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Multimodal Action and Selective Toxicity of Zerovalent Iron Nanoparticles against Cyanobacteria Blahoslav Marsalek,† Daniel Jancula,† Eliska Marsalkova,† Miroslav Mashlan,‡ Klara Safarova,‡,§ Jiri Tucek,‡,§ and Radek Zboril*,‡,§ †

Institute of Botany, Academy of Sciences of the Czech Republic, Lidická 25/27, 657 20 Brno, Czech Republic Centre for Nanomaterial Research, Faculty of Science, and §Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, Slechtitelu 11, 783 71 Olomouc, Czech Republic

‡

S Supporting Information *

ABSTRACT: Cyanobacteria pose a serious threat to water resources around the world. This is compounded by the fact that they are extremely resilient, having evolved numerous protective mechanisms to ensure their dominant position in their ecosystem. We show that treatment with nanoparticles of zerovalent iron (nZVI) is an effective and environmentally benign method for destroying and preventing the formation of cyanobacterial water blooms. The nanoparticles have multiple modes of action, including the removal of bioavailable phosphorus, the destruction of cyanobacterial cells, and the immobilization of microcystins, preventing their release into the water column. Ecotoxicological experiments showed that nZVI is a highly selective agent, having an EC50 of 50 mg/L against cyanobacteria; this is 20−100 times lower than its EC50 for algae, daphnids, water plants, and fishes. The primary product of nZVI treatment is nontoxic and highly aggregated Fe(OH)3, which promotes flocculation and gradual settling of the decomposed cyanobacterial biomass.

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INTRODUCTION The evolution of oxygen-producing cyanobacteria approximately 2.5 billion years ago had a profound impact on the Earth’s atmosphere, changing its early reducing composition to its current oxygen-rich one.1 As primary producers of organic compounds, cyanobacteria play crucial roles in aquatic and terrestrial ecosystems. They fulfill a number of key functions, including CO2 and N2 fixation, oxygen evolution, biomass production, and active colonization of substrates during primary and secondary succession in both terrestrial and aquatic ecosystems.1−7 Despite their importance in the maintenance and evolution of ecosystems, cyanobacteria can also pose many serious environmental and health risks, depending on their abundance, which has been observed to be increasing in various waters around the world. Several hypotheses have been put forward to explain this increased abundance; it is generally accepted that the two most important factors in the increased formation of large cyanobacterial blooms are global climate change8 and dramatic increases in the quantities of bioavailable nutrients in surface waters.9−11 Cyanobacteria produce structurally diverse toxins (microcystins, nodularins, saxitoxins, anatoxins, cylindrospermopsin), which can pose a significant health hazard in drinking water; among their potentially fatal effects are liver damage (including liver cancer), imunotoxicity, embryotoxicity, cytotoxicity, and neurotoxicity.12−18 In recent years, various technologies for the elimination and removal of cyanobacterial water blooms have been developed. © 2012 American Chemical Society

These methods differ in terms of their mechanism and selectivity of action, efficiency, large-scale applicability, environmental acceptability, financial cost, and technological sophistication. The most widely used methods are designed to reduce phosphorus loads in catchment and sediments.10,19 Because of their adverse ecological impacts and relatively brief duration of action, direct chemical methods based on the use of algaecides are not considered to be useful in advanced restoration projects.20 Other approaches that have been considered involve the use of flocculants or coagulants,21,22 oxidative techniques (e.g., ozonation, hydrogen peroxide application, chlorination), 23−25 and physical methods such as ultrasound technologies.26 The most modern methods are based on various ecotechniques such as lake destratification, food-web manipulation,27 or biotic interactions such as those between cyanobacteria (and their toxins) and bacteria or macrophytes.28 However, all of these technologies have several general disadvantages, including low selectivity and adverse environmental impact. Moreover, oxidative and ultrasonic methods do not destroy or inactivate already-released toxins. The effects of algaecides and flocculants are temporary; treatment with these agents does not preclude the future occurrence of cyanobacterial blooms. And while ecotechniques and methods based on Received: Revised: Accepted: Published: 2316

September 9, 2011 January 9, 2012 January 11, 2012 January 11, 2012 dx.doi.org/10.1021/es2031483 | Environ. Sci. Technol. 2012, 46, 2316−2323

