gelidium floridanum W.R. Taylor is distributed along the Brazilian coastline from Espirito Santo State (191200 10″S; 401200 16″W) to Rio Grande do Sul State (301010 59″S; 511130 48″W), Brazil. As a source of agar extraction throughout the world, this species has achieved significant importance, as agar is utilized in diverse products with gelling and bacteriological properties (Armisen et al., 1995). Gelidium J.V. Lamouroux species are known to produce highquality agar with low sulfate content; as such, suppliers can command high prices (Sousa-Pinto et al., 1999). Considering the effects of heavy metals on seaweeds, the present study aimed to evaluate and compare the biological effects of three different heavy metals (Cd, Cu, and Pb) on physiological responses and ultrastructure features in the red macroalga G. floridanum by

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studying metal bioabsorption, morphological features, growth rate, photosynthetic pigments, flavonoid contents, ultrastructure characteristics, and elementary chemical composition. This study can serve to support subsidized environmental impact reports and monitoring programs. 2. Materials and methods 2.1. Algal material Individual G. floridanum samples were collected from Sambaqui Beach (271290 18.8″S; 481320 18.9″W), Florianopolis-SC, Brazil, in January 2012, during the summer season. The algal samples were collected from the intertidal zone during low tide and transported at ambient temperature in dark containers to LABCEVUFSC (Plant Cell Laboratory) (Macroalgae Laboratory, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil). Macroepiphytes from macroalgal samples were meticulously eliminated by cleaning with a brush and filtered seawater. Apical portions were maintained in culture medium with filtered seawater plus von Stosch enrichment solution at half strength (VSES/2; Edwards, 1970) and cultivated under laboratory-controlled conditions during 14 days (experimental acclimation period) before experimental treatment with Cd, Cu, and Pb. 2.2. Culture conditions and experimental treatments Apical thallus portions were selected (72.0 g) from the acclimated G. floridanum plants and cultivated for 7 days under the experimental treatments with heavy metals in Erlenmeyer flasks containing 500 mL of natural sterilized seawater, 34 practical salinity units (p.s.u.), and enriched with VSES/2 (without EDTA, ethylene diamine tetraacetic acid). Laboratory-controlled conditions were 2472 1C, continuous aeration, 8075 mmol photons. m  2 s  1 (Philips C-5 Super 84 16 W/840 fluorescent lamps; LI-COR light meter 250, USA), and 12 h photocycle (starting at 8 h). Experimental treatments were carried out with a control (without metal addition) and Cd, Cu and Pb supplied individually as CdCl2, CuCl2 or PbCl2 at 50 and 100 mM for each metal. Eight replicates were made for each experimental group (seven treatments). 2.3. Concentration of Cd, Cu and Pb in seawater and algal samples The concentrations of Cd, Cu and Pb in water and algal samples (initial and end of experiment) were analyzed by inductivity-coupled plasma atomic emission spectrometry (ICP-AES, ARCOS from M/s. Spectro, Germany), using the following analyte line: Cd 214.438 nm, Cu 324.754 nm and Pb 220.353 nm plasma view-axial, with a detection limit of 0.001 ppm for Cd, Cu and Pb. After 7 days of exposure to Cd, Cu and Pb, algal samples (300 mg) were washed in distilled water, dried at 65 1C and digested in nitric acid. Water samples (50 mL) were digested using concentrated nitric acid. Total metal absorption was expressed in percent, calculated through (mg of Cd/Cu and Pb in 500 mL of water by mg of Cd in 750 mg of wet weight algae). The Bioconcentration Factor (BCF) was calculated as remaining metal concentration and wet weight plant biomass (expressed in ppm) divided by initial concentration of metal added in the culture medium. All analyses were performed in quadruplicate. 2.4. Growth rates (GRs) Growth rates were calculated using the following equation: GR (%.day  1)¼ [(Wt/Wi)1/t  1]  100, where Wi¼ initial wet weight, Wt ¼wet weight after 7 days, and t¼ experimental time in days (Penniman et al., 1986); where Wi¼initial wet weight, Wt¼ wet weight after 7 days, and t¼ experimental time in days (Penniman et al., 1986). 2.5. Pigments analysis The contents of photosynthetic pigments (chlorophyll a, phycobiliproteins and carotenoids) and flavonoids of G. floridanum were analyzed from frozen fresh samples (n ¼4) kept at  40 1C until use. Chlorophyll a was extracted from approximately 1 g of algal material in 3 mL of dimethylsulfoxide (DMSO, Merck, Darmstadt, FRG) at 40 1C, during 30 min, using a glass tissue homogenizer (Hiscox and Israelstam, 1979; Schmidt et al., 2010a, 2010b). The homogenates were centrifuged at 2000 g for 20 min, and the supernatant containing the pigment was quantified spectrophotometrically, according to Wellburn (1994). Phycobiliproteins were extracted from about 1 g of algal material ground to a powder with liquid nitrogen and extracted at 4 1C in darkness in 0.1 M phosphate buffer, pH 6.4. The homogenates were centrifuged at 2000 g for 20 min.

