Characterization of ZnO particles

In this study, analytical grade ZnO NPs suspended in 40 % butylglycol (Sigma-Aldrich^™, USA) and pharmaceutical grade ZnO particles (SMPs) in powder form (Farmacia Paris, México) were used. The butylglycol was eliminated by centrifuging 1 mL of the suspension at 18 000 g for 20 min and washing the NPs three times with de-ionized water. The dry NPs and SMPs were diluted in de-ionized water 1:100 (v:v) and sonicated (Ultrasonicador, Branson 1800^™, Connecticut, USA) at 70 W and 70 Hz for 10 min. Morphology, roundness index (largest diameter/smallest diameter) and average size of both types of particles were examined with an transmission electron microscope (TEM) (Tecnai 2 Spirit, Fei Company, USA), operated at 120 Kv and, with the image processor TIA (Tecnai, Imagining & Analysis, New York USA) version 4.7 SP3 (1994-2014). The average diameter of the SMPs was 238±30.7 nm and that of NPs was 26.7±1 nm.

Biological material and experiment setup

Azolla filiculoides plants were obtained from the solarium of the area of Microbiology of the Graduate Program in Edaphology, Colegio de Postgraduados, campus Montecillo (19.52° N, 98.88° W, altitude 2250 m). One gram of fresh plants was placed in each plastic tray (12´6.5´4 cm) containing 200 mL nutritive solution, pH 5.5 (Yoshida et al., 1971) and kept in a greenhouse with an average diurnal temperature of 26 °C and nocturnal temperature of 19 °C.

The treatments exposure of fern to two particle sizes (NPs and SMPs) and three concentrations of ZnO particles (100, 200, and 400 mg L^-1); the control contained no particles. The eight treatments were distributed in a completely randomized experimental design. The nutrient solution with NPs was sonicated for 10 min to disperse the particles before introducing the fern. The variables were measured after 6 d of exposure to the treatments.

Relative growth rate and quantification of photosynthetic pigments

The relative growth rate (RGR; g g·d^-1) was calculated with the equation:

RGR=Lniw-Lnfw/(∆t)

where Ln _iw : natural logarithm of initial weight (iw); Ln _fw : natural logarithm of final weight (fw); t: incubation time (d).

The photosynthetic pigments were extracted in 80 % acetone and the content of chlorophyll a (Chla), chlorophyll b (Chlb) and xanthophylls+carotenoids (x+c) were measured with UV/vis spectroscopy using the formula of ^{Lichtenthaler and Wellburn (1983)}.

Total antioxidant activity and total soluble phenolic compounds

Total antioxidant activity was determined by the bleaching test of 1,1-diphenyl-2picrylhydrazyl radical (DPPH) (^{Matthäus, 2002}). Extraction was obtained from 150-200 mg A. filiculoides fresh weight with 1 mL 80 % methanol at 4 °C; the extracts were centrifuged 15 min at 17000 g. To 30 mL extract, 235 mL DPPH solution was added. Initial absorbance readings and final readings (15 min later) were taken at a wavelength of 515 nm with a spectrophotometer (Synergy 2, Biotek Instruments^™, USA). Antioxidant activity was calculated by applying aliquots of Trolox and known concentrations of 1,1-diphenyl-2picrylhydrazyl (DPPH) solution. The results were expressed in equivalent micromoles of Trolox per gram of fresh tissue (mM Trolox g^-1 FW).

Content of total soluble phenolic compounds was measured with the Folin-Ciocalteu reactive test using chlorogenic acid as the standard. A sample of 150-200 mg of fresh frond was macerated with 1 mL methanol (80 %) at 4 °C and centrifuged 15 min at 17 000 g. The mixture of 30 mL extract+90 mL Na₂CO₃ and 150 mL of Folin-Ciocalteau reagent was placed on microplates and 30 min later, absorbance was read at 725 nm with a spectrophotometer UV/vis (Synergy 2, Biotek Instruments^™, USA). The results were expressed in equivalent micrograms of chlorogenic acid per gram of fresh tissue (mg g^-1 FW).