Environmental Science & Technology

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All experiments were performed in triplicate; the toxicity of nZVI was expressed as the effective concentration (EC50) required to cause a 50% decrease in the biomass (50% decrease in the concentration of chlorophyll a) in the water column after 24 h of incubation. The term toxicity relates to all factors responsible for decrease of chlorophyll a. Daphnia magna. D. magna bioassays were performed according to the international standard method (ISO 6341, 1996). Organisms were obtained from RECETOX (Masaryk University, Brno, Czech Republic). Juveniles of D. magna (30 individuals per variant, not older than 24 h) were transferred into separate polystyrene plates using the standard exposure solution (ISO 6341, 1996). The tested concentrations of nZVI were 100, 500, 1000, 2500, and 5000 mg/L. The dispersions were stirred rapidly for 30 s and then left to stand for 48 h. The temperature was kept at 20(±2) °C during the exposure. Daphnids were inspected after 24 and 48 h of exposure. All experiments were performed in triplicate; the toxicity of nZVI was expressed in terms of the effective concentration (EC50) required to cause the immobilization of 50% of all individuals after 48 h of interaction. Lemna minor. Duckweed (L. minor) bioassays were performed according to the international standard method (OECD 221, 2002). Testing was performed in 250-mL flasks containing 100 mL of medium (Steinberg medium). The tested concentrations of nZVI were 5, 10, 50, 100, 500, and 1000 mg/ L. The so-formed dispersions were stirred rapidly for 30 s and then left to stand for 7 days at 24(±1) °C under continuous illumination (50 μmol/m2·s) by fluorescent lamps (Narva). All experiments were performed in triplicate. The frond numbers and dry weight after 7 days were used as measures of growth inhibition when determining the EC50. Sinapis alba. Seeds of S. alba were placed in Petri dishes (20 seeds per dish) with diameters of 10 cm and filter paper on the bottom. Aqueous dispersions of nZVI were added to the cultivation media (ISO 7346) to achieve final concentrations of 50, 100, 500, and 1000 mg/L. The so-formed dispersions were stirred rapidly for 30 s and then left to stand for 72 h at 25(±1) °C. All experiments were performed in triplicate; the root length after 72 h was used as the end point when determining the EC50. Desmodesmus subspicatus. The growth assay with the green alga D. subspicatus was performed in flasks with sample volume of 50 mL/flask, with three replicates for each sample (ISO 8692). The initial concentration of algae was 10 000 cells per milliliter. The tested concentrations of nZVI were 5, 10, 50, 100, 500, and 1000 mg/L. The so-formed dispersions were stirred rapidly for 30 s and then left to stand for 72 h at 24(±1) °C under continuous illumination (50 μmol/m2·s) by fluorescent lamps (Narva). The EC50 was determined by monitoring the growth rate of the green alga. Poecilia reticulata. The acute toxicity of nZVI to the fish P. reticulata was measured according to the CSN EN ISO 7346-2 guideline. A homogeneous population of fish (3−4 months old) was treated in accordance with legislation for the protection of animals against cruelty. Suspensions of nZVI nanoparticles of predefined concentrations were added to all jars aside from the controls. On the basis of preliminary results, concentrations of 0 (control), 750, 1500, and 3000 mg/L were tested. Immediately after the addition of nZVI nanoparticles, 10 fish were transferred into each jar. The temperature of the baths in the jars was maintained at 22(±1) °C, the initial pH was 8.0, and the initial dissolved oxygen concentrations exceeded 8 mg/

biotic interactions have considerable potential, those that have been reported to date are somewhat inefficient and have limited large-scale applicability.29 In this paper, we have identified zerovalent iron nanoparticles as a potentially useful multipronged weapon for use against cyanobacterial water blooms. This environmentally friendly technology is the first known method that allows for the selective removal of cyanobacteria, and has four principal modes of action. The preventive mode is based on its ability to remove bioavailable phosphorus. The destructive mode relates to the precipitation of iron-containing solids within the cell and the oxidative stress imposed by nZVI; this stems from the production of oxidizing radicals during the reaction of nZVI with water and from the active uptake of Fe(II) ions by cyanobacterial cells. Immobilization prevents the release of free microcystin into the water; it becomes sorbed on the large, positively charged surface of the nontoxic and environmentally benign ferric hydroxide produced by the oxidation of nZVI. In this context, it is also possible that nZVI might promote the partial decomposition of microcystin. Finally, the physical mode of action involves the formation of a compact waste product consisting of the destroyed cyanobacterial cells and highly aggregated ferric hydroxide. Because the product is highly flocculated and prone to particle agglomeration, the organic− inorganic aggregates formed by nZVI treatment do not present any hazard to the ecosystem; they are not nanoscale materials. These aggregates settle within a few hours, leading to the complete cleansing and decontamination of the water column.

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MATERIALS AND METHODS Zerovalent Iron Nanoparticles Used. For the testing, we used commercially available product Nanofer25 (obtained from Nanoiron, Ltd., Rajhrad, Czech Republic), which is a reactive aqueous dispersion of nanoparticles stabilized solely by an inorganic modifier. The content of the solid phase in the dispersion has been found to be 20% (by weight). The material contains >90% Fe(0) in the solid phase (as proved by the analysis of X-ray powder diffraction pattern and roomtemperature Mössbauer spectrum with Fe3O4 and FeO being impurities). The average particle size of iron nanoparticles is ∼70 nm (derived from the analysis of transmission electron microscopy images), and the particle specific surface area is ∼25 m2/g (calculated from the nitrogen adsorption/desorption curve employing Brunauer−Emmett−Teller (BET) theory; see Supporting Information, Figure S1). Ecotoxicological Bioassays with nZVI. For all bioassays, aqueous dispersions of nZVI were added to the cultivation media to achieve various final concentrations depending on the tested biological species (see below). Microcystis aeruginosa. These bioassays were carried out in 100-mL flasks with a sample volume of 75 mL. Tests were conducted using natural water sample from a eutrophic pond (Splaviska, Brno, Czech Republic) inoculated with a cyanobacterial bloom of predominantly M. aeruginosa at a concentration of 250 μg/L (in terms of the concentration of chlorophyll a). The tested concentrations of nZVI were 5, 10, 50, 100, and 500 mg/L. The so-formed dispersions were stirred rapidly for 30 s, after which the cyanobacteria were incubated with the nZVI particles for 24 h at 24(±1) °C under continuous illumination (50 μmol/m2·s) by fluorescent lamps (Narva). Chlorophyll a concentrations were determined using a FluoroProbe instrument (BBE Moldaenke) in samples taken from the middle of the water column after 24 h of incubation. 2317

dx.doi.org/10.1021/es2031483 | Environ. Sci. Technol. 2012, 46, 2316−2323


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Figure 1. Ecotoxicological bioassays demonstrate the potent and selective activity of nZVI against cyanobacteria along with its low ecotoxicity to selected relevant organisms. Toxicity units: 1/EC50.

