Nanotoxicology, 2010; Early Online, 1–13

Effect of silver nanoparticle surface coating on bioaccumulation andreproductive toxicity in earthworms (Eisenia fetida)

WILLIAM A. SHOULTS-WILSON1, BRIAN C. REINSCH2, OLGA V. TSYUSKO1,PAUL M. BERTSCH1, GREGORY V. LOWRY2, & JASON M. UNRINE1

1Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, and 2Department of Civil andEnvironmental Engineering, Carnegie Mellon University, Pittsburg, PA, USA

(Received 11 August 2010; accepted 29 October 2010)

AbstractThe purpose of this study was to investigate the effect of surface coating on the toxicity of silver nanoparticles (Ag NPs) soil.Earthworms (Eisenia fetida) were exposed to AgNO3 and Ag NPs with similar size ranges coated with either polyvinylpyrro-lidone (hydrophilic) or oleic acid (amphiphilic) during a standard sub-chronic reproduction toxicity test. No significant effectson growth or mortality were observed within any of the test treatments. Significant decreases in reproduction were seen inearthworms exposed to AgNO3, (94.21 mg kg-1) as well as earthworms exposed to Ag NPs with either coating (727.6 mg kg-1

for oleic acid and 773.3 mg kg-1 for polyvinylpyrrolidone). The concentrations of Ag NPs at which effects were observed aremuch higher than predicted concentrations of Ag NPs in sewage sludge amended soils; however, the concentrations at whichadverse effects of AgNO3 were observed are similar to the highest concentrations of Ag presently observed in sewage sludge inthe United States. Earthworms accumulated Ag in a concentration-dependent manner from all Ag sources, with more Agaccumulating in tissues from AgNO3 compared to earthorms exposed to equivalent concentrations of Ag NPs. No differenceswere observed in Ag accumulation or toxicity between earthworms exposed to Ag NPs with polyvinylpyrrolidone or oleic acidcoatings.

Keywords: Eisenia fetida, silver nanoparticles, reproductive toxicity, bioaccumulation, X-ray absorption spectroscopy

Introduction

Silver nanoparticles (Ag NPs) have become an inte-gral component in a wide array of consumer products(Klaine et al. 2008; Luoma 2008). Products currentlybeing produced that utilize Ag NPs include medicaldevices, food-storage containers, soaps and sanitizers,wound dressings, and fabrics; Ag NPs are frequentlyutilized in polymeric, colloidal, spun and powderforms (Luoma 2008). With greater Ag NP productionand incorporation into consumer products, it isexpected that Ag NPs will provide an increasingcontribution of anthropogenic Ag into the environ-ment (Blaser et al. 2008). Much of the Ag fromfabrics is expected to enter the wastewater streamand eventually treatment plants in dissolved ornano-particulate form (Benn and Westerhoff 2008)where an estimated 90% will be removed from thewaste stream and partitioned to sewage sludge(Blaser et al. 2008; Mueller and Nowack 2008;

Gottschalk et al. 2009). The sludge-associated AgNPs may be disposed of through incineration, land-filling or application to terrestrial environments asbiosolids, depending on the prevailing practices ofthe country or region (Mueller and Nowack 2008). Inthe United States and much of Europe, approximately60% of sewage sludge is applied to agricultural landsas biosolids (United States Environmental ProtectionAgency [USEPA] 2009). Thus, this represents apotential exposure pathway to terrestrial organismsof high significance.Despite increased production of Ag NPs, relatively

little is known about their environmental fate andpotential effects, particularly in terrestrial environ-ments (Klaine et al. 2008; Unrine et al. 2008). Thetoxicity of Ag NPs has thus far been mainly studied inthe aquatic environment, with toxicity demonstratedfor bacteria (Morones et al. 2005; Panacek et al. 2006;Choi et al. 2008; Choi and Hu 2008), yeast(Panacek et al. 2009), paramecia (Kvitek et al.

Correspondence: Dr Jason Unrine, Department of Plant and Soil Sciences, University of Kentucky, N212-N Agricultural Science Center North, Lexington, KY40546, USA. Tel: +1 859 257 1657. Fax: +1 859 257 3655. E-mail: jason.unrine@uky.edu

ISSN 1743-5390 print/ISSN 1743-5404 online � 2010 Informa UK, Ltd.DOI: 10.3109/17435390.2010.537382

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2009), algae (Griffitt et al. 2008; Navarro et al. 2008),daphnia (Griffitt et al. 2008), Japanese medaka(Chae et al. 2009), fathead minnow embryos(Laban et al. 2010), and various zebrafish life stages(Lee et al. 2007; Asharani et al. 2008; Griffitt et al.2008; Yeo and Kang 2008; Bar-Ilan et al. 2009). Inthe terrestrial environment, Ag NPs have not beeninvestigated to such an extent and Ag toxicity itselfis not well studied (Ratte 1999; Nahmani et al.2007b). The soil nematode Caenorhabditis elegans(Rhabditidae, Maupas 1900) has been used to assessAg NP toxicity but these experiments were performedin K-medium (NaCl and KCl solution) rather than insoils (Roh et al. 2009). To address the need for anunderstanding of the terrestrial toxicity of Ag NPs, thecurrent study investigated the toxicity of Ag NPs insoil to a model soil organism, the epigeic earthwormEisenia fetida (Lumbricidae, Savigny 1826).Another aspect of Ag NP toxicity that has not been