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Phycobiliprotein levels [allophycocyanin (APC), phycocyanin (PC), and phycoerythrin (PE)] were determined by UV–vis spectrophotometry (Multireader Infinite M200, TECAN), and calculations were performed using the equations of Kursar et al. (1983). Carotenoids were extracted from 1 g of algal material by using hexane:acetone (1:1, v/v) containing 100 mg.L  1 tert-butyl hydroxytoluene (BHT). The homogenates were filtered through a cellulose membrane to remove particles, and the organosolvent was evaporated under N2 flux. The residues were dissolved in hexane (3 mL), and carotenoid contents were determined through UV-visible spectrophotometry (SF200ADV, Bel), by measuring the absorbance at 450 nm (3 lectures per sample) (Multireader Infinite M200, TECAN). Carotenoid quantification was based on β-carotene standard curve (1–50 μg mL  1; y ¼0.055x; r2 ¼0.999).

2.10. Data analysis Data were analyzed by unifactorial Analysis of Variance (ANOVA) and Tukey's a posteriori test using the Statistica software (Release 6.0), considering p r0.05. Statistical comparisons were performed to evaluate the variations in growth rates, concentration of photosynthetic pigments, flavonoid contents, elementary composition, and bioabsorption under Cd, Cu and Pb treatments.

3. Results 3.1. Cadmium, copper and lead uptake

2.6. Total flavonoid assay Flavonoids were extracted from 1 g fresh frozen samples (n¼4) using 10 mL 80% aqueous methanol. The homogenates were filtered through a cellulose membrane to remove particles, and one aliquot (2 mL) of the extract was centrifuged for 5 min at 4000 rpm. The total flavonoid content was determined by the aluminum chloride colorimetric method (Zacarias et al., 2007). Briefly, an aliquot of 0.5 mL of extracts was added to 2.5 mL of ethanol and 0.5 mL of 2% aluminum chloride hexahydrate (AlCl3  6H2O). After incubation at room temperature for 1 h, the reaction mixture absorbance was measured at 420 nm (Hitachi, Model 100–20). Flavonoid quantification was based on the quercetin standard curve (10–200 μg mL  1; y ¼ 0.010x; r2 ¼ 0.999).

The analysis of Cd, Cu and Pb uptake showed absence of these metals in the control samples (Supplementary Table 1). Samples treated with Cd showed a percentage of bioabsorption of 3.48% for 50 μM and 1.92% for 100 μM. Copper showed the highest percentage of bioabsorption at 68.53% for 50 μM and 67.18% for 100 μM. Lead-treated plants showed bioabsorption of 32.86% for 50 μM and 27.73% for 100 μM. The bioconcentration factor was up to 1500 for Cu-treated plants (Supplementary Table 1). A high bioconcentration factor was also verified for Pb-treated plants.

2.7. Confocal laser scanning microscopy (CLSM)

3.2. Growth rates (GRs)

Ultrastructural features of G. floridanum were investigated by laser scanning confocal microscopy (Leica TCS SP-5, Wetzlar, Germany) and an Argon laser at excitation of 440, 488 and 514 nm. A Leica HCX PLAPO lambda 63  /1.4–0.6 oil immersion objective was fitted on the inverted fluorescent microscope. The autofluorescence of the chlorophyll was used for visualization of the chloroplast structure (Schmidt et al., 2012b). The LAS-AF Lite program (Leica) was also used for final processing of the confocal images.