Figure 2. (a) SEM images of cyanobacteria before treatment, (b) unused nZVI particles, (c) highly deformed cells after brief exposure to nZVI, and (d) completely destroyed cells surrounded by ferric oxide aggregates.

m2·s) by fluorescent lamps (Narva). Aqueous dispersions of nZVI were added to the cultivation media to achieve final concentrations of 10, 50, 100, and 500 mg/L. The so-formed dispersions were stirred rapidly for 30 s and then incubated for 24 h. A negative control sample was prepared using cultivation media containing the same amount of cyanobacteria but without nZVI. Similarly, a sample prepared in the same way but using the herbicide Paraquat (Ehrenstorfer GmbH) at a concentration of 50 mg/L in place of nZVI was used as a positive control. After 24 h of incubation, the solid biomass was removed by filtration through a GF/C Whatman filter and the concentration of microcystin (MC) in the solution was determined by highly sensitive competitive indirect ELISA

L (O2). The mortalities of the fish after 96 h of exposure to nZVI nanoparticles were evaluated. Testing on fishes was performed by ALS Laborator Group (ALS Czech Republic, Ltd.). Measurements of Microcystin Release after nZVI Application. The experiments were carried out using water inoculated with a M. aeruginosa laboratory strain that remains in the colonial form (CCT12/2008). The concentration of cyanobacteria (measured in terms of the concentration of chlorophyll a) in the water was 250 μg/L. Laboratory experiments were performed in triplicate in 100-mL beakers containing 75 mL of cultivation medium with cyanobacterial cells at 24(±1) °C under continuous illumination (50 μmol/ 2318



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(LOD = 0.125 μg MC/L) using monoclonal antibodies against MC-LR. Microscopic Observations and Mö ssbauer Spectra. Transmission 57Fe Mössbauer spectra of dispersions frozen at 270 K were acquired using a Mössbauer spectrometer of 512 channels in constant acceleration mode with a 57Co(Rh) source. To confirm the magnetic state of the nZVI oxidation product, in-field Mössbauer spectra were recorded at 5 K in an external magnetic field of 5 T, applied parallel to the γ-rays’ direction of propagation, using an Oxford Instruments cryomagnetic system. The isomer shift values are reported relative to α-Fe at room temperature. Transmission electron microscopy (TEM) images were obtained using a JEM2010 microscope operated at 200 kV with a point-to-point resolution of 1.9 Å. Scanning electron microscopy (SEM) images were acquired using a field-emission scanning electron microscope (Hitachi) operating at 0.7 and 4.0 kV. Phosphorus Determination. Dissolved orthophosphate was determined by the method described in European standard EN ISO 6878, which is based on spectrophotometric quantification at 680 nm using ammonium molybdenate. The amount of particulate phosphorus (cell-bound phosphates in algae or bacteria) was determined by the same method, after filtration of biomass using a 0.22 μm Milipore filtration membrane. Before analysis, the cells separated by filtration were disintegrated using an ultrasonic disintegrating device (Bandelin Sonoplus UW2070).

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RESULTS AND DISCUSSION The results of ecotoxicological bioassays showing the effects of nZVI on six representative organisms, including the cyanobacterium M. aeruginosa, are summarized in Figure 1. It was found that nZVI exhibited selective toxicity: it was very potent against the cyanobacterium, but its toxicity to other ecotoxicologically important organisms such as algae, daphnids, plants, and fishes was 2−3 orders of magnitude lower. To be specific, the effective nZVI concentration required to inhibit 50% of organisms (EC 50 ) for D. magna (an aquatic invertebrate) and P. reticulata (a fish) were greater than 1000 and 2500 mg/L, respectively, while that for M. aeruginosa was 50 mg/L. Similarly, the toxicity of nZVI toward aquatic and terrestrial plants and algae was also found to be very low (S. alba, L. minor, and D. subspicatus). This unique selectivity and high toxicity is presumably related to specific chemical interactions between nZVI and the cyanobacterial cells. Cyanobacteria are Gram-negative; in this context, it is interesting that nZVI has also been shown to have potent bactericidal effects against the Gram-negative bacterium Escherichia coli.30 While its precise mechanism of action remains to be determined, nZVI has been observed to cause significant physical disruption of the cell membranes of E. coli. It was suggested that this bactericidal activity was probably due to the reaction of dissolved Fe(II) with intracellular oxygen or hydrogen peroxide, which can induce oxidative stress by producing reactive oxygen species.30 This stress can result also from the disturbance of the electronic and/or ionic transport chains due to the strong affinity of the nanoparticles for the cell membrane.31 The importance of using highly reactive nZVI was demonstrated by Li et al.,32 who found that aged nZVI material containing high levels of iron oxide nanoparticles is relatively benign to bacteria. It should be noted that this disruption of the cell membranes is unique to nZVI; it is not observed with other iron-based species.30 The selective effects of nZVI toward

Figure 3. (a) TEM image of cyanobacterial cells after brief (5 min) exposure to nZVI at a concentration of 50 mg/L, (b) Mössbauer spectrum of cyanobacterial cells after brief (5 min) exposure to nZVI at a concentration of 50 mg/L, and (c) Mössbauer spectrum of the nZVI sample of the same concentration (50 mg/L) stored under the same conditions for the same length of time in cultivation media without cyanobacteria. The inset in the TEM image shows a detail of the compact cell membrane in cells that have not been treated with nZVI.