extensively examined is the role that the NP surfacechemistry (coating) plays in toxicity. Several studieshave shown differences in toxicity (Griffitt et al. 2008;Navarro et al. 2008; Bar-Ilan et al. 2009; Kvitek et al.2009; Panacek et al. 2009) and transcriptomicresponses (Roh et al. 2009) between Ag NP andAgNO3, with ionic forms typically exhibiting greatertoxicity. However, Ag NPs have different surfacechemistries depending on coating or functionaliza-tion, which may alter their toxicity and/or mode ofaction. For instance, Ahamed et al. (2008) found thatAg NP particles coated with polysaccharidescaused greater damage to mammalian cell linesthan similarly-sized Ag NP that were uncoated. Pre-vious studies also suggest that Ag NPs stabilized withsurfactants cause greater toxicity to microorganismsthan unmodified Ag NPs (Kvitek et al. 2009;Panacek et al. 2009). In contrast, zerovalent ironnanoparticles have been shown to be less toxic tobacteria when coated with polymers or natural organicmatter (Li et al. 2010).It is unclear if these differences in surface chemistry

are important determinants of toxicity in environmen-tally realistic exposure scenarios, such as in soil. Forexample, it is widely acknowledged that naturallyoccurring colloids in soil environments are coatedwith substances such as iron oxohydroxides and nat-ural organic matter (Bertsch and Seaman 1999).Therefore, engineered surface coatings may be exten-sively modified in the natural environment, and theproperties of pristine coatings may become lessimportant in terms of bioavailability and toxicity.Furthermore, if toxicity is primarily related to therelease of free Ag ions, then surface coatings mayonly be important as they relate to oxidation anddissolution.

The objectives of this study were: (i) To determineconcentrations of Ag (both ionic and nanoparticulate)that caused adverse effects on E. fetida growth, mor-tality and reproduction, and (ii) to determine whetherAg NP toxicity could be influenced by surface chem-istry. In order to achieve this, we performed standard-ized reproductive toxicity tests (Organisation forEconomic Cooperation and Development [OECD]2004) on E. fetida exposed to various concentrationsof ionic Ag and AgNPs in artificial soil media. The AgNPs were coated with either polyvinyl pyrolidone(PVP) or oleic acid (OA). The PVP-coated Ag NPsare hydrophilic, primarily forming stable suspensionsin polar solvents (Yan et al. 2009), while OA-coated Ag NPs are amphiphilic, forming stable sus-pensions in polar solvents, non-polar solvents orpolar/non-polar interface layers depending on pH(Li et al. 2002; Seo et al. 2008). The log Kow valuesof PVP and OA are �3.4 and 7.73, respectively(BASF 2008; International Programme on ChemicalSafety [IPCS] 2010; in press). Therefore, comparingthe two should provide an understanding of theimportance of surface chemistry in Ag NP toxicityto E. fetida under an environmentally relevant expo-sure scenario.

Materials and methods

Material characterization

Silver powders, each with a stated nominal sizedistributions of 30–50 nm, with polyvinylpyrolidone(PVP) and oleic acid (OA) coatings were purchasedfrom NanoAmor (Nanostructured & AmorphousMaterials, Inc; Houston, TX, USA). The powderswere both high purity (>95% Ag), which we verifiedby dissolving in concentrated, ultra-high purityHNO3 and analyzing for metallic impurities usinginductively coupled plasma mass spectrometry (ICP-MS; see description of methods below). Primaryparticle diameter distributions were determinedusing transmission electron microscopy (TEM) asdescribed in detail in (Shoults-Wilson et al. 2010;in press). A Philips BioTWIN 12 Transmission Elec-tron Microscope (FEI Company; Hillsboro, OR,USA) was used to examine the particles, at23,000–6,800� magnification and an acceleratingpotential of 100 kV. Particle diameter determina-tions via TEM were statistically analyzed based onmeasurements of at least 100 particles per materialfrom at least three separate micrographs using imageanalysis software (NIS Elements, Nikon, Tokyo,Japan). The hydrodynamic radii of the particles instock suspensions (in deionized water) used to dosethe soils were determined by dynamic light scattering

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(DLS) using a Malvern Zeta-Sizer Nano-ZS (Mal-vern Instruments; Worcestershire, UK). Intensityweighted distributions were transformed to volumeweighted distributions using the complex refractiveindex components 0.135 (real) and 3.99 (imaginary).The same instrument was used to determine appar-ent zeta potential in stock suspensions using phaseanalysis light scattering (PALS). The mass weightpercentage of surface coating material was deter-mined using thermogravimetric analysis (TGA;SDTQ600, TA Instruments, New Castle, DE,USA). A correction for moisture content wasmade by determining mass loss upon heating to120�C. The mass loss due to the coatings was deter-mined after heating to 700�C.

Characterization of Ag speciation in soil

X-ray absorption spectroscopy (XAS; specificallyextended X-ray absorption fine structure, or EXAFS)was conducted at Stanford Synchrotron RadiationLaboratory (SSRL; Menlo Park, CA USA) wigglermagnet beamline 11-2. Compounds and methodsused to perform XAS analysis of Ag NPs in soilhave been previously described in detail (Reinschet al. 2010; Shoults-Wilson et al. 2010). Briefly, AgK-edge (25.514 keV) XAS spectra were collected overthe energy range 25.284–26.635 keV at room tem-perature. Data were analyzed using SixPACK soft-ware version 0.67 (Webb 2005). X-ray absorptionscans of related samples were calibrated for changesin assigned monochromator energy to 25.514 eV byusing the first derivative of the Ag calibration standardand then averaging multiple scans. Background sub-traction was performed using SixPACK with E0