After 7 days of experimentation, control, Cd-, Cu-, and Pb-treated G. floridanum showed particular morphological features with reduction in the amount of branches, noticeable discoloration, and algal necrosis (Supplementary Fig. 1). Evident bleaching and depigmentation of the apical segments were observed after exposure to 50 μM Cd (Supplementary Fig. 1B), 100 μM Cd (Supplementary Fig. 1C), 50 μM Cu (Supplementary Fig. 1D), 100 μM Cu (Supplementary Fig. 1E), and 100 μM Pb (Supplementary Fig. 1G) when compared to control (Supplementary Fig. 1A). Treatments with 50 μM Pb showed no depigmentation (Supplementary Fig. 1F) and color similar to that of control (Supplementary Fig. 1A). Cd-, Cu- and Pb-treated plants at 50 and 100 mM showed significantly lower GRs than control, except for 50 mM of Pb (Supplementary Table 2). Control plants showed the highest GRs of 1.48% day  1 with natural red coloration and increasing formation of lateral pigmented branches at the end of the experiment (Supplementary Fig. 1A). Lower GRs were observed at higher concentrations of the heavy metals.

2.8. Transmission electron microscopy (TEM) For observation under TEM, samples approximately 5 mm in length were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) plus 0.2 M sucrose overnight as described in Schmidt et al. (2009, 2012c). The material was post-fixed with 1% osmium tetroxide for 4 h, dehydrated in a graded acetone series and embedded in Spurr0 s resin following Schmidt et al. (2010c). Ultrathin sections were stained with aqueous uranyl acetate followed by lead citrate, according to Reynolds (1963). Four replicates were made for each experimental group; two samples per replication were then examined and photographed under TEM JEM 1011 (JEOL Ltd., Tokyo, Japan, at 80 kV). Similarities based on the comparison of individual treatments with replicates suggested that the ultrastructural analyses were reliable. 2.9. Scanning electron microscope (SEM)

3.3. Photosynthetic pigments and flavonoids

Samples for SEM observations were fixed using procedures identical to those used for TEM. The samples were dehydrated with ethanolic series, dried on Critical point EM-CPD-030 (Leica, Heidelberg, Germany), and then sputter-coated with gold prior to examination, according to Schmidt et al. (2012a). The samples were examined under SEM JSM 6390 LV (JEOL Ltd., Tokyo, Japan, at 10 kV). Cadmium, copper and lead were analyzed in the cell wall using SEM (NORAN System 7, Thermo Scientific Instruments) coupled to an energy dispersive X-ray spectrometer (SEM-EDX), without post-fixing the samples in osmium tetroxide or coating with gold.

Apical samples of Cd-, Cu- and Pb-treated G. floridanum showed modifications in the content of photosynthetic pigments (Table 1). Chlorophyll a showed the largest reduction after exposure to 100 mM of Cu (43% compared to control), followed by 35% for 100 mM of Cd. The amounts of phycobiliproteins (APC, PC, and PE) were significantly reduced in G. floridanum exposed to both concentrations of Cd, Cu and Pb. On the other hand, plants exposed to

Table 1 Photosynthetic pigments (mg.g-1 FW; Chl a, chlorophyll a; APC, allophycocyanin; PC, phycocyanin; PE, phycoerythrin; Car, carotenoids) and flavonoid contents (Flav) of G. floridanum cultivated with 50 μM and 100 μM of Cd, Cu and Pb over a period of 7 days. Treatments

Chl a

APC

PC

PE

Car

Flav

Control 50 μM Cd 100 μM Cd 50 μM Cu 100 μM Cu 50 μM Pb 100 μM Pb

1997 1.38a 1967 0.54b 1287 1.20c 1967 1.13b 1127 0.75d 1977 0.29ab 1977 0.20ab

325 7 0.25a 1807 0.45d 1807 0.18d 1757 0.85e 1157 0.25f 295 7 0.35b 1957 0.25c

1407 0.35a 807 0.20d 757 0.20e 707 0.25f 457 0.55g 1357 0.65b 857 0.25c

455 7 0.25a 3007 0.25d 260 7 0.20e 1857 0.25f 1307 0.35g 4357 1.10b 3007 0.50c

1.017 0.01d 0.96 7 0.02c 1.077 0.00e 0.477 0.02f 0.53 7 0.02g 1.247 0.01b 1.29 7 0.01a

0.57 70.01ab 0.48 70.06b 0.69 70.03a 0.19 70.02d 0.31 70.06c 0.55 70.07b 0.6770.03ab

Data are means 7 SD (n¼ 4). Letters indicate significant differences according to Tukey's test (p r0.05).