Figure 4. The release of microcystins into the water column after treatment with different concentrations of nZVI nanoparticles. For comparative purposes, the effects of treatment with Paraquat, which destroys cells but does not remove microcystins, are also shown.

bacteria and cyanobacteria is probably related to the cell membrane character, as also supported by the confirmed high toxicity against Gram-negative Pseudomonas fluorescens bacteria, while no toxic effect is reported against the fungus Aspergillus 2319



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formed iron(III) species.36−40 The second one relates to the precipitation of separate iron phosphate41−43 in mineral form, as proved by an independent experiment (see Supporting Information, Figure S2). These results demonstrate that nZVI has the potential to be a powerful preventative agent, being able to simultaneously remove both phosphate ions and particulate phosphorus. It thus combines the effects of common Fe-based flocculants, which target phosphate ions, and modern preventive ecotechniques, which are effective against particulate phosphorus. We also studied the mechanism by which nZVI interacts with cyanobacteria. The nZVI particles were found to have a high affinity for the cyanobacterial cells; the association of the two resulted in the formation of compact assemblies in the water column within a few minutes of treatment with nZVI. It is possible that this is a consequence of purely physical processes based on electrostatic interactions between the positively charged nZVI surface and the negative charges on the outer surfaces of the cyanobacterial cell membranes. However, it is likely that this aggregation is also favored by chemical processes induced by the reaction of nZVI with water. Scanning electron microscopy (SEM) experiments provided visual evidence of the gradual cell deformation and ultimate cell destruction induced by nZVI (see Figure 2). For comparative purposes, healthy untreated cells of M. aeruginosa are shown in Figure 2a; unused nZVI particles are shown in Figure 2b. Within a few minutes of exposure to nZVI, the cells can be seen to have deformed significantly and are surrounded by a shell of iron(III) hydroxide aggregates formed by oxidation of nZVI (see Figure 2c). The composition of these shells was subsequently confirmed by Mössbauer spectroscopy (vide infra). Thus, cyanobacterial cells treated with nZVI rapidly acquire a coating of iron and iron(III) hydroxide that causes lethal shading of the light-harvesting complexes of photosystem II. As shown in Figure 2d, extended treatment with nZVI results in the destruction of most of the cells; this is the key mechanism by which nZVI eliminates cyanobacteria. The cell membrane disruption caused by nZVI treatment is probably a consequence of the oxidative stress induced by the reaction of water with the nanoscale iron, as suggested by Lee et al. on the basis of their studies with E. coli.30 The selective toxicity of nZVI toward cyanobacteria could conceivably be due to the production of •OH radicals, which are known to be an order of magnitude more toxic to cyanobacteria than to algae due to their different membrane compositions.24 However, our experimental and theoretical results strongly suggest that the observed destruction of cyanobacterial cells by nZVI is primarily due to the active transport of ferrous ions, produced by reaction of nZVI with water, across the cyanobacterial cell membrane. It is well-known that dissolved iron is, in addition to phosphorus and nitrogen, another key nutrient for cyanobacteria.44 We hypothesize that ferrous ions are transported into the cyanobacterial cells, where they react with oxygen and hydrogen peroxide via Fenton-like reactions that produce reactive oxygen species, resulting in severe cell damage. Furthermore, at the pH and redox potential encountered in the cytoplasm, the importation of large quantities of Fe(II) would result in massive precipitation of iron(III) hydroxide nanoparticles and secondary cell destruction. The formation of such precipitates in cyanobacterial cells treated with nZVI was demonstrated by transmission electron microscopy (TEM). The TEM micrograph shown in Figure 3a depicts cyanobacterial cells after a few minutes of treatment with nZVI and thus

Figure 5. (a) Photograph obtained immediately after the dispersal of nZVI (50 mg/L) in the sample with cyanobacterial cells (left) and after 24 h of treatment (right), demonstrating the decomposition and gradual sedimentation of the cyanobacterial biomass. (b) The SEM image shows a sample of the settled and decomposed biomass. The results of an EDX analysis of the sediment are shown in the inset. Al originates from the substrate for microscopic analysis.

versicolor when treated with the same nanoparticles in the same manner.33 To understand the mechanisms by which nZVI acts against bacteria, we performed detailed chemical, microscopic, and microbiological analyses of nZVI−cyanobacteria systems both during and after treatment. In particular, we focused on evaluating the ability of nZVI to eliminate phosphorus, to induce cell destruction, and to immobilize microcystin produced by the cyanobacteria. Microscale zerovalent iron was proved to be potentially applicable for phosphorus removal if applied in a permeable reactive barrier.34 We tested this hypothesis with nZVI under conditions suitable for cyanobacterial growth, in cultivation media with an ecologically relevant initial phosphate concentration of 0.2 mg/L. It was found that nZVI has two different modes of action, depending on the nature of the phosphoruscontaining species. The iron particles react with dissolved phosphate ions to form insoluble iron phosphate and also remove particulate phosphorus tied up in plankton biomass, e.g., in bacterial and algal cells. At the lowest nZVI concentration examined (0.15 mg/L), 80% of the soluble phosphate in the tested system was removed within 2 h. The same concentration of nZVI (0.15 mg/L) also removed 75% of the particulate phosphorus fixed in bacterial cells. Concerning the mechanism of phosphorus removal, two different routes should be taken into account.35 The first one involves the formation of the surface complexes of phosphate with the 2320



