EFFECTS OF SILVER NANOPARTICLES ON ZEBRAFISH (DANIO RERIO) AND ESCHERICHIA COLI (ATCC 25922): A COMPARISON OF TOXICITY BASED ON TOTAL SURFACE AREA VERSUS MASS CONCENTRATION OF PARTICLES IN A MODEL EUKARYOTIC AND PROKARYOTIC SYSTEM CHRISTOPHER R. BOWMAN, y FRANK C. BAILEY, y MATTHEW ELROD-ERICKSON, y ARIANNE M. NEIGH, z and RYAN R. OTTER *y yDepartment of Biology, Middle Tennessee State University, Murfreesboro, Tennessee, USA znanoComposix, Inc, San Diego, California, USA (Submitted 3 February 2012; Returned for Revision 7 March 2012; Accepted 30 March 2012) Abstract —Silver nanoparticles (Ag NPs) have been classified as the most abundant NP found in commercial products. In the present study, zebrafish (Danio rerio) and bacteria (Escherichia coli; ATCC 25922) were used to test the size-dependent toxicological effects of Ag NPs, the effects of ionic silver versus Ag NPs, and Ag NP effects on mortality using mass concentration (mg/L) compared with total surface area (nm 2 /L). Several diameters of Ag NPs (20, 50, 110 nm) as well as AgNO 3 were chosen as experimental treatments. Treated zebrafish embryos exhibited anomalies of the heart, namely, slower heart rates and pericardial edema. A size-dependent response was not observed in zebrafish when viewing mortality across all Ag NP treatments, although 20 nm elicited the highest incidence of abnormal motility and induced slower development. An Ag NP dose- and size-dependent response was observed in treated bacteria using mass concentration, with 20-nm Ag NP producing the highest mortality rate. In both zebrafish and bacteria, AgNO 3 was shown to be more toxic than Ag NPs at equivalent concentrations. When total surface area of Ag NPs was used to gauge bacterial mortality, a total surface area-dependent, but not size-dependent, response was observed for all three Ag NPs used in the present study, with nearly 100% mortality observed once a total surface area of approximately 1E þ 18 nm 2 /L was reached. This trend was not apparent, however, when measuring total surface area for zebrafish mortality. Environ. Toxicol. Chem. 2012;31:1793–1800. # 2012 SETAC Keywords —Silver nanoparticles Total surface area Zebrafish Microbial toxicology Nanotoxicology INTRODUCTION Nanoparticles (NPs) are identified as particles with dimen- sions of 1 to 100 nm [1]. Nanoparticles have been shown to exhibit highly reactive behavior in biological systems because of a relatively large surface-to-volume ratio, resulting in a high percentage of atoms on the surface of the NPs compared with larger structures [2]. The dynamic nature of these particles has proved most useful in many technological applications, including delivering drugs to the cells of cancer patients [3], increasing the efficiency of solar panels [4], and even lowering the emissions and improving the performance of biodiesel engines [5]. It is estimated that by 2015 the nanotechnology economy will be valued at more than $1 trillion [6], but very little is known about the toxicological effects in biological systems. Previous research indicates that NPs may pose a serious risk to people and to the environment [7]. Studies performed in vivo show that NPs can be deleterious to model organisms (e.g., bacteria, algae, invertebrates, and fish) in aquatic systems [8]. Traditionally, researchers have gauged organismal toxicity by evaluating the mass concentration (e.g., mg/L) of various chemicals and compounds, although it has been suggested that mass concentration may not be the most appropriate dose metric for NPs [9]. Given that surface area may play an integral role in NP toxicity, perhaps there is more relevance in quantifying total surface area of NPs in suspension (e.g., nm 2 /L), instead of using only mass concentration. Indeed, in one study investigating the inhalation of ultrafine and fine carbon NPs in rats, researchers found significantly different results when gauging toxicity on a mass concentration basis compared with total surface area [10]. In addition, Oberdorster [11] treated rats with ultrafine and fine TiO 2 NPs and discovered that ultrafine NPs induced a greater neutrophil response when using equivalent mass concentra- tions. However, when toxicity was gauged using total surface area, the toxic response for both ultrafine and fine TiO 2 fit the same dose–response curve. The author of the latter study surmised that total surface area might be a more valid dose metric for assessing toxicity when testing different-sized NPs with the same surface chemistry. Furthermore, researchers studying equivalent mass concentrations of different NPs (e.g., Au, Ag, TiO 2 ) found the corresponding total surface area of these NPs to differ by orders of magnitude [12]. Silver nanoparticles (Ag NPs) have been classified as being the most common nanomaterials to be engineered for commer- cial use [13]. Because of their antimicrobial effects, Ag NPs are frequently used in medicine, water-purification systems, cosmetics, and many consumer products, including washing machines, food containers, and clothing [14]. One study has shown that Ag NPs can be leached into wastewater during clothes washing [15], possibly contaminating bacteria in waste- water treatment facilities. Such leaching could consequently place a risk on aquatic organisms in receiving streams and lakes. Research performed on model organisms, such as zebrafish and Japanese medaka embryos, have shown Ag NPs to induce mortality in a size-dependent manner and to delay hatching [16,17]. Silver nanoparticles have been observed in the brain, testis, liver, and blood throughout embryogenesis of zebrafish Environmental Toxicology and Chemistry, Vol. 31, No. 8, pp. 1793–1800, 2012 # 2012 SETAC Printed in the USA DOI: 10.1002/etc.1881 * To whom correspondence may be addressed (Ryan.Otter@mtsu.edu). Published online 9 May 2012 in Wiley Online Library (wileyonlinelibrary.com). 1793