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50 and 100 mM of Pb showed an increase of carotenoid content compared with control and other metal treatments. Flavonoid contents showed significant decrease only in Cu-treated plants when compared with control and other treatments. 3.4. Confocal laser scanning microscopy When observed in confocal microscopy, control cells of G. floridanum showed a large quantity of chloroplasts with reddish color (Fig. 1A) and high autofluorescence intensity (Fig. 1B). In contrast, plants exposed to 50 and 100 mM of Cd showed changes in chloroplast morphology, with green color (Fig. 1C and E) and reduction of autofluorescence intensity (Fig. 1D and F). After treatment with 50 and 100 mM of Cu, similar changes in

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chloroplast morphology and green color (Fig. 1G and I), in addition to low chloroplast autofluorescence, were observed (Fig. 1H and J). The chloroplast of plants treated with 50 and 100 mM of Pb appeared with more reddish color (Fig. 1K and M) and more autofluorescence intensity (Fig. 1L and N) than Cd- and Cu-treated samples, but still lower than control. 3.5. Transmission electron microscopy When observed by transmission electron microscopy, control samples of G. floridanum showed cortical cells to be somewhat vacuolated, mostly filled with chloroplasts and a large quantity of floridean starch grains close to the chloroplasts (Fig. 2A). These cells were surrounded by a thick cell wall (Fig. 2B). The chloroplasts

Fig. 1. Confocal microscopy images of Gelidium floridanum. A and B—control samples. (A) observe a large quantity of chloroplasts with reddish color. (B) note the chloroplasts with high autofluorescence intensity. C and D—samples treated with 50 μM of Cd. (C) observe the green color of the chloroplasts. (D) note the reduction in autofluorescence intensity of the chloroplasts. E and F—samples treated with 100 μM of Cd. (E) observe the green color of the chloroplasts. (F) note the reduction in autofluorescence intensity of the chloroplasts. G and H—samples treated with 50 μM of Cu. G: sample with green color, indicating loss of accessory pigments. (H) observe that few cells present fluorescence in the chloroplasts. I and J—samples treated with 100 μM Cu. (I) sample with green color, indicating loss of accessory pigments. ( J) observe the reduction in autofluorescence intensity of the chloroplasts. K and L—samples treated with 50 μM Pb. (K) samples showed more intensity of reddish color when compared with control samples. (L) observe the high fluorescence of the chloroplasts. M and N—samples treated with 100 μM Pb. (L) observe the increased intensity in reddish color. (N) sample with more autofluorescence intensity in the chloroplasts compared to control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Transmission electron microscopy (TEM) micrographic images of control samples of G. floridanum. (A) overview of cortical (CC) and subcortical cells (SC) showing numerous floridean starch grains and chloroplasts embedded in thick cell wall. (B) detail of cortical cell with floridean starch grains (S) and thick cell wall (CW). (C) detail of chloroplast (C), showing the thylakoids (arrows) and plastoglobuli (p). Observe the mitochondria (M). (D) note the association of some organelles and ribosomes (arrows head) with chloroplast. Thylakoids (arrows), rough endoplasmic reticulum (RER), and mitochondria.