Nitrogenase activity and chlorophyll fluorescence

Nitrogenase activity was assessed indirectly by determining the concentration of ethylene produced by 1 g of fresh A. filiculoides, floating on 200 mL nutritive solution (of the four NP and SMP treatments) in a l-L recipient hermetically sealed with teflon tape. From the recipient, 10 % of the air was extracted and substituted with an equivalent volume of acetylene gas. After 2 h, 5 mL gas was extracted and put into vacutainer tubes for gas chromatography analysis (HP 5890 Series II PLUS GC with FID and TCD, Agilent Technologies), using an ethylene standard of 20 ppm N2 balance (INFRA de México, S. A. de C. V.). Based on the area under the curve, concentrations of problem samples were adjusted. The results were expressed in micromoles of ethylene per gram of fresh tissue (mmol ethylene g^-1 FW).

Chlorophyll fluorescence was evaluated with a portable fluorometer (OS-30p, Chlorophyll Fluorometer, USA). The fronds were preconditioned in darkness for 5 min before measuring the maximum efficiency of the PSII (Fv/Fm) system photochemical potential following the procedure of ^{Küpper et al. (2002)}.

Zn microanalysis with EDS-ESM

Energy dispersive X ray spectroscopy (EDS) coupled with scanning electron microscopy (SEM), allow to identify and quantify element composition in specific areas of a sample. After 6 d of treatment, unwashed A. filiculoides fronds were dried at 60 °C until constant weight. The dried fronds were mounted on Cu sample holder, with double-sided adhesive copper tape and coated with gold/palladium (80/20) with a metal evaporator (Fine Coat, Jeol, Japan). The abaxial and adaxial surfaces of the frond were analyzed with EDS (INCA x-ACT, Oxford Instruments, UK) attached to a SEM (Jeol JSM-6390, Japan) operating at 20 Kv. The relative concentration of Zn element was expressed in percentages of weight and mass.

Experimental design and statistical analysis

The experimental design was completely randomized, the treatments were two sizes of particles, small (NPs 26.7±1 nm) and large (SMPs 238±30.7 nm), three ZnO particle concentrations (100, 200 and 400 mg L^-1) and one control without particles; each treatment was replicated three times, and the experiment was repeated three times (n=9). The data were analyzed with an ANOVA and treatment means were compared with the Tukey test (p≤0.05), using SAS (Statistical Analysis System Institute Inc., Cary NC, USA, 2002). Values of each variable were expressed as averages ± standard error of the mean (SE).

Characterization of ZnO particles

Analytical grade NPs and observed with TEM, had an average size of 26.7±1.0 nm, roundness index of 1.09±0.058, isodiametric shape and smooth texture. The most abundant NP diameter was 21 to 30 nm (Figure 1 A and C). Pharmaceutical grade SMPs had an average diameter of 238±30.7 nm and a roundness index of 3.2±0.54; the most abundant submicrometric size was between 201-300 nm (Figures 1 B and D). The SMPs were nine times larger and three times longer than the NPs.

Figure 1 Morphometric analysis of ZnO particles using Transmission Electron Microscopy (TEM). A-B, NPs morphology and sizes; C-D, SMPs submicrometric particles morphology and sizes, (n=80). 

Effect of ZnO NPs and SMPs on growth

The relative growth rate of A. filiculoides was not affected by the presence of the particles, regardless of the size or concentration (Figure 2). This contrasts with Arabidopsis thaliana, whose growth decreased with 100 mg L^-1 ZnO NPs (^{Landa et al., 2012}).

Figure 2 Relative growth rate (RGR) of Azolla filiculoides exposed to nanoparticles (NPs) and submicrometric particles (SMPs) of ZnO for 6 d. There were no significant differences between treatments (n=9), (Tukey, p≤0.05); PS: dry weight. 