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into the aquatic system. However, the experimental data indicate that nZVI treatment did not cause any measurable increases in microcystin concentration (see Figure 4). Even when nZVI was used at a concentration of 500 mg/L1 order of magnitude higher than its EC50 valuethe concentration of microcystin in the medium was similar to that observed in the control sample. For comparative purposes, treatment of cyanobacteria with the common herbicide Paraquat (which also destroys cyanobacterial cells) at a concentration of 50 mg/ L causes a 10-fold increase in microcystin concentration. This unique ability of nZVI to immobilize the released microcystin may be attributable to several mechanisms. We propose that the microcystins released from damaged cells may be (i) sorbed on the large surface area of the ferric hydroxide reaction products and (ii) oxidatively decomposed by the evolved radicals. In this context, it should be noted that the viability of immobilizing microcystins on nanomaterials with a sufficiently high surface area and/or on porous materials that act as sorbents and steric barriers to the cyanobacterial toxins has already been demonstrated using nanofiltration membranes.49 This hypothesis is also consistent with the high sorption of microcystin-LR on activated carbon.50 Similarly, high sorption of nodularin, a cyclic pentapeptide hepatotoxin produced by the bloom-forming cyanobacterium Nodularia spumigena, on finegrained nanosediments from the Baltic has been reported.51 Recently, various other natural sediments and clay minerals have been proved to adsorb microcystin with a high efficiency.52 We should emphasize that the sorption properties of ferric hydroxide would considerably contribute not only to the microcystin immobilization but also to the phosphorus removal.53 A final factor that makes zerovalent iron nanoparticles an attractive and potentially powerful tool for the treatment of waters contaminated by cyanobacteria is that they are environmentally benign. In particular, it has been demonstrated that nZVI particles larger than approximately 30 nm require no more regulatory scrutiny than does bulk iron.54 Moreover, nZVI is already produced commercially on a large scale because of its widespread use as an environmentally friendly agent for the in situ treatment of groundwater. In this context, nZVI is popular because of its ability to induce reduction and precipitation reactions that effectively transform more than 70 toxic substances, including polychlorinated hydrocarbons, phosphates, nitrates, and species containing arsenic, uranium, and heavy metals.55−57 One outstanding potential issue with using the results discussed above to justify the use of nZVI for the treatment of cyanobacterial blooms stems from the fact that the ecotoxicological assays reported herein were conducted under conditions designed to ensure efficient contact between the nanoparticles and the cyanobacteria. In particular, given the expected sedimentation of nZVI in real-world use, one might wonder whether the various anticyanobacterial mechanisms identified would operate quickly enough to make the nZVI effective. We therefore conducted a series of experiments in cylinders to study the sedimentation process and the nature of the decomposed cyanobacterial biomass, including the oxidized iron. An nZVI concentration equal to its measured EC50 value (50 mg/L) for cyanobacteria was used in these experiments. Figure 5a shows a photograph of a green dispersion of untreated cyanobacteria at an ecologically relevant concentration of 250 μg/L that had been left to stand for 24 h (left) alongside an image of an otherwise-identical dispersion after 24

shows an early stage in the process, before complete cell destruction has occurred. Even at this early stage, the rapid disintegration of the membrane is readily apparent, as is the formation of iron(III) hydroxide particles inside the cell and on its surface. For comparative purposes, the compact cell membranes of untreated cyanobacterial cells are shown in the inset. This intracellular formation of iron oxide resembles the intracellular formation of akaganeite (β-FeOOH) nanorods observed by Brayner et al.45 in cells of the cyanobacteria Anabaena sp. and Calothrix sp. and the green alga Klebsormidium sp. grown under high-iron conditions. A mechanism involving the formation of iron−siderophore complexes was proposed to explain this observation and was compared to the iron biomineralization process observed in magnetotactic bacteria.46 In the light of these results, it is tempting to suggest that cyanobacteria may be unable to effectively regulate their uptake of iron in environments where it is highly abundant and may thus be prone to “selfdestruction” under such circumstances. This hypothesis is consistent with the well-known high affinity of cyanobacteria for iron and the efficiency with which they take up ferrous ions from their surroundings.47 In order to identify and quantify the iron-containing products arising from the interaction of nZVI with cyanobacterial cells, we used Mössbauer spectroscopy to study a sample of cyanobacteria that had been treated with nZVI for 5 min (see Figure 3b). The samples were frozen at 270 K prior to analysis to prevent secondary oxidation of the nZVI during the measurement. In addition to the typical sextet with a hyperfine magnetic field of ∼33 T that was attributed to residual α-Fe, the spectrum also contained a central doublet whose hyperfine parameters (isomer shift of 0.43 mm/s and quadrupole splitting of 0.77 mm/s) were consistent with those expected for highspin iron(III) in ferric hydroxide.48 Since many (super)paramagnetic iron(III) oxides(oxyhydroxides) exhibit similar hyperfine parameters in their room temperature zero-field spectra, the sample’s in-field Mössbauer spectrum (not shown) was also measured at 5 K in an external magnetic field of 5 T and compared with that recorded at the same temperature without the applied magnetic field. These comparative measurements and unchanged line intensities clearly prove the speromagnetic nature of the Fe(III) phase, which is the specific behavior of nearly amorphous iron oxides like Fe(OH)3 and Fe2O3.48 Remarkably, analysis of the areas of the different peaks in the Mössbauer spectrum revealed that iron(III) hydroxide accounted for 48% of the total iron in the cyanobacterial sample after only 5 min of exposure to nZVI; for comparative purposes, Fe(OH)3 accounted for only 12% of the iron in an nZVI sample of the same concentration (50 mg/ L) stored under the same conditions for the same length of time in cultivation media without cyanobacteria (see Figure 3c). This key finding strongly suggests the existence of a specific chemical reaction between nZVI and cyanobacterial cells or radicals produced within them. We believe that this would be a result of an intensive iron uptake through the cell membrane and consequent precipitation of ferric hydroxide. Anyway, it is worth mentioning that observed differences in Mössbauer spectra would be also due to different conditions for heterogeneous nucleation (i.e., rates of oxide production) in the presence and without the presence of cyanobacteria in the system. One would expect that the damage nZVI causes to cyanobacterial cells would result in the release of cyanotoxins 2321