defined as 25.530 keV, and R-background = 1.0 Å.The resulting spectra were converted to frequency (k)space and weighted by k3 (Webb 2005). The Linearleast-squares combination fitting (LCF) procedureemployed was adapted from Kim et al. (2000)and Reinsch et al. (2010) whereby only componentsthat contributed significantly (a > 10% decrease infitting correlation based on reduced Chi squaredvalue) to the fit were included.EXAFS spectra were collected for a library of

model compounds including, Ag0, Ag2S, AgO,Ag2O, AgPO4, AgCO3, AgSO4, and AgCH3CO2

and were used in LCF analysis. To help constrainthe possible number of Ag species, we also analyzedboth stock Ag NP powders for total S content using ahigh temperature combustion sulfur analyzer. Themethod was verified when we observed excellentagreement between theoretical and measured valuesfor high purity Ag2S and AgSO4 samples.

Sub-chronic and reproductive toxicity test

The design of the earthworm reproduction, bioaccu-mulation and subchronic lethality assays followedpublished guidelines (OECD 2004). The artificialsoil medium consisted of 69.58% dry mass quartzsand, 0.43% dry mass crushed limestone, 20% drymass kaolin clay, and 10% dry mass sphagnum peatmoss (sieved to < 2 mm). We selected a range of AgNP exposure concentrations based on range findingstudies where citrate coated Ag NPs did not causesignificant mortality in E. fetida at concentrations ashigh as 40 mg kg-1 (Unrine et al. unpublished data).To treat dried soils, NPs were first suspended in18.2 MW DI water through sonication. We sonicated1 L of 4 g L-1 of the particles in an ultrasonic bath in1 L glass media bottles FS20 Ultrasonic Cleaner,Fisher Scientific, Fairlawn, NJ, USA). The resultantsuspensions were applied to soils at a rate of 50% ofthe moisture holding capacity (approximately 26% v/w). The nominal concentrations of the soils were 10,100 and 1000 mg Ag kg-1 dry soil. A separate treat-ment added dissolved AgNO3 to nominal concentra-tions of 10 and 100 mg Ag kg-1 dry soil. Threereplicate exposure containers of soil (750 g) wereprepared for each treatment and allowed to equili-brate in an incubator overnight.Ten adult earthworms with fully developed clitella

were added to each exposure chamber. Earthwormmasses ranged from 0.243–0.752 g and averaged0.450 ± 0.101 g after being gently cleaned on moist-ened paper towels. The animals were maintained inan environmental chamber at 20�C with 12 h of lightper day to mimic a realistic diurnal cycle and facilitatereproduction. After 24 h of exposure, 5 g of a 50/50mixture of dehydrated alfalfa and commercially avail-able chicken feed was added to the surface andhydrated with 5 mL of DI water. This feed mixturehad previously been shown to allow rapid, sustainedgrowth in the earthworms. At the end of each week ofexposure, earthworms were observed for mortalityand another 4 g of feed mixture was spread on thesurface and hydrated. Container weights were mon-itored and water was added on a biweekly basis tomaintain weight. After 28 days of exposure, earth-worms were observed for mortality, gently cleaned onpaper towels, massed and placed on wet filter paperinside Petri dishes. The earthworms remained in thedishes for 24 h to void their guts and were thensacrificed. An additional 5 g of food and 5 mL ofwater were added to the containers and they wereincubated for an additional 28 days, with weekly soilmoisture replenishment. The soil was manuallysearched and cocoons were collected and counted.All cocoons were immediately placed in water and

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whether or not they floated [a method of determininghatching success (OECD 2004)] was recorded.

ICP-MS analyses

To determine Ag concentration, whole earthwormsand ~ 0.25 g soil samples were digested in concen-trated trace metal grade nitric acid using aMARS Express microwave digestion system (CEM,Matthews, NC, USA) according to U.S. EPA method3052 (USEPA 1996). Total Ag concentrations in thedigestates were determined by ICP-MS followingU.S. EPA method 6020A (USEPA 1998), using anAgilent 7500 CX (Agilent Technologies; Santa Clara,CA, USA). Digestion sets included standard refer-ence tissues (DOLT-4, National Research Council ofCanada; Ottawa, ON, Canada) or soils directly spikedwith a known mass of the OA and PVP Ag NPs.Duplicate digestions and reagent blanks were alsoincluded with each digestion set. Analytical runsincluded duplicate dilutions, spike recovery samples,and inter-calibration/cross-calibration verificationsamples. The instrument was recalibrated after thefirst 10 samples and every 20 samples thereafter.Previous experiments demonstrated that the AgNPs dissolved in concentrated HNO3 at room tem-perature (‡ 95% recovery). Recovery of Ag afterdirectly adding Ag NP powder to soils in the digestionvessels was 94.6 ± 2.1%. Mean recovery of tissue Agwas 117.3% from DOLT-4. Spike recovery averaged98.5% and the mean relative percentage difference(RPD) between replicate subsamples of soil was1.4%. Concentrations in earthworms exposed toclean soil as controls did not exceed the methodquantification limit (MQL: 0.217 ng mL-1). Threeearthworms exposed to low (10 mg kg-1 nominal)concentrations of Ag also did not exceed the MQLand were not considered for data analysis. All con-centrations are expressed in terms of Ag.