Photosynthetic pigments

The NPs significantly reduced levels of Chla, Chlb and total Chl by 33.3 %, 71.8 % and 49.1 %, relative to the SMPs (Table 1), whereas application of 400 mg NPs decreased the pigments by 47.3 %, 10.4 % and 20.8 %, relative to the control (Table 1). These results are similar to those observed in Salvinia natans exposed to 1, 10, 20, 50 mg L^-1 25 nm ZnO NPs (^{Hu et al., 2013}). NPs can produce peroxidation of the chloroplast membrane, altering accumulation of pigments and of photosynthesis (^{Ma et al., 2013}; ^{Hu et al., 2013}). The ZnO NPs reduced total concentration of carotenoids and xanthophylls (c+x) by 29.1 % in the fern fronds; application of 400 mg of NPs reduced the content of c+x by 24.3 %, relative to the control (Table 1). The c+x are important molecules that participate in detoxification of several forms of activated oxygen produced in photosynthetic complexes. Thus b-carotene quenchs the state of excitation of the chlorophyll triplet, which reduces the formation of singlet oxygen (^{Foyer and Shigeok, 2011}). In this sense, the small particles (ZnO NPs) are more toxic than SMPs because they reduce the levels of x+c in A. filiculoides. This response is similar to that observed when Oryza sativa L. was exposed to 25 nm Ag NPs, which reduced the levels of photosynthetic pigments and caused greater toxicity than larger particles (Prakash et al., 2014; Yin et al., 2013). Toxicity of small NPs was observed in in vitro cultures of bacteria, yeasts, algae, crustaceans and mammal cells (^{Ivask et al., 2014}). NP toxicity is associated with their high surface/volume ratio ability to enter the cells and chloroplasts, destabilizing plant photosynthesis and antioxidant system. The effect of concentration, of both particle sizes, was significant at 400 mg L^-1 , which reduced the level of all the photosynthetic pigments studied (Table 1).

Total antioxidant activity, total phenolic compounds, nitrogenase activity and chlorophyll fluorescence

Total antioxidant activity and total soluble phenolic compound contents in A. filiculoides significantly decreased in the presence of NPs, but increased in the presence of SMPs (Figure 3). Greater toxicity of the small particles (NPs) may be explained by its small size that favors rapid absorption, particle solubility and liberation of Zn⁺ ions (^{Reed et al., 2012}). Stress caused by heavy metals raises the number of reactive oxygen species (ROS) and, in response, the plant produces more antioxidant compounds to inhibit oxidative degradation of biological molecules (^{Foyer and Noctor, 2005b}; ^{Sheng et al., 2008}; ^{Hernández et al., 2012}). An increase in the antioxidant catalase (CAT) and glutathione (GSH) was observed in Fagopyrum esculentum in presence of ZnO NPs (^{Lee et al., 2013}). Our study is the first of its kind to address the effect of two ZnO particle sizes on physiological variables in aquatic plants, particularly A. filiculoides, and its relationship to tolerance and attenuation of oxidative stress. Some phenolic compounds can sequester free radicles (Gulcin et al., 2003) and act as electron donors as a detoxification mechanism (^{Sánchez et al., 2010}; ^{Forni et al., 2012}). The decrease in antioxidant activity and total phenolic compound content in the presence of NPs (Figure 3) was similar to the effect produced by cadmium and uranium in Azolla and Lemna minor (^{Sela et al., 1988}; ^{Forni et al., 2012}). The effect of exposure to 100 mg L^-1 ZnO NPs on A. thaliana gene expression was studied with micro-arrays; after 7 d, the induced genes were associated with oxidative stress, the response to lesions and electron transport (^{Landa et al., 2012}). Azolla filiculoides forms symbiosis with Anabaena azollae cyanobacteria, which fixes atmospheric nitrogen by means of the activity of the enzyme nitrogenase, which facilitates N assimilation and fern growth (^{Reddy, 1987}; ^{Vessey, 1994}).

Figure 3 Total antioxidant activity and total phenols in Azolla filiculoides exposed to NPs (26.6±1 nm) and SMPs (268±30 nm) at three concentrations (100, 200 and 400 mg particles L^-1) for 6 d. Mean ± standard error; n=9. Different letters indicate significant differences between treatments (Tukey, p≤0.05); PF: fresh weight. 

Our results are the first to reveal the effect of ZnO NPs on nitrogenase activity in the symbiosystem Azolla-Anabaena and show the response of the symbiont to the particles. Nitrogenase activity in the A. filiculoides-Anabaena symbiosystem decreased significantly (82 %) as of the lowest concentration (100 mg L^-1) (Figure 4). NPs reduced this enzymatic activity by 23.5 %, relative to SMPs (Table 2). The reduction was 79 % and 83 % in concentrations of 100-400 mg L^-1, relative to the control (Figure 4). These results indicate a clear alteration in N metabolism of cyanobacteria and, consequently, a reduction in assimilable N for the plant. ^{Sood et al. (2012)} compared the phytoremediating potential of aquatic macrophytes (Elodea, Eichhornia, Lemna, Pistia, Salvinia, Ceratophyllum and Azolla) and underlined the phytoremediating capacity of Azolla because it grows rapidly, fixes nitrogen and hyper-accumulates metals.