assumed the typical internal organization of red algae with unstacked, evenly spaced thylakoids (Fig. 2C). In the chloroplasts, a few plastoglobuli were observed between the thylakoids (Fig. 2C). Small mitochondria, free ribosomes and rough endoplasmic reticulum (RER) were present in association with the chloroplasts and nuclei (Fig. 2D). After culturing G. floridanum in a concentration of 50 and 100 μM of Cd for 7 days, cortical cells appeared more vacuolated (Fig. 3A) with increasing cell wall thickness, exhibiting concentric layers of microfibrils (Fig. 3B). The presence of a large amount of electron-dense vesicles about to form a new cell layer was also verified (Fig. 3C). Chloroplasts showed few changes in ultrastructural organization (Fig. 3C E). The number of plastoglobuli increased in the chloroplasts (Fig. 3D,E). However, some thylakoids were grouped with irregular morphology (Fig. 3F,G). Floridean starch grains were not observed. Some mitochondria were swollen (Fig. 3G). Copper treatments caused more dramatic ultrastructure changes than Cd in G. floridanum, with cortical cells showing a large reduction in cytoplasmic cell volume (Fig. 4A,B). The cell wall showed increasing thickness with deposition of concentric layers of microfibrils (Fig. 4C,D) and appeared with spots of black deposits, most likely Cu (Fig. 4E). Some smaller floridean starch grains were observed in the cytoplasm (Fig. 4F,G). The chloroplasts were degenerated and disrupted (Fig. 4H), and the presence of plastoglobuli was observed (Fig. 4H). Mitochondria were disrupted and swollen (Fig. 4I). In some cells, a large vacuole with translucid content was observed (Fig. 4J,K), together with numerous electron-dense precipitates, probably Cu (Fig. 4J,K). After Pb treatment, the cortical cells of G. floridanum showed a few changes in shape and increased cell wall thickness (Fig. 5A,C). In the cortical cells, an increase of vacuole volume (Fig. 5B) could be observed, together with numerous electron-dense precipitates, probably Pb (Fig. 5A,C). The presence of a large amount of electron-dense vesicles was also observed in the cytoplasm, suggesting the presence of electron-dense precipitates, most likely

lead (Fig. 5D,E). In addition, chloroplasts showed no changes in ultrastructural organization (Fig. 5D,E). The number of plastoglobuli increased in the chloroplasts (Fig. 5H), and some starch grains were observed near the chloroplasts (Fig. 5B). 3.6. Scanning electron microscopy The images captured with SEM showed the rough surface of the control sample (Supplementary Fig. 2A). Treatments with Cd, Cu and Pb showed different results. Less surface roughness was observed in algae treated with 50 μM of Cd (Supplementary Fig. 2B), whereas treatment with 100 μM of Cd (Supplementary Fig. 2C) caused surface roughness to increase relative to control. Both treatments of Cu (Supplementary Fig. 2D,E) showed decreased surface roughness compared to control samples. When treated with Pb (Supplementary Fig. 2F,G), the surface roughness gradually decreased according to the increasing of concentration of Pb. The results of X-ray microanalysis of G. floridanum was qualitative (Supplementary Table 3). X-ray microanalysis of the control cell wall surface revealed the presence of different percentages of such elements as carbon, nitrogen, oxygen, sodium and potassium in all samples. Cd, Cu and Pb were not detected on the surface of the control cell wall, but the presence of these metals in their respective treatments was detected (Supplementary Table 3).

4. Discussion Heavy metals affect the biology and development of diverse aquatic organisms, including macroalgae, in multiple ways. Copper, cadmium and lead are among the most toxic heavy metals that pollute aquatic coastal environments. The present study showed that equal concentrations of Cd, Cu and Pb induce different levels of physiological, chemical and cell organization responses in the red macroalga G. floridanum.

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Fig. 3. TEM micrographic images of G. floridanum treated with Cd. (A) detail of cortical cell treated with 50 μM of Cd showing a large vacuole (V). Note the chloroplast (C) and cell wall (CW). (B) detail of cell wall of the cortical cell treated with 100 μM of Cd. Observe an increase in thickness of cell wall with deposition of concentric microfibril layers (arrow). C–D–E: detail of cortical cells treated with 50 μM of Cd. C: magnification of cortical cell showing vesicles containing matrix of cell walls (arrowheads) and increase in cell wall thickness. Observe the rupture in thylakoids into the chloroplast (arrow). Mitochondria (M). (D) observe the irregularity of thylakoids (arrows) and the increase in plastoglobuli (P). (E) chloroplast with increasing plastoglobuli (P) and vacuoles (V). Note the rupture of thylakoids (arrow). Observe the mitochondria. F–G: cortical cells treated with 100 μM of Cd. (F) detail of the chloroplast showing alterations in thylakoids (arrows) and internal plastoglobuli. (G) magnification of the chloroplast; observe the rupture of thylakoids (arrow). Note the vesicles with content matrix of cell walls (ve). Observe the mitochondria.