Statistical analyses

All data were log transformed before analysis toimprove normality and homogeneity of variance. Nor-mality and homogeneity of variance of the data weretested using Shapiro-Wilk’s test and Levene’s test,respectively. Significant differences of a treatment ongrowth, mortality, bioaccumulation and reproductionbetween treatments and controls were tested using aone-way analysis of variance (ANOVA) if normal andhomogenous. Post-hoc multiple comparisons forANOVA were performed using Duncan’s MultipleRange test. Non-normal data were tested using the

Wilcoxon Rank-Sum test. Accumulation of Ag as afunction of Ag concentration in exposure soil wasmodeled by transforming the data in order to producea linear relationship. Linear relationships were thenanalyzed for significance (a = 0.05) using ANOVA.Bioaccumulation factors (BAFs) were calculated bydividing concentrations of Ag found in earthwormtissues by concentrations in exposure soils and com-pared using ANOVA. All calculations were per-formed using Statistical Analysis Software (SASv9.1, SAS Institute, Cary NC, USA).

Results

Material characterization

Primary particle diameter was determined by mea-suring images of the particles taken using TEM. ThePVP coated particles had a slightly larger mean diam-eter (56.35 ± 1.16 nm) than the particles coated withOA (50.60 ± 1.02 nm). The two particles had verysimilar particle size distributions (Figure 1A, 1B) withthe majority of the particles within 10 nm of thenominal (30–50 nm) size range. Dynamic light scat-tering (DLS) was used to determine the size distri-bution of particles in the stock suspensions that wereused to dose the exposure soils. This technique pro-vided similar size distributions to TEM size analysisfor the PVP-coated particles (Figure 1A, 1C). How-ever, the particles coated in OA had a greater per-centage of aggregates in suspension which were aslarge as 200 nm in diameter (Figure 1B, 1D). Theelectrophoretic mobility of particles was found to be�1.9 ± 0.0 mM cm V-1 s-1 for the particles coated inPVP and�2.4 ± 0.1 mMcmV-1 s-1 for particles coatedin OA. The apparent zeta potentials estimated usingthe Hückel approximation (Fka = 1.5) were �35.9 ±0.8 mV for the PVP-coated particles and �45.4 ±1.1 mV for the OA-coated particles. No metallicimpurities were detected in the stock suspensions.The coatings accounted for 6.4 and 1.5 wt % ofthe mass of the PVP and OA powders, respectively.

Characterization of Ag speciation

Over the course of 28 d, Ag NPs did not appreciablyoxidize while in the artificial soil (Table I). Silvernanoparticles exposed only to air were found to bepartially oxidized (Table I, Figure 2A, 2C), withapproximately 14% as Ag2O and 86% as Ag0 forPVP NPs. For OA coated NPs, only the Ag0 com-ponent was statistically significant. Total S analysisruled out significant sulfidation of the particles since

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the PVP Ag NP sample contained only 0.009% S andthe OA Ag NP sample only contained 0.011% S.Approximately 20% of the initial OA coated particlespectrum (Figure 2C) was left as unidentified oxi-dized Ag, because after determining the Ag0 foil wasthe initial and dominant component no other modelcompound, when applied with Ag0, lowered thereduced chi squared value of the overall fit below10% of the reduced chi squared-value of the fit thatonly included Ag0. After aging for 28 d in soil, similarspeciation was observed with the PVP Ag NP soilcontaining 89% Ag0, and the OA Ag NP soil

containing approximately the same percentage ofAg0 as the particles exposed to air alone.

Growth and mortality

There were no significant differences in growthbetween any treatments and the control (Table II).There were no significant differences in growthbetween E. fetida exposed to PVP- or OA-coatedAg NPs. Significant increase in mortality was onlyfound in one treatment (9.331 ± 0.873 mg kg-1 PVP

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Figure 1. Histograms of size classes by frequency percent of total particle volume, as well as cumulative percentage of size classes as part of allnanoparticles. The individual graphs demonstrate particles size of PVP-coated (A) and OA-coated (B) particles counted using transmissionelectron microscope; PVP-coated (C) and OA-coated (D) particle sizes calculated using dynamic light scattering.

Table I. A summary of EXAFS analysis of the chemical form of two Ag NPs with different surface coatings. Ag NPs were analyzed directlyfrom their packaging (exposed only to air) or after aging for 28 d in artificial soil. No oxidation occurred in the soil. Chi2 and reduced Chisquared (RC2) values are provided as a means to quantitatively compare relative goodness of fit between samples.

Coating Exposure %Ag0 %Ag2O %Ag2S Unidentified Ag species Chi square RC2

PVP Air 86 ± 3% 14 ± 3% ND ND 6.656 0.0257

(30–50 nm) Soil 89 ± 3% 11 ± 3% ND ND 430.5 2.290

Oleic acid Air 79.5 ± 0.4% ND ND 20.5% ± 0.4% 2.035 0.0078

(30–50 nm) Soil 78.2 ± 0.5% ND ND 21.8% ± 0.5% 59.96 0.0362

ND, not determined.

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NPs at 28 d; Table III). This concentration was thelowest concentration for that Ag NP and two con-centrations which were one and two orders-of-magnitude higher demonstrated no significant

mortality. Also, the majority of the mortality observedoccurred in only one replicate container. Therefore,this increase in mortality is most likely random andnot the result of Ag toxicity.

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Figure 2. Extended X-ray absorption fine structure spectroscopy (EXAFS) spectra (solid black line) linear combination fitting results (greyline), with the uncertainty for the last digit in parenthesis, for 30–50 nm PVP-coated silver nanoparticles (Ag NPs) exposed to air only (A), andaged for 28 d in artificial soil (B); results for 30–50 nm oleic acid-coated Ag NPs exposed to air only (C) and aged for 28 d in artificial soil (D).Fitted models are a combination of spectra from Ag0 (dashed line) and Ag2O (dotted line) standards. The fitted model in Panel C is dashed tobetter show the underlying data. The quantitative fitting results are also displayed, including goodness of fit analyses, in Table III.

