pubs.acs.org/Langmuir © 2010 American Chemical Society
ZnO Nanoparticles: Synthesis, Characterization, and Ecotoxicological Studies Roberta Brayner,*,† Si Amar Dahoumane,† Claude Yepremian,‡ Chakib Djediat,‡ Micha€el Meyer,† Alain Coute,‡ and Fernand Fievet† †
Universit e Paris Diderot (Paris 7), CNRS, UMR 7086, Interfaces, Traitements, Organisation et Dynamique des Syst emes (ITODYS), 15 rue Jean de Baı¨f, F-75205 Paris Cedex 13, France, and ‡Mus eum National d’Histoire Naturelle, D epartement RDDM, USM 505, 57 rue Cuvier, F-75005 Paris, France Received October 22, 2009. Revised Manuscript Received February 17, 2010
The potential ecotoxicity of nanosized zinc oxide (ZnO), synthesized by the polyol process, was investigated using common Anabaena flos-aquae cyanobacteria and Euglena gracilis euglenoid microalgae. The photosynthetic activities of these microorganisms, after addition of ZnO nanoparticles, varied with the presence of protective agents such as trin-octylphosphine oxide (TOPO) and polyoxyethylene stearyl ether (Brij-76) used to control particle size and shape during the synthesis. In the case of Anabaena flos-aquae, the photosynthetic activity, after addition of ZnO, ZnO-TOPO, and ZnO-Brij-76, decreased progressively due to stress induced by the presence of the nanoparticles in the culture medium. After contact with ZnO-TOPO nanoparticles, this decrease was followed by cell death. On the other hand, after 10 days, a progressive increase of the photosynthetic activity was observed after contact with ZnO and ZnO-Brij-76 nanoparticles. In the case of Euglena gracilis, cell death was observed after contact with all nanoparticles. Transmission electron microscopy (TEM) analyses of ultrathin sections of microorganisms showed that polysaccharides produced by Anabaena flos-aquae avoid particle internalization after contact with ZnO and ZnO-Brij-76 nanoparticles. On the other hand, nanoparticle internalization was observed after contact with all nanoparticles in the presence of Euglena gracilis and also with ZnO-TOPO nanoparticles after contact with Anabaena flos-aquae.
Introduction To produce unique products with novel properties, we need to manipulate materials at the nanoscale level.1 In the world, manmade nanoparticles and materials are being rapidly produced in large quantities, and it was shown, in the past ten years, that nanomaterials have different toxicity profiles compared with larger particles because of their small size and also their high reactivity. In this moment, the toxicological and environmental effects of direct or indirect exposure to these manufactured nanomaterials are not completely elucidated.2 ZnO nanoparticles have been applied in broad fields, including sunscreens, biosensors, food additives, pigments, rubber manufacture, and electronic materials.3 Many studies showed the influence of ZnO nanoparticles on microorganisms.4-7 For example, (i) ZnO had significant growth inhibition on E. coli4,5 and (ii) ZnO had a good bacteriostasis effect based on Streptococcus agalactiae and Staphylococcus aureus, which are two etiological agents of several infective diseases in humans.7 Physicochemistry is essential to understanding the fate and behavior of nanoparticles in the environment, as well as uptake and distribution within organisms, and the interactions of nanoparticles with other pollutants. It has been reported that when the particle size decreases, there is a
tendency to increase the toxicity, even if the same material is relatively inert in bulk form (e.g., SiO2, carbon black, TiO2, ZnO).8,9 The nanoparticles, due to their nanoscale, shape, and consequently huge surface area, may interact more efficiently with biological systems, producing important toxicity. In addition, the surface area is directly correlated to many other physicochemical properties such as chemical reactivity, surface adsorption ability, surface charge, and so on. All these factors strongly dominate nanotoxicological behavior in vivo.8,9 In addition, the toxicological impact of nanoparticles depends also on the biological target used. In this new multidisciplinary field, there are many challenges ahead and some controversies, but knowledge transfer from biology, toxicology, colloid chemistry, as well as material and geological sciences will enable us to improve (nano)ecotoxicology studies. The present work is focused on (i) the study of the physicochemical properties of ZnO nanoparticles prepared without and with protective agents such as TOPO and Brij-76 before and after contact with the culture media and also (ii) the study of the ecotoxic impact of these nanoparticles on Anabaena flos-aquae and Euglena gracilis photosynthetic microorganisms.
Experimental Section
*To whom correspondence should be addressed. E-mail: roberta.brayner@ univ-paris-diderot.fr.
ZnO nanoparticles were synthesized in diethylene glycol (DEG) medium by forced hydrolysis of zinc acetate.10-13 The general procedure involves addition of zinc acetate to 80 mL of polyol and
(1) Gerber, C.; Lang, H. P. Nat. Nanotechnol. 2006, 1, 3. (2) Guzman, K. A. D.; Taylor, M. R.; Banfield, J. F. Environ. Sci. Technol. 2006, 40, 1401. (3) Ji, S. L.; Ye, C. H. J. Mater. Sci. Technol. 2008, 24, 457. (4) Adams, L. K.; Lyon, D. Y.; Alvarez, P. J. Water Res. 2006, 40, 3527. (5) Zhang, L.; Jiang, Y.; Ding, Y.; Povey, M.; York, D. J. Nanopart. Res. 2007, 9, 479. (6) Roselli, M.; Finamore, A.; Garaguso, I.; Britti, M. S.; Mengheri, E. J. Nutr. 2003, 133, 4077. (7) Huang, Z.; Zheng, X.; Yan, D.; Yin, G.; Liao, X.; Kang, Y.; Yao, Y.; Huang, D.; Hao, B. Langmuir 2008, 24, 4140.