Bioabsorption assays showed a greater accumulation of Cu when compared with Cd and Pb, while Cd showed less bioaccumulation. The percentages of absorption of all samples treated with 50 μM and 100 μM of Cd, Cu and Pb are correlated with factor of bioabsorption of each treatment, respectively. X-ray analysis with SEM-EDS carried out on samples of G. floridanum detected the presence of Cd, Cu and Pb in all treatments, with the highest percentage for the presence of Cu on the surface. However, only samples treated with 100 μM of Cu showed significantly greater accumulation than samples exposed to treatment with 50 μM Cu, and these results differed from those of Cd and Pb accumulations, which showed no difference between the two concentrations used. These data suggest that the interaction between Cu and the agar polysaccharide composition of cell wall matrix is greater than its interaction with either Cd or Pb. This evidence suggests, in turn, that sequestration of Cu results in the formation of complexes between Cu and negative groups of agar. Belenikina (2008) also observed that agarophytes are bioaccumulators of heavy metals because of the presence of agar. After the experimental period, the control samples of G. floridanum presented a significant increase in biomass, whereas Cd-, Cu- and Pb-treated plants showed less increase in biomass. Negative growth rate was quite obvious under 100 μM of Cu. In Pb-treated plants, the increase in biomass was also less than that of control samples. To account for these results, it can be hypothesized that the metabolic routes were most likely diverted to protect the photosystem apparatus, and cells treated with metals, and cell wall growth, suggesting a physicochemical strategy to chelate to prevent the entry of metal into the cell. Therefore, the deviation of metabolic pathways must have altered growth rates of the heavy metal-treated G. floridanum samples. After the experimental period, the control samples of G. floridanum presented branches, and their initial reddish color remained intact. However, samples treated with Cd showed loss of reddish color

and dichotomous branches when compared with the control. These responses were less intense with lower concentrations of Cd. Among all treatments, thallus treated with Cu showed the highest depigmentation response and morphological deformations. Unlike Cu and Cd, Pb treatments showed no morphological differences with 50 μM compared to the control, but with treatment of 100 μM, bleaching occurred at the apices of the thallus. Moreover, the growth rates of these treatments were greatest for each concentration in comparison with the treatments with Cu and Cd, respectively. The loss of reddish color observed in samples treated with Cd and Cu, as well as the samples treated with 100 μM of Pb, is corroborated by the decrease of phycobiliprotein accessory pigments (APC, PC and PE) in G. floridanum. These pigments are responsible for the red color of the thallus. They are located in structures called phycobilisomes associated with the surface of thylakoids and are responsible for capturing light energy at complementary wavelength than chlorophyll a and transfer it to PSII (Xia et al., 2004). Phycobiliproteins were degraded by decreasing their quantity, probably as a mechanism deployed to prevent the excess of excitation energy that causes serious oxidative damage to several biological compounds, such as lipids, proteins and nucleic acids. It is likely that the loss in accessory pigments in G. floridanum samples treated with metals caused a reduction in fluorescence once the accessory pigments had transferred light energy for PSII. Several studies have shown that Cd causes modifications to accessory pigments, including the work of Xia et al. (2004), who treated Gracilaria lemaneiformis (Bory de SaintVincent) Greville, with Cd for 4 days, Bouzon et al. (2012a) who treated H. musciformis with Cd for 7 days, and Santos et al. (2012) who treated G. domingensis with Cd for 7 days. Quantification of chlorophyll a showed a decrease for treatments with 100 μM of Cd and Cu, while Pb treatments had no significant effects. To explain, treatments with Cd and Cu most likely promoted a

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Fig. 4. TEM micrographic images of G. floridanum treated with Cu. (A) detail of the cortical cell (CC) treated with 50 μM of Cu showing a thick cell wall (CW). (B) detail of the cortical cell treated with 100 μM of Cu showing numerous starch grains and thick cell wall. C: detail of increasing thickness of cortical cell wall treated with 50 μM of Cu. Observe vesicles of deposition in the cell wall (arrow) and metal deposition (arrowhead). D: cell wall thickness in cortical cell treated with 100 μM of Cu. E–F: cortical cell treated with 50 μM of Cu. (E) detail of vacuoles with metal deposits (arrow). (F) detail of numerous, small starch grains. G–H: cortical cell treated with 100 μM of Cu. (G) detail of cortical cell showing numerous, small starch grains (arrows). Note the thickness of cell wall. (H) Note the disrupted chloroplast (C) with the presence of plastoglobuli (P). (I) detail of vacuole of cortical cell treated with 50 μM of Cu. Note the altered mitochondria (M). J–K: detail of cortical cell treated with 100 μM of Cu. J: observe the vacuole with metal deposit (arrow) and starch grains (S). (K) detail of vacuole (V) with reticulated content.