Table II. Measurements of earthworm growth at different exposure concentrations expressed as mean values plus or minus standard error.

Ag form Surface coating [Ag]soil (mg kg-1) Initial mass (g) Final mass (g) Growth (g)

None NA 0 0.477 ± 0.014 0.674 ± 0.053 0.197 ± 0.045

Ionic NA 8.38 ± 1.26 0.454 ± 0.007 0.766 ± 0.024 0.313 ± 0.021

94.12 ± 3.21 0.438 ± 0.028 0.578 ± 0.028 0.140 ± 0.015

NP PVP 9.33 ± 0.87 0.417 ± 0.011 0.688 ± 0.032 0.271 ± 0.021

79.45 ± 1.02 0.494 ± 0.026 0.718 ± 0.052 0.224 ± 0.030

773.3 ± 11.2 0.416 ± 0.011 0.680 ± 0.037 0.264 ± 0.035

NP OA 8.03 ± 0.30 0.457 ± 0.031 0.720 ± 0.010 0.264 ± 0.021

81.62 ± 2.90 0.446 ± 0.010 0.683 ± 0.020 0.238 ± 0.031

727.6 ± 34.0 0.430 ± 0.019 0.693 ± 0.035 0.262 ± 0.023

NP, Nanoparticle; PVP, Polyvinylpyrolidone; OA, Oleic acid.

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Bioaccumulation

Tissue concentrations of Ag measured at the end ofthe experiment in earthworms exposed to AgNO3 andAg NPs were Ag concentration-dependent (Figure 3).E. fetida exposed to Ag at similar concentrationsaccumulated significantly higher concentrations ofAg when exposed to AgNO3 compared to either ofthe Ag NPs. Bioaccumulation factors were also higherfor AgNO3 than for Ag NPs (Figure 4). The BAFsrecorded varied depending on the exposure concen-tration, with lower BAFs typically resulting fromgreater exposures. At all of the concentrations, accu-mulation was very similar between the two Ag NPs,and at comparable exposure concentrations therewere no significant differences in tissue concentration

or BAF between the two surface functionalizedmaterials.

Reproduction

The number of cocoons produced per earthwormdecreased in a concentration-dependent mannerfrom exposure to AgNO3 and both AgNPs (Figure 5).Exposure to 94.12 ± 5.56 mg kg-1 AgNO3 caused asignificant decrease in cocoon production that wasnot observed in treatments of similar concentrationsof Ag NPs. Only at concentrations as high as 773.3 ±11.2 mg kg-1 (PVP Ag NPs) and 727.6 ± 34.0 (OA AgNPs) was a significant decrease in cocoon productionobserved for organisms exposed to Ag NPs. At

Table III. Mortality of E. fetid a over the course of the experiment. Values that are significantly different (p < 0.05) than that of the control aremarked by *.

Ag form Surface coating [Ag]soil (mg kg-1)% Survival

Day 7% SurvivalDay 14

% SurvivalDay 21

% SurvivalDay 28

None NA 0 100% 100% 100% 93.0%

Ionic NA 8.38 ± 1.26 100% 100% 100% 96.7%

94.12 ± 3.21 100% 96.7% 96.7% 96.7%

NP PVP 9.33 ± 0.87 95.0% 90.0% 90.0% 85.0%*

79.45 ± 1.02 100% 96.3% 96.3% 89.3%

773.3 ± 11.2 100% 100% 100% 100%*

NP OA 8.03 ± 0.30 100% 100% 100% 90.0%

81.62 ± 2.90 100% 100% 100% 100%

727.6 ± 34.0 100% 100% 100% 100%*

NP, Nanoparticle; PVP, Polyvinylpyrolidone; OA, Oleic acid.

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Figure 3. Ag accumulated by E. fetida after 28 days as a function of the exposure concentration of Ag in the soil. Tissue and soil concentrationsof Ag were transformed to provide a significant (p < 0.05) linear relationship in Panel A. The untransformed data are shown in panel B. All errorbars represent standard error. . = Ag+ (r2 = 0.9875; m = 0.2900);! = Ag NP (PVP) (r2 = 0.9937; m = 0.2226);r = Ag NP (OA) (r2 = 0.9842;m = 0.2196). a–e: Indicates groups of values that are significantly (p < 0.05) different from groups marked with a different letter.

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no concentration was cocoon production significantlydifferent between earthworms exposed to the twodifferent Ag NPs. Rate of cocoon hatching was notsignificantly affected by any Ag treatment when com-pared to the controls.