(8) Herve-Bazin, B.,Ed. Les nanoparticles: un enjeu majeur pour la sante au travail? EDP Sciences: 2007. (9) Kumar, C. S. S. R., Ed. Nanomaterials - Toxicity, Health and Environmental Issues; Wiley-VCH: Weinheim, 2006. (10) Jezequel, D.; Guenot, J.; Jouini, N.; Fievet, F. J. Mater. Res. 1995, 10(1), 77. (11) Feldmann, C. Adv. Func. Mater. 2003, 13(2), 101. (12) Brayner, R.; Ferrari-Iliou, R.; Brivois, N.; Djediat, C.; Benedetti, M. F.; Fievet, F. Nano Lett. 2006, 6(4), 866. (13) Brayner, R. Nano Today 2008, 3(1-2), 48.
6522 DOI: 10.1021/la100293s
Published on Web 03/02/2010
Langmuir 2010, 26(9), 6522–6528
Brayner et al.
Article
Figure 1. TEM micrographs of ZnO nanoparticles synthesized in DEG medium by varying the hydrolysis ratio (H): (a) ZnO without addition of protective agents, (b) ZnO-TOPO, and (c) ZnO-Brij. H2O to reach a final concentration between 0.06 and 0.6 mol 3 L-1. The hydrolysis ratio (H = nH20/nZn2þ) was varied from 10 to 80. To control particle size and shape, protective agents such as TOPO and Brij-76 were added to zinc acetate and polyol solution with concentrations between 10-2 and 10-1 M. The mixture was then heated at 180 °C during 1 h under vigorous stirring. All photosynthetic microorganisms were selected on the basis of previous reports demonstrating that they are able to synthesize some metallic nanoparticles (Au, Ag, Pt, and Pd) by an enzymatic route.14 In addition, they remain alive for more than 3 months in the presence of these nanoparticles.14 In addition, Anabaena flosaquae can avoid particle internalization due to the presence of polysaccharides surrounding the cell wall, while Euglena gracilis can internalize nanoparticles by endocytosis. Anabaena flos-aquae planktonic prokaryotic cyanobacteria, strain ALCP B24, and Euglena gracilis eukaryotic euglenoid came from MNHN Culture Collection. Anabaena flos-aquae was grown in 250 mL erlenmeyer flasks, in sterile Bold’s basal medium (BB culture medium) and buffered with 3.5 mM phosphate buffer at a control temperature of 20.0 ( 0.5 °C and luminosity (30-60 μmol m-2 s-1 photosynthetic photon flux (PPF)) under ambient CO2 conditions. The pH of the medium was adjusted to 7.0 using 1 M NaOH solution. Euglena gracilis was grown in 250 mL Erlenmeyer flasks, in mineral medium (M medium) at a control temperature of 20.0 ( 0.5 °C and luminosity (70-100 μmol m-2 s-1 PPF) under ambient CO2 conditions. Before addition of 10-3 M ZnO nanoparticles in DEG, the culture was transferred (20% (v/v) of inoculum) and grown for 4 weeks. Nanoparticles were characterized using X-ray diffraction (XRD), UV-visible spectroscopy, dynamic light scattering (DLS), zeta potential, scanning electron microscopy (SEM) coupled with X-ray energy dispersive spectrometry (EDS), and transmission electron microscopy (TEM). Microorganisms were characterized using optical microscopy. The maximum quantum efficiency of photosystem II (Fv/Fm) that corresponds to the photosynthetic activity of these microorganisms was measured using a pulsed (14) Brayner, R.; Barberousse, H.; Hemadi, M.; Djediat, C.; Yepremian, C.; Coradin, T.; Livage, J.; Fievet, F.; Coute, A. J. Nanosci. Nanotechnol. 2007, 7, 2696.
Langmuir 2010, 26(9), 6522–6528
Figure 2. (a) XRD patterns of ZnO nanoparticles (H = 10); (b) HRTEM of ZnO nanorod (growth orientation along c-axis). amplitude modulation (PAM) fluorometer. TEM analyses of microorganism thin sections were used to study the biocide action of ZnO nanoparticles. In this case, the samples were fixed in a mixture containing 2.5% glutaraldehyde, 1.0% acrolein, and 0.1% ruthenium red in a phosphate S€ orengen buffer (0.1 M, pH 7.4). Live/dead tests were conducted with trypan blue molecules. In this test, when the cells are dead, the color changes from green to blue.
Results and Discussion Morphology, Structure, and Surface Charge of ZnO Nanoparticles. Figure 1 shows TEM micrographs of ZnO nanoparticles obtained by varying the hydrolysis ratio (H). There are two factors that influence the size and shape of ZnO materials synthesized by forced hydrolysis in polyol medium: (i) the nature of the protective agent added during ZnO formation and (ii) the hydrolysis ratio. For ZnO prepared without addition of protective agent, using H=2, spherical submicrometer-sized nanoparticles (d = 0.2 ( 0.05 μm), formed by coalescence of spherical ZnO nanoparticles, were observed. After increase of the hydrolysis ratio from 10 to 30, nanorods were formed and the length of these nanorods varies with the hydrolysis ratio (30 < length < 100 nm). At H = 300, particles with crown morphology were also observed. For ZnO prepared with TOPO, using H = 2, very small spherical nanoparticles were obtained (d = 2.0 ( 0.4 nm). At H=10, spherical nanoparticles with narrow size distribution were observed (d=15.0 ( 0.7 nm). For 30