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h of treatment with nZVI (right). The water in the treated column is clear and free of live cyanobacteria; the decomposed biomass, which contains heavy iron(III) hydroxide, has undergone sedimentation. Microscopic (SEM) analyses of the filtered and air-dried sediment indicated that the reaction product was not a nanodimensional material. Instead, the inorganic−organic composite forms compact flocks whose sizes range from tens of micrometers to millimeters (see the scale in the SEM image in Figure 5b) and which contain highly aggregated ferric hydroxide and organic fractions. Energy dispersive spectroscopy (EDS) analysis of the material shown in the SEM image (see Figure 5b) indicated that the flocks contain elements consistent with the formation of iron(III) hydroxide (Fe, O), the destruction of cyanobacterial cells (C), and the removal of phosphorus from the water (P). The other elements identified originate from the cultivation solution. It should be emphasized that neither the organic carbon (which accounts for the bulk of the decomposed cyanobacterial cells) nor the highly aggregated ferric hydroxide (which occurs naturally in various waters in the form of ferrihydrite) presents any significant ecological hazard. More to the contrary, ferric (oxy)hydroxides like ferrihydrite are able to remove various toxic substances including arsenic and heavy metals from water due to their high sorption capacity.58

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REFERENCES

(1) Rasmussen, B.; Fletcher, I. R.; Brocks, J. J.; Kilburn, M. R. Reassessing the first appearance of eukaryotes and cyanobacteria. Nature 2008, 455, 1101−U9. (2) Savage, D. F.; Afonso, B.; Chen, A. H.; Silver, P. A. Spatially ordered dynamics of the bacterial carbon fixation machinery. Science 2010, 327, 1258−1261. (3) Tripp, H. J.; Bench, S. R.; Turk, K. A.; Foster, R. A.; Desany, B. A.; Niazi, F.; Affourtit, J. P.; Zehr, J. P. Metabolic streamlining in an open-ocean nitrogen-fixing cyanobacterium. Nature 2010, 464, 90−94. (4) Zehr, J. P.; Bench, S. R.; Carter, B. J.; Hewson, I.; Niazi, F.; Shi, T.; Tripp, H. J.; Affourtit, J. P. Globally distributed uncultivated oceanic N-2-Fixing cyanobacteria lack oxygenic photosystem II. Science 2008, 322, 1110−1112. (5) Kump, L. R.; Barley, M. E. Increased subaerial volcanism and the rise of atmospheric oxygen 2.5 billion years ago. Nature 2007, 448, 1033−1036. (6) Montoya, J. P.; Holl, C. M.; Zehr, J. P.; Hansen, A.; Villareal, T. A. Capone DG high rates of N-2 fixation by unicellular diazotrophs in the oligotrophic Pacific Ocean. Nature 2004, 430, 1027−1031. (7) Falkowski, P. G.; Katz, M. E.; Knoll, A. H.; Quigg, A.; Raven, J. A.; Schofield, O.; Taylor, F. J. R. The evolution of modern eukaryotic fytoplankton. Science 2004, 305, 354−360. (8) Johnk, K. D.; Huisman, J.; Sharples, J.; Sommeijer, B.; Visser, P. M.; Stroom, J. M. Summer heatwaves promote blooms of harmful cyanobacteria. Glob. Change Biol. 2008, 14, 495−512. (9) Tilman, D.; Fargione, J.; Wolff, B.; D’Antonio, C.; Dobson, A.; Howarth, R.; Schindler, D.; Schlesinger, W. H.; Simberloff, D.; Swackhamer, D. Forecasting agriculturally driven global environmental change. Science 2011, 292, 281−284. (10) Conley, D. J.; Paerl, H. W.; Howarth, R. W.; Boesch, D. F.; Seitzinger, S. P.; Havens, K. E.; Lancelot, C.; Likens, G. E. Controlling eutrophication: Nitrogen and phosphorus. Science 2009, 323, 1014− 1015. (11) Schindler, D. W.; Hecky, R. E.; Findlay, D. L.; Stainton, M. P.; Parker, B. R.; Paterson, M. J.; Beaty, K. G.; Lyng, M.; Kasian, S. E. M. Eutrophication of lakes cannot be controlled by reducing nitrogen input: Results of a 37-year whole-ecosystem experiment. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 11254−11258. (12) Funari, E.; Testai, E. Human health risk assessment related to cyanotoxins exposure. Crit. Rev. Toxicol. 2008, 38, 97−125. (13) Feurstein, D.; Kleinteich, J.; Heussner, A. H.; Stemmer, K.; Dietrich, D. R. Investigation of microcystin congener-dependent uptake into primary murine neurons. Environ. Health. Perspect. 2010, 118, 1370−1375. (14) Cox, P. A.; Banack, S. A.; Murch, S. J.; Rasmussen, U.; Tien, G.; Bidigare, R. R.; Metcalf, J. S.; Morrison, L. F.; Codd, G. A.; Bergman, B. Diverse taxa of cyanobacteria produce beta-N-methylamino-Lalanine, a neurotoxic amino acid. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 5074−5078. (15) Cox, P. A.; Banack, S. A.; Murch, S. J. Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 13380−13383. (16) Ding, W. X.; Shen, H. M.; Ong, C. N. Microcystic cyanobacteria extract induces cytoskeletal disruption and intracellular glutathione alteration in hepatocytes. Environ. Health. Perspect. 2000, 108, 605− 609. (17) Zegura, B.; Sedmak, B.; Filipic, M. Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2. Toxicon 2003, 41, 41−48. (18) Hitzfeld, B. C.; Hoger, S. J.; Dietrich, D. R. Cyanobacterial toxins: Removal during drinking water treatment, and human risk assessment. Environ. Health. Perspect. 2000, 108, 113−122. (19) McCormick, P. V.; Shuford, R. B. E.; Chimney, M. J. Periphyton as a potential phosphorus sink in the Everglades Nutrient Removal Project. Ecol. Eng. 2006, 27, 279−289. (20) Garcia-Villada, L.; Rico, M.; Altamirano, M.; Sanchez-Martin, L.; Lopez-Rodas, V.; Costas, E. Occurrence of copper resistant mutants in