Discussion

Toxicity of silver to Eisenia fetida

The present study found no concentration-dependentchanges in growth or mortality in E. fetida causedby AgNO3 exposure up to 94.12 ± 5.56 mg kg-1or

Ag NP up to 791.7 mg kg-1, although in the case ofAgNO3, there was a non-significant decrease ingrowth. In any case, the concentrations of AgNO3

used in the present study were not sufficient to causesignificant mortality in E. fetida. We did find signifi-cant, concentration dependent Ag accumulation andreproductive toxicity was observed in earthwormsexposed to AgNO3 and Ag NPs at the highest con-centrations tested for each treatment. The concentra-tions at which we observed reproductive toxicity wasorders of magnitude higher than currently predictedconcentrations of Ag NPs in sewage sludge (up to6.24 mg kg-1; Gottschalk et al. 2009). However, thehighest total Ag concentrations found in sewage sludgein the United States (82�195 mg kg-1; USEPA 2009)are similar to the Ag concentrations from AgNO3 atwhich we observed reproductive impairment. Actualconcentrations in agricultural soil would be somewhatlower depending on the actual application rate ofsewage sludge biosolids and plowing practices. Inforested soils, there may be little or no mixing ofbiosolids into the soil profile. Ag may also accumulatein the soil over long periods of repeated application.Silver ions have long been known to be toxic to a widevariety of aquatic organisms, with fewer studiesinvestigating toxicity to terrestrial organisms (Ratte1999). Earlier studies with the earthworm speciesLumbricis terrestris (Lumbricidae, Linnaeus 1751)demonstrated decreased growth but no significantmortality or even bioaccumulation of silver (Ratte1999). The studies reviewed by Ratte (1999) appearto imply that earthworms do not accumulate or suffertoxic effects from Ag.One previous study also found a significant rela-

tionship between total concentrations of Ag in con-taminated soil and E. fetida bioaccumulation, growth,mortality and reproduction (Nahmani et al. 2007a).The highest Ag concentration that the organisms wereexposed to in that study was 64.4 mg kg-1, which isless than the highest concentration used in the presentstudy. However, the soils investigated by Nahmaniet al. (2007a) varied widely in composition and con-tained multiple potentially toxic metals, potentiallysupplementing Ag toxicity. The study itself found astrong correlation between soil characteristics (suchas soil pH and % sand), other metals (such as Pb andCd) and toxic effects (Nahmani et al. 2007a). There-fore, it is difficult to determine whether this previouswork provides unambiguous evidence for the toxicityof Ag to E. fetida. A related study found a significantcorrelation between Ag concentration in soil pore-water and Ag uptake constants (Nahmani et al. 2009).However, the kinetics of Ag uptake by E. fetida couldnot be well described by multiple regression models.Overall, the results of Nahmani et al. (2007a, 2009)

[Ag] kg mg-1 1

Co

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rm

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7

AgNO3Ag NP (PVP)Ag NP (OA)Control MeanControl SE

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*

**

100010010

Figure 5. The number of cocoons produced per individual E. fetidaat the end of the 28-day exposure period at each exposure con-centration. The mean cocoons/earthworm produced by controlanimals is provided as a benchmark. All error bars representstandard error. . = Ag+; ! = Ag NP (PVP); r = Ag NP (OA).*Indicates a mean values that are significantly (p < 0.05) differentthan those of the controls.

0.00

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0.10

0.12AgNO3

Ag NP (PVP)Ag NP (OA)

a

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e e

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F (

[Ag

] Tis

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/[A

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oil)

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Nominal [Ag]Soil (mg kg-1)

bb

Figure 4. Eisenia fetida bioaccumulation factors (BAFs) for Agcompared to exposure concentrations. Because BAF values canvary with exposure, values are grouped by nominal exposure con-centration. a–e: Indicates groups of values that are significantly(p < 0.05) different from groups marked with a different letter.

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agree with our finding that earthworms exposed to Agaccumulate it and suffer toxic effects.

Toxicity of silver ions versus silver nanoparticles

In the present study, AgNO3 was found to be sig-nificantly more toxic to E. fetida than two Ag NPs.E. fetida exposed to AgNO3 accumulated signifi-cantly more Ag and in some cases had significantlydecreased reproduction than those exposed tocomparable concentrations of Ag NPs. This findingis similar to the conclusions of previous aquatictoxicity studies. Kvitek et al. (2009) found thatAgNO3 was more toxic to Paramecium caudatumthan several different Ag NPs stabilized by surfac-tants. A similar study found that Ag NPs stabilizedby surfactants had antifungal activity similar to thatof AgNO3 but that ionic AgNO3 was more toxic thannon-stabilized Ag NPs (Panacek et al. 2009). AgNO3

also caused a greater decrease of photosynthesis inthe algae Chlamydomonas reinhardtii than Ag NPs(Navarro et al. 2008). With regards to metazoans,dissolved AgNO3 has been shown to be more toxicthan some Ag NPs to adult zebrafish (Danio rerio)and adult Daphnia pulex, while the reverse was truefor juvenile D. rerio and neonate Ceriodaphnia dubia(Griffitt et al. 2008). This latter study also concludedthat dissolved Ag could not account for all Ag NPtoxicity. Another study that used D. rerio embryos asa model found a lower LC50 for AgNO3 than AgNPs of various sizes (Bar-Ilan et al. 2009). Similarly,a study on the embryos of fathead minnows(Pimephalas promelas) found AgNO3 to be more toxicthan Ag NPs (Laban et al. 2010) although onceagain, dissolved Ag could not account for all AgNP toxicity. Overall, the body of evidence fromaquatic toxicity studies supports our finding thatAg ions are more toxic than Ag NPs.Some studies have drawn the opposite conclusions.