Figure 4 Nitrogenase activity in Azolla filiculoides exposed to nanoparticles (NPs) and submicrometric particles (SMPs) of ZnO for 6 d. Mean ± standard error; (n=6). Different letters indicate significant differences between treatments of the same particle size (Tukey, p≤0.05); PF: fresh weight. 

The particles did not affect fern growth for 6 d, possibly suggesting remediation potential, but the toxicity observed for the symbiont compromises growth in the medium term.

Chlorophyll fluorescence decreased when exposed to both particle sizes of ZnO and in all of the concentrations tested (Figure 5). This reduction was 15.3 % on average, relative to the control (Table 2). Chlorophyll fluorescence was used to assess toxicity of CuO NPs in Lemna gibba (^{Perrault et al., 2010}). Moreover, heavy metals have a toxic effect for most plants (^{Viehweger, 2014}). Thus, exposure of A. filiculoides and A. caroliniana to copper ions (2 mM Cu²⁺) significantly depress Fv/Fm levels after 35 h of exposure (^{Sánchez et al., 2010}). In our study, exposure of A. filiculoides to 100 mg NPs L^-1 reduced Fv/Fm by 22 %, compared with the SMPs (238±30.7 nm), which reduced fluorescence by only 15 % (Figure 5). Yield of PSII (Fv/Fm) measures photochemical potential and electron transport efficiency of PSII to PSI.

Figure 5 Chlorophyll fluorescence, PSII yield of Azolla filiculoides exposed to nanoparticles (NPs) and submicrometric particles (SMPs) of ZnO for 6 d. Mean ± standard error; n=9. Different letters indicate significant differences between treatments of the same particle size (Tukey, p≤0.05). 

This variable is used as an indicator of stress in plants and allows characterization of the effects and mechanisms of different types of environmental stress by heavy metals, herbicides and detergents in aquatic systems (^{Ralph et al., 2007}).

Maximum Fv/Fm values measured in healthy plants are above 0.7 (^{Butler and Kitajima, 1975}; ^{Sánchez-Viveros et al., 2010}). Although there are no reports on the effect of ZnO NPs on PSII photochemical efficiency, inhibition of this variable in diverse plants and species of Azolla is associated with the presence of heavy metals, for example, application of 2 mM Cu²⁺ (^{Michalak, 2006}; ^{Sánchez-Viveros et al., 2010}; ^{Viehweger, 2014}). In our study, at 100 mg L^-1 NPs reduced Fv/Fm by 22 %, relative to the control, and 15 %, compared with the SMPs (Figure 5).

Reduction in chlorophyll fluorescence indicates lower yield of the reaction centers of plastocyanin to catalyze electron transfer efficiently between PS II (cytochrome b₆f) and PS I (^{Küpper et al., 2002}; ^{Letelier et al., 2010}). A reduction in Fv/Fm caused by ZnO NPs may be partially explained by the toxic effect of the Zn²⁺ ion on the PSII photochemical reaction, which blocks electron transport (^{Padua et al., 2010}). Expression of genes associated to electron transport was suppressed in A. thaliana exposed to 100 mg L^-1 ZnO NPs (^{Landa et al., 2012}). According to ^{Ivask et al. (2014)}, there is an effect of Ag NP size on toxicity for bacteria, algae, crustaceans and mammal cells. Our study contributes new information on the response of the photosynthetic reaction centers to two particle sizes of ZnO, nanometric and submicrometric, in A. filiculoides.

Microanalysis of Zn with EDS-ESM

The X ray signal of Zn element was produced from agglomerates and crystals adhered to the abaxial epidermis of the frond. In the treatments with NPs, Zn accumulation increased proportionally with concentration. These observations were similar to those from a study on Fagopyrum sculentum, in which ZnO NPs accumulated mostly on the root surface and accumulation increased in function of NP concentration (^{Lee et al., 2013}). In contrast, in treatments with SMPs, the higher the concentration of SMPs in the solution, the lower the accumulation of Zn on the frond. The appearance of precipitates in the nutritive solution at 200 and 400 mg L^-1 suggests that, because of their size, these particles precipitated and the Zn did not accumulate on the leaf. In order to better understand Zn toxicity, further studies are required to understand mobility of this element in plants.