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S Supporting Information *

Additional material on the physicochemical characterization of the used nZVI sample (X-ray powder diffraction pattern, particle size distribution, room-temperature Mössbauer spectrum, nitrogen adsorption/desorption isotherms) and X-ray powder diffraction pattern of the nZVI sample after the reaction with KH2PO4 as an evidence for formation of iron(III) phosphate. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +420585634947; fax: +420585634958; e-mail: [email protected].

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ACKNOWLEDGMENTS The authors gratefully acknowledge the support of the Operational Program Research and Development for InnovationsEuropean Development Fund (Project No. CZ.1.05/ 2.1.00/03.0058 of the Ministry of Education, Youth, and Sports of the Czech Republic). This work was also supported by the Ministry of Education of the Czech Republic (Project Nos. 1M6198959201, 1M0571, and MSM6198959218), the Ministry of Industry of the Czech Republic (Project No. FR-TI3/196), the Ministry of Agriculture of the Czech Republic (Project No. QH81012 of the National Agency for Agricultural Research), and the Academy of Sciences of the Czech Republic (Project Nos. KAN115600801 and AVOZ60050516). The authors thank Dr. D. Jancik, Dr. J. Filip, and Dr. K. Siskova (all from Regional Centre of Advanced Technologies and Materials, Palacky University, Olomouc, Czech Republic) for assistance in the microscopic analyses, X-ray powder diffraction measurement, and additional experiments, respectively. The authors also thank the Nanoiron Co. (Rajhrad, Czech Republic) for the assistance in nZVI testing. 2322



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the toxic cyanobacteria Microcystis aeruginosa: Characterisation and future implications in the use of copper sulphate as algaecide. Water Res. 2004, 38, 2207−2213. (21) Dixon, M. B.; Richard, Y.; Ho, L.; Chow, C. W. K.; O’Neill, B. K.; Newcombe, G. Integrated membrane systems incorporating coagulation, activated carbon and ultrafiltration for the removal of toxic cyanobacterial metabolites from Anabaena circinalis. Water Sci. Technol. 2011, 63, 1405−1411. (22) Chow, C. W. K.; Drikas, M.; House, J.; Burch, M. D.; Velzeboer, R. M. A. The impact of conventional water treatment processes on cells of the cyanobacterium Microcystis aeruginosa. Water Res. 1999, 33, 3253−3262. (23) Annadotter, H.; Cronberg, G.; Aagren, R.; Lundstedt, B.; Nilsson, P. A.; Strobeck, S. Multiple techniques for lake restoration. Hydrobiologia 1999, 396, 77−85. (24) Drabkova, M.; Admiraal, W.; Marsalek, B. Combined exposure to hydrogen peroxide and lightSelective effects on cyanobacteria, green algae, and diatoms. Environ. Sci. Technol. 2007, 41, 309−314. (25) Daly, R. I.; Ho, L.; Brookes, J. D. Effect of chlorination on Microcystis aeruginosa cell integrity and subsequent microcystin release and degradation. Environ. Sci. Technol. 2007, 41, 4447−4453. (26) Zhang, G. M.; Zhang, P. Y.; Fan, M. H. Ultrasound-enhanced coagulation for Microcystis aeruginosa removal. Ultrason. Sonochem. 2009, 16, 334−338. (27) Becker, A.; Herschel, A.; Wilhelm, C. Biological effects of incomplete destratification of hypertrophic freshwater reservoir. Hydrobiologia 2006, 559, 85−100. (28) Nimptsch, J.; Wiegand, C.; Pflugmacher, S. Cyanobacterial toxin elimination via bioaccumulation of MC-LR in aquatic macrophytes: An application of the “Green Liver Concept”. Environ. Sci. Technol. 2008, 42, 8552−8557. (29) Hulot, F. D.; Lacroix, G.; Lescher-Moutoue, F. O.; Loreau, M. Functional diversity governs ecosystem response to nutrient enrichment. Nature 2000, 405, 340−344. (30) Kim, J. Y.; Park, H. J.; Lee, C.; Nelson, K. L.; Sedlak, D. L.; Yoon, J. Inactivation of Escherichia coli by nanoparticulate zerovalent iron and ferrous ion. Appl. Environ. Microb. 2010, 76, 7668−7670. (31) Auffan, M.; Achouak, W.; Rose, J.; Roncato, M. A.; Chaneac, C.; Waite, D. T.; Masion, A.; Woicik, J. C.; Wiesner, M. R.; Bottero, J. Y. Relation between the redox state of iron-based nanoparticles and their cytotoxicity toward Escherichia coli. Environ. Sci. Technol. 2008, 42, 6730−6735. (32) Li, Z. Q.; Greden, K.; Alvarez, P. J. J.; Gregory, K. G.; Lowry, G. V. Adsorbed polymer and NOM limits adhesion and toxicity of nanoscale zerovalent iron to E. coli. Environ. Sci. Technol. 2010, 44, 3462−3467. (33) Diao, M. H.; Yao, M. S. Use of zero-valent iron nanoparticles in inactivating microbes. Water Res. 2009, 43, 5243−5251. (34) McCobb, T. D.; LeBlanc, D. R.; Massey, A. J. Monitoring the removal of phosphate from ground water discharging through a pondbottom permeable reactive barrier. Ground Water Monit. Rem. 2009, 29, 43−55. (35) Mino, T.; Van Loosdrecht, M. C. M.; Heijnen, J. J. Microbiology and biochemistry of the enhanced biological phosphate removal process. Water Res. 1998, 32, 3193−3207. (36) Zeng, L.; Li, X. M.; Liu, J. D. Adsorptive removal of phosphate from aqueous solutions using iron oxide tailings. Water Res. 2004, 38, 1318−1326. (37) Chitrakar, R.; Tezuka, S.; Sonoda, A.; Sakane, K.; Ooi, K.; Hirotsu, T. Phosphate adsorption on synthetic goethite and akaganeite. J. Colloid Interface Sci. 2006, 298, 602−608. (38) Luengo, C.; Brigante, M.; Antelo, J.; Avena, M. Kinetics of phosphate adsorption on goethite: Comparing batch adsorption and ATR-IR measurements. J. Colloid Interface Sci. 2006, 300, 511−518. (39) Zach-Maor, A.; Semiat, R.; Shemer, H. Synthesis, performance, and modeling of immobilized nano-sized magnetite layer for phosphate removal. J. Colloid Interface Sci. 2011, 357, 440−446. (40) Zach-Maor, A.; Semiat, R.; Shemer, H. Adsorption-desorption mechanism of phosphate by immobilized nano-sized magnetite layer:

Interface and bulk reactions. J. Colloid Interface Sci. 2011, 363, 608− 614. (41) Smith, S.; Takacs, I.; Murthy, S.; Daigger, G. T.; Szabo, A. Phosphate complexation model and its implications for chemical phosphorus removal. Water Environ. Res. 2008, 80, 428−438. (42) Zhang, T.; Ding, L. L.; Ren, H. Q.; Guo, Z. T.; Tan, J. Thermodynamic modeling of ferric phosphate precipitation for phosphorus removal and recovery from wastewater. J. Hazard. Mater. 2010, 176, 444−450. (43) De Gregorio, C.; Caravelli, A. H.; Zaritzky, N. E. Performance and biological indicators of a laboratory-scale activated sludge reactor with phosphate simultaneous precipitation as affected by ferric chloride addition. Chem. Eng. J. 2010, 165, 607−616. (44) Masse, E.; Salvail, H.; Desnoyers, G.; Arguin, M. Small RNAs controlling iron metabolism. Curr. Opin. Microbiol. 2007, 10, 140−145. (45) Brayner, R.; Yepremian, C.; Djediat, C.; Coradin, T.; Herbst, F.; Livage, J.; Fievet, F.; Coute, A. Photosynthetic microorganismmediated synthesis of akaganeite (beta-FeOOH) nanorods. Langmuir 2009, 25, 10062−10067. (46) Faivre, D.; Schuler, D. Magnetotactic bacteria and magnetosomes. Chem. Rev. 2008, 108, 4875−4898. (47) Fujii, M.; Dang, T. C.; Rose, A. L.; Omura, T.; Waite, T. D. Effect of light on iron uptake by the freshwater cyanobacterium Microcystis aeruginosa. Environ. Sci. Technol. 2011, 45, 1391−1398. (48) Machala, L.; Zboril, R.; Gedanken, A. Amorphous iron(III) oxideA review. J. Phys. Chem. C 2007, 111, 4003−4018. (49) Teixeira, M. R.; Rosa, M. J. Integration of dissolved gas flotation and nanofiltration for M. aeruginosa and associated microcystins removal. Water Res. 2006, 40, 3612−3620. (50) Lee, J.; Walker, H. W. Effect of process variables and natural organic matter on removal of microcystin-LR by PAC-UF. Environ. Sci. Technol. 2006, 40, 7336−7342. (51) Torunska, A.; Bolalek, J.; Plinski, M.; Mazur-Marzec, H. Biodegradation and sorption of nodularin (NOD) in fine-grained sediments. Chemosphere 2008, 70, 2039−2046. (52) Wu, X. Q.; Xiao, B. D.; Li, R. H.; Wang, C. B.; Huang, J. T.; Wang, Z. W. Mechanisms and factors affecting sorption of microcystins onto natural sediments. Environ. Sci. Technol. 2011, 45, 2641−2647. (53) Bjerrum, C. J.; Canfield, D. E. Ocean productivity before about 1.9 Gyr ago limited by phosphorus adsorption onto iron oxides. Nature 2002, 417, 159−162. (54) Auffan, M.; Rose, J.; Bottero, J. Y.; Lowry, G. V.; Jolivet, J. P.; Wiesner, M. R. M. Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective. Nat. Nanotechnol. 2009, 4, 634−641. (55) Tratnyek, P. G.; Johnson, R. L. Nanotechnologies for environmental cleanup. Nano Today 2006, 1, 44−48. (56) Li, L.; Fan, M. H.; Brown, R. C.; Van Leeuwen, J. H.; Wang, J. J.; Wang, W. H.; Song, Y. H.; Zhang, P. Y. Synthesis, properties, and environmental applications of nanoscale iron-based materials: A review. Crit. Rev. Environ. Sci. Technol. 2006, 36, 405−431. (57) Kanel, S. R.; Manning, B.; Charlet, L.; Choi, H. Removal of arsenic(III) from groundwater by nanoscale zero-valent iron. Environ. Sci. Technol. 2005, 39, 1291−1298. (58) Tonkin, J. W.; Balistrieri, L. S.; Murray, J. W. Modeling metal removal onto natural particles formed during mixing of acid rock drainage with ambient surface water. Environ. Sci. Technol. 2002, 36, 484−492.

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