Exposure to certain Ag NPs has been shown toproduce significantly more inhibition of nitrifyingbacteria (Choi et al. 2008) and a slight decrease inthe reproduction of nematodes (Caenorhabditis ele-gans) compared to AgNO3 (Roh et al. 2009).C. elegans also showed up-regulation of differentgenes when exposed to ionic Ag than when exposedto Ag NPs, indicating again that Ag NP toxicity maynot result entirely from exposure to dissolved Ag.However, this C. elegans study was conducted inK-medium containing high Cl- concentrations (83mM) that would have caused precipitation of Ag asAgCl, limiting Ag ion bioavailability. Such high Cl-

concentrations are not representative of the majorityof soil solutions. A study by Chae et al. (2009) found

increased mortality in Japanese medaka (Oryziaslatipes) exposed to AgNO3 than Ag NPs at similarconcentrations, although they did not quantify dis-solved Ag in the AgNP exposures which was mixed by1 h of sonication and two weeks of stirring. Longperiods of oxygen-saturated suspension in water canresult in significant dissolution of Ag NPs (Liu andHurt 2010). Japanese medaka also exhibited differentpatterns of biomarker responses between AgNO3 andAg NP exposures, supporting the theory that Ag NPshave a mode of toxicity separate from dissolved Ag(Chae et al. 2009). Taking all available informationinto consideration, while dissolved Ag is typicallymore toxic than Ag NPs, it is unclear whether itaccounts for all Ag NP toxicity.The varying responses to Ag ions and Ag NPs may

be the result of comparing endpoints in different testspecies, in different exposure media. It may also bedue to the different sizes and surface chemistries ofthe Ag NPs under investigation. In studies that do notanalyze dissolved Ag in Ag NP exposures, toxicitybetween the two sources of Ag may also be conflated.Few previous studies have been conducted in soilmedia or on organisms other than bacteria, makingcomparisons with the present study difficult. Inenvironmentally realistic exposure scenarios, whereaggregation and dissolution are expected to play animportant role and particle surfaces are extensivelymodified, particle specific toxicity may be less impor-tant. Our EXAFS studies have demonstrated thatonly 11�20% of the Ag was oxidized after 28 d inthe experimental soil for PVP-Ag NPs and OA-Ag NPs, respectively. This is almost identical to thestate of the particles prior to introduction to the soil,making it apparent that little additional oxidation tookplace during the exposure.Scheckel et al. (2010) found that both coated and

uncoated Ag NPs exhibited similar rates of oxidationand sorption in a kaolin reaction system over18 months. They also observed a lack of transforma-tion in the presence of NaNO3, but in the pre-sence of Cl-, significant transformation occurredover 18 months. The approximately 8-fold greatertoxicity of AgNO3 appears to correlate with the11�20% of the Ag NPs that are oxidized to Ag2O.This suggests that oxidation and the possible releaseof free Ag ions plays an important role in toxicity,although a role of intact oxidized particles cannot beruled out. For example, the EXAFS data suggestedthat the PVP-Ag NP containing soil contained 11%Ag2O, however, it is possible that this fraction ofAg2O may dissolve in the earthworm gut or mucous,resulting in either dietary or dermal exposure. Fur-thermore, intact particles may have been taken upwith subsequent dissolution within the tissues

Effects of silver nanoparticles on earthworms 9

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(Unrine et al. 2008). Concentrations of dissolved Agions in pore waters were too low to be measured usingeither ion specific electrodes or ultrafiltration (3 kDa)followed by ICP-MS (MDL = 0.05 mg L-1).E. fetida accumulated higher concentrations of Ag

when exposed to AgNO3 than when exposed to AgNPs. However, bioaccumulation does not appear tobe a function solely of Ag ions. If only the oxidizedportion of Ag NPs aged in test soils, one would expectat least 9- to 12.5-fold less accumulation of Ag NPsrelative to AgNO3. On the contrary, the present studyobserved only 2.5- to 5.5-fold less accumulation fromAg NP-treated soils relative to AgNO3 treated soils.This indicates that dissolved ions cannot entirelyaccount for the Ag accumulated by earthwormsexposed to Ag NPs, even when accounting fordecreasing BAFs with increasing exposure concentra-tion. While the principle route of metal uptake byearthworms is dermal absorption of dissolved ions,studies have found that a significant amount of metalis taken up through the digestive tract (Saxe et al.2001; Vijver et al. 2003). Other recent studies haveindicated that earthworms may take up intact metalnanoparticles (Unrine et al. 2008, 2010). Therefore,while toxicity of Ag NPs may be primarily related toAg oxidation and dissolution, earthworms may accu-mulate particles which increase their tissue concen-trations of Ag without appreciably contributing totoxicity. However, Petersen et al. (2008) have esti-mated that any BAF below 0.0315 ± 0.0001 calculatedfor E. fetida could result from incomplete voiding ofgut contents over 24 h. Because none of the BAFs forAg NPs calculated in the present study exceeded0.0315, it may be difficult to distinguish Ag NPbioaccumulation from Ag NPs retained in the gutfollowing voiding of gut contents. Future studies ofAg NP toxicity and accumulation should account foroxidation and dissolution of the Ag NPs in order toexplain potential toxicity from Ag ions and accumu-lation of Ag ions and Ag NPs.