reduction in enzymatic activity (Stobart et al., 1985; Xia et al., 2004) or promoted the deficiency of Mg and Fe in the biosynthesis of chlorophyll a (Greger and Ogren, 1991; Xia et al., 2004). The analysis G. floridanum samples under confocal microscopy showed reddish color in surface cells of control samples, but loss of reddish color in Cd- and Cu-treated samples. Pb treatments showed reddish color in surface cells. The images of chloroplast autofluorescence of control and heavy metal-treated G. floridanum showed that samples treated with Cd and Cu presented a reduction in autofluorescence intensity, while treatment with Pb presented only minor reduction of chloroplast autofluorescence. The amount of carotenoids in G. floridanum did not change in treatments with Cd. However, it was altered by Cu and Pb treatments. In treatments with Cu, a decrease of 50% in the amount of carotenoids was observed. Moreover, Pb treatments presented an increase in the amount of carotenoids, suggesting that carotenoids could act as an antioxidant mechanism for this metal. Carotenoids are synthesized and stored in chloroplasts as a lipophilic antioxidant (Romer et al., 2002), and the increase of this pigment may be interpreted as a protective acclimation mechanism to prevent the toxic effects of Pb. Gouveia et al. (2013) observed the same results for G. domingensis treated with Pb. Collen et al. (2003) and Pinto et al. (2003) obtained the same results for Gracilaria tenuistipitata C.F.Chang and B.M. Xia treated with Cd and Cu. According to Gouveia et al. (2013), the production of carotenoids is a strategy to prevent the effects of ROS. The amount of flavonoids decreased in the treatments of Cu, but did

not significantly change with treatments of 50 μM of Pb and 50 μM Cd, although an increase in the amount of flavonoids in treatments of 100 μM of Cd and 100 μM of Pb was noted. According to Collen et al. (2003), flavonoids are synthesized with the purpose of preventing oxidative stress. In Porphyra acanthophora var. brasiliensis E.C. Oliveira and Coll, Bouzon et al. (2012b) observed that the amount of flavonoids in ambient samples was greater than that of either control or UVB-treated samples. It is possible that the action of flavonoids as an antioxidant is not an effective mechanism for G. floridanum under metal stress, as these compounds were not synthesized in greater amounts. Analyses carried out with TEM showed the presence of electron-dense particles in the cell wall with all treatments, most likely representing deposits of Cd, Cu and Pb, as well as deposition vesicles of cell wall substances were observed. According to Talarico (2002), Cd-treated A. saviana showed similar deposition of electron-dense particles in the cell wall, and this was attributed to Cd. The same phenomenon was observed in H. musciformis treated for 7 days with Cd (Bouzon et al., 2012a) and G. domingensis treated for 7 days with Cd (Santos et al., 2013) with increasing cell wall thickness. The images of the external cell wall surface of the samples analyzed by SEM show the surface roughness of the control sample. The addition of Cd treatment showed loss of roughness and depression, which may be associated with altering the outermost layer of the cell wall observed in TEM analysis. Treatment with 100 μM of Pb showed the greatest loss of roughness among all metal treatments.

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Fig. 5. TEM micrographic images of G. floridanum treated with Pb. (A) cortical cell (CC) treated with 50 μM of Pb showing numerous starch grains and chloroplast embedded in thick cell wall (CW). The deposition of metals is visible (D). (B) cortical cell treated with 100 μM of Pb showing starch grains (S), vacuole (V), deposit of metals and chloroplast (C) embedded in thick cell wall. C–D: detail of cortical cell treated with 50 μM of Pb. C: magnification of cortical cell. Observe the vacuole with granular content. Observe an increase in thickness of cell wall with deposition of concentric microfibril layers (arrow). (D) observe increase of starch grains, deposition of metal and thylakoid organization (arrow). E–H: detail of the cortical cell treated with 100 μM of Pb. E: observe the chloroplast with increase of plastoglobuli, thylakoids (arrow), metal deposits, and starch grains.

The increased thickness of the cell wall is one chelation strategy, in which sulfated compounds are present in cell walls of algae, and these chelate metals (Diannelidis and Delivopoulos, 1997). Accomplished with red algae treated with Cd obtained the same conclusion as the increase in cell wall thickness (Bouzon et al., 2012a; Santos et al., 2013). The increase of the cell wall is a strategy to prevent the entry of metal into the cell, once sulfate compounds have chelated the metals (Santos et al., 2013). Since the cell wall bioabsorbs heavy metals, bioaccumulation also occurs within the cell. Favero et al. (1996) evaluated the importance of Ulva rigida (C. Agradht) as a bioindicator of heavy metals in the environment since heavy metal accumulation occurs inside the cells of this alga. TEM analysis showed increased vacuolization and deposition of metals in both cell wall and vacuole in treatments with Cu and Pb. The deleterious effects of the treatments of Cd, Cu and Pb in samples of G. floridanum suggest that the chelation of metals by cell walls is insufficient. Transport proteins uptake heavy metals in the plasma membranes. The fact that Cu was more absorbed can be related to these protein channels located in all cell membranes (Yruela et al., 2005). More specifically, cadmium can be transported by IRT1, an iron transporter gene belonging to the ZIP family, as well as ATNramp3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency, and still be transported by calcium transporters (Benavides et al., 2005). According to Yruela (2005), many Cu transporters have already been characterized in plant cells, such as those in the cell membrane (COPT1), cytosol (copper chaperones) and chloroplasts (COPT3, PAA1, PAA2 and CpCCS), mitochondria (COX17) and Golgi body (RAN1, chaperones and COPT5). Yruela (2005) also reports the existence of Pb transporter proteins. Because of the presence of transporters in the plasma membrane, heavy metals can easily enter the cell, promoting

alterations in organelles, as well as proteins, such as pigments. According to Collen et al. (2003), heavy metals also promote the production of reactive oxygen species (ROS), and ROS can induce changes in several cellular components, such as lipids, proteins and nucleic acids. TEM analyses showed cell control samples with chloroplast and free parallel thylakoids, the typical organization of red algae, and a few plastoglobuli. Samples treated with the respective metals showed ultrastructural changes. In contrast to the chloroplasts of Cd- and Pb-treated samples, the chloroplasts of Cu-treated samples were more dramatically altered. While the treatments with Cu underwent degeneration, other metal treatments suffered disruptions in thylakoids, except samples treated with Pb, which suffered fewer disruptions in the thylakoids. The increasing promotion of ROS for heavy metals induces peroxidation and destabilization of chloroplast membranes (Li et al., 2006). In experiments with Cd in red algae, structural deformation of the chloroplast was also observed (Talarico, 2002; Bouzon et al., 2012a; Santos et al., 2013). Another feature that caught our attention was the large increase in the number of plastoglobuli in treatments with Pb. It is likely that cells use this mechanism for heavy metal detoxification. The plastoglobuli are interpreted as lipidic material (Bouzon et al. (2012a, 2012b)), and an increase in the amount of plastoglobuli was also observed in Ceramium ciliatum (J.Ellis) Ducluzeau exposed to Cd (Diannelidis and Delivopoulos, 1997).

5. Conclusion The present study showed that treatment with Cd, Cu and Pb is toxic to G. floridanum, inducing a variety of changes in bioabsoption,

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morphological characteristics, growth rate, pigments and flavonoids, and ultrastructural features. Serious damage to the photosynthetic apparatus is evidenced by discoloration, necrosis, pigment reduction, chlorophyll autofluorescence, ultrastructure chloroplast organization, and starch biosynthesis. Cell wall thickness could be associated with chelating properties of agar amorphous composition of cell wall induced to increase metal concentration exposure and coupled to vacuolization of the cells. Cadmium and Pb promote the increase of carotenoid synthesis, probably as chemical antioxidant defense against reactive oxygen species. The toxicity of Cu was more dramatic for G. floridanum than other metals, followed by Cd toxicity, while Pb exposure resulted in potential adaptation to metal toxicity. These results indicate the susceptibility of G. floridanum to different metals and concentrations and, as such, can be used to as data to support the subsidization of programs to monitor environmental impact.

Acknowledgments The authors would like to acknowledge the staff of the Central Laboratory of Electron Microscopy (LCME), Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil, for the use of their scanning and transmission electron microscopes. This study was supported, in part, by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil). Éder C. Schmidt holds a postdoctoral fellowship from CAPES. Zenilda L. Bouzon is a CNPq fellow. Fungyi Chow is a FAPESP fellow.

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