Influence of surface coating on silver nanoparticle toxicity

The present study found no difference in toxicityendpoints between organisms exposed to similarly-sized Ag NPs coated with polyvinylpyrrolidone (PVP)and oleic acid (OA). The primary difference betweenthe two surface coatings investigated in the presentstudy was in their hydrophobicity, with PVP providinga hydrophilic coating as opposed to more hydropho-bic behavior of OA-coated NPs (Li et al. 2002;Seo et al. 2008; Yan et al. 2009). Because hydropho-bicity is often correlated to bioavailability, Ag NPbioaccumulation and toxicity would be predicted to

differ based on coating properties. Previous studieshave primarily made comparisons between non-coated Ag NPs and those functionalized by materialsthat produce stable suspensions (e.g., Ahamed et al.2008; Kvitek et al. 2009; Panacek et al. 2009). Forinstance, Ag NPs modified with a surfactant(TWEEN 80) and PVP caused more toxicity to Par-amecium caudatum than unmodified particles, whileparticles modified with polyethylene glycol causedsimilar toxicity compared to the unmodified particles(Kvitek et al. 2009). Likewise, Panacek et al. (2009)found increased antifungal activity from Ag NPsstabilized by surfactants and PVP than from AgNPs that were not stabilized. The authors spec-ulated that by disrupting cell membranes, surfactantsprovided Ag NPs with an easier entry into cells, thusincreasing their toxicity over unmodified particles.Similarly, a study using mammalian cell lines foundevidence for greater apoptosis and DNA damage incells exposed to polysaccharide (acacia gum) coatedAg NPs and those that were uncoated (Ahamed et al.2008).Surface coating has been shown to influence tox-

icity and uptake of Cd/Se and Cd/Te quantum dots bya murine macrophage cell line (Clift et al. 2008).Quantum dots coated in a hydrophobic mixture oforganic compounds caused significant toxicity thatwas not caused by carboxyl and amino polyethyleneglycol coated particles. A study conducted on Fe0

NPs has likewise demonstrated that uncoated parti-cles were more toxic to Escherichia coli than coatedparticles due to fewer direct interactions betweenbacterial cells and coated particles (Li et al. 2010).They also found some differences in toxicity betweenNPs with different coatings, with coatings that pro-duced thicker layers around the NP being less toxic.These studies indicate that the surface modification ofnanoparticles plays a large role in interaction withcells and, therefore, toxicity. However, all involvedsimplified, primarily aqueous systems and assessedtoxicity using unicellular organisms or mammaliancell cultures. It is not clear that surface coatings wouldhave such a strong effect on toxicity in complexenvironmental systems, especially when nanoparticleswould be expected to be coated in organic matter orclay minerals through natural processes (Bertsch andSeaman 1999; Li et al. 2010).One oversight of many previous studies of surface

coating effects on Ag NP toxicity is a failure tocharacterize Ag dissolution. This is an importantconsideration, because surface coating may affectthe rates of oxidation and dissolution of Ag. Giventhe known toxicity of Ag ions it seems prudent toattempt to disprove the hypothesis that Ag NP toxicityis primarily caused by release of free Ag ions before

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claiming that Ag NPs cause particle-specific toxicity.Because Ag ions are readily sorbed to reactive mineralsurfaces and high molecular weight organic matter,we were unable to directly measure Ag dissolution inthe soils; however, solid-state EXAFS analysis of thebulk particles and soils suggested that the two parti-cles were not chemically equivalent. The PVP-coated particles were oxidized to a lesser extentthan OA-coated particles. This indicates that surfacefunctionalization of Ag NPs can impact the chemicalstate of the Ag NPs. While in theory this should affectthe rate of Ag ion release from the particles andtherefore toxicity, our study found no significantdifference between the two. If Ag ions are the causeof most toxicity, parameters that affect Ag dissolutionsuch as soil pH, soil organic matter and gut pH orpassage time would be expected to be consistentbetween the treatments and therefore lead to similartoxicity. In any event, it is possible that particlesurface coatings may influence fate and transport ofAg NPs, including partitioning of Ag particles tosewage sludge, which could ultimately influenceexposure scenarios. Therefore, studies investigatingthe toxicity of Ag NPs should continue to examine therole of surface functionalization in environmentallyrelevant exposures.

Conclusions

We have shown that the earthworm E. fetida canbioaccumulate Ag from both both AgNO3 and AgNPs. We have also demonstrated that AgNO3 isnearly an order-of-magnitude more toxic to E. fetidathan the two surface functionalized Ag NPs that weexamined. EXAFS analyses found only 11–20% oxi-dized Ag(I) and no further oxidation over time, andcould not rule out a role for Ag ions in toxicity of AgNPs to earthworms. Finally, as a corollary to theprevious conclusion, the relative hydrophobicity ofthe Ag NP surface coatings used in this study didnot appear to affect toxicity or bioaccumulation of Agin E. fetida exposed in soil. This further lends supportto the idea that the observed Ag NP toxicity wasprimarily related to the release of Ag ions and notto nanoparticle-specific toxicity. More research intoAg NP toxicity in terrestrial systems is warranted, inparticular the effects of long-term accumulation andaging processes on Ag NP toxicity in soil are needed.

Acknowledgements

The authors wish to acknowledge the assistance of M.Lacey, E. Harding, J. Backus, J. Song, O. Zhurbich

and two anonymous reviewers. Portions of thisresearch were carried out at the Stanford SynchrotronRadiation Laboratory, a national user facility operatedby Stanford University on behalf of the U.S. Depart-ment of Energy, Office of Basic Energy Sciences. Wethank Dr C. Kim and the members of his Environ-mental Geochemistry Lab of Chapman University forsample preparation and analysis support. Also, thanksto the staff and beamline scientists at SSRL.

Declaration of interest: Major funding for thisresearch was provided by the United States Environ-mental Protection Agency (U.S. EPA) through Sci-ence to Achieve Results Grant # RD 833335 andthrough support of the National Science Foundation(NSF) and U.S. EPA for the Center for Environmen-tal Implications of Nanotechnology (CEINT). It wasalso funded in part by the National Science Founda-tion (BES-0608646 and EF-0830093), and the U.S.EPA R833326. The authors report no conflict ofinterest. The authors alone are responsible for thecontent and writing of the paper.

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