Particle size, surface charge and concentration ...

Science of the Total Environment 468–469 (2014) 968–976

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Particle size, surface charge and concentration dependent ecotoxicity of three organo-coated silver nanoparticles: Comparison between general linear model-predicted and observed toxicity Thilini Silva a,1, Lok R. Pokhrel a,1,2, Brajesh Dubey b,⁎, Thabet M. Tolaymat c, Kurt J. Maier a, Xuefeng Liu d a

Department of Environmental Health, College of Public Health, East Tennessee State University, Johnson City, TN 37614, United States Environmental Engineering Program, School of Engineering, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada c USEPA, Office of Research and Development, National Risk Management Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH 45224, United States d Department of Biostatistics and Epidemiology, College of Public Health, East Tennessee State University, Johnson City, TN 37614, United States b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Nanotoxicity predicted based on particle properties for three organo-coated AgNPs against Escherichia coli and Daphnia magna. • Particle size, surface charge, and concentration dependent AgNP toxicity were observed for both organisms. • General linear model showed interactive effects of primary particle size and surface charge which explained AgNP toxicity.

a r t i c l e

i n f o

Article history: Received 13 July 2013 Received in revised form 2 September 2013 Accepted 2 September 2013 Available online xxxx Editor: Damia Barcelo Keywords: Organo-coated silver nanoparticles General linear model Nanotoxicology Nanoparticle characteristics Escherichia coli Daphnia magna

a b s t r a c t Mechanism underlying nanotoxicity has remained elusive. Hence, efforts to understand whether nanoparticle properties might explain its toxicity are ongoing. Considering three different types of organo-coated silver nanoparticles (AgNPs): citrate-coated AgNP, polyvinylpyrrolidone-coated AgNP, and branched polyethyleneimine-coated AgNP, with different surface charge scenarios and core particle sizes, herein we systematically evaluate the potential role of particle size and surface charge on the toxicity of the three types of AgNPs against two model organisms, Escherichia coli and Daphnia magna. We find particle size, surface charge, and concentration dependent toxicity of all the three types of AgNPs against both the test organisms. Notably, Ag+ (as added AgNO3) toxicity is greater than each type of AgNPs tested and the toxicity follows the trend: AgNO3 N BPEI-AgNP N Citrate-AgNP N PVPAgNP. Modeling particle properties using the general linear model (GLM), a significant interaction effect of primary particle size and surface charge emerges that can explain empirically-derived acute toxicity with great precision. The model explains 99.9% variation of toxicity in E. coli and 99.8% variation of toxicity in D. magna, revealing satisfactory predictability of the regression models developed to predict the toxicity of the three organo-coated AgNPs. We anticipate that the use of GLM to satisfactorily predict the toxicity based on nanoparticle physico-chemical

⁎ Corresponding author. Tel.: +1 519 824 4120x52506. E-mail address: [email protected] (B. Dubey). 1 These first co-authors contributed equally to this work. 2 Current address: US Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, 200 SW 35th St., Corvallis, OR 97333, United States. 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.09.006

T. Silva et al. / Science of the Total Environment 468–469 (2014) 968–976

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characteristics could contribute to our understanding of nanotoxicology and underscores the need to consider potential interactions among nanoparticle properties when explaining nanotoxicity. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Envisioned as a promising technology touted to offer greater benefits to mankind, nanotechnology has emerged as a cross-disciplinary science of the twenty first century encouraging collaboration among interdisciplinary scientists in a way never witnessed before (Pokhrel and Dubey, 2012). Progress in tuning engineered nanomaterial (ENM) functionality for desired applications has extended ENMs' fields of applications (Costanza et al., 2011), and therefore, a rapid commercialization of nano-enabled products is taking place (www.nanotechproject.org; Tolaymat et al., 2010; National Academy of Sciences, 2012). As nanoenabled products can release chemical(s) in ‘nano’ and/or ionic form into the environment (Impellitteri et al., 2009; Benn et al., 2010; Tolaymat et al., 2010), concerns over environmental contamination and subsequent hazard to the receptor organisms have often been raised (Klaine et al., 2008; Vecitis et al., 2010; Yamashita et al., 2011; Pokhrel et al., 2012; Pokhrel and Dubey, 2012, 2013a; Rahaman et al., 2012). Because aquatic systems can be regarded a major sink for environmental contaminants, systematic investigation of the potential aquatic toxicity of the most common nanomaterial types, such as silver nanoparticles (AgNP), may provide insights into if and how nanoparticles would cause toxicity should the exposure occur. The mechanistic basis of AgNP toxicity to the biotic receptors—both prokaryotic and eukaryotic organisms—has remained less clear as has been the case for other (in)organic nanomaterials (Vecitis et al., 2010; El Badawy et al., 2011; Yamashita et al., 2011; Pokhrel et al., 2012; Barceló et al., 2013). Nonetheless, recent evidence of oxidative stress via generation of reactive oxygen species (Choi and Hu, 2008), direct physical contact leading to membrane perturbation or cell-pitting (Choi and Hu, 2008; Fabrega et al., 2009; El Badawy et al., 2011), and DNA damage (Ahamed et al., 2008) have been documented in the literature following exposure to AgNPs. Less well understood is whether nanoparticles or the associated ionic form is more toxic than the other (Xiu et al., 2012; Pokhrel and Dubey, 2013a), and whether there is any combined effects of the two forms (Pokhrel et al., 2012). Identifying factors enabling our understanding of nanoparticle toxicity has long remained challenging (Xiu et al., 2012; Pokhrel et al., 2013, in review; Pokhrel and Dubey, 2013b). Unclear is how nanoparticle characteristics would interact with the biologic receptor characteristics at the nano-bio interface, as such interaction can potentially influence the toxicity (Vecitis et al., 2010; El Badawy et al., 2011). Amongst the factors identified to mediate nanoparticle toxicity, primary particle size has generally remained central (Choi and Hu, 2008; Jiang et al., 2008; Park et al., 2011); however, it is beginning to be understood that particle dissolution into toxic ions, particle state of aggregation, other transformations that could potentially occur once nanoparticles enter different environments, and surface charge/coating agent could also influence the toxicity and therefore studies suggest considering them during toxicity assessment (Liu and Hurt, 2010; El Badawy et al., 2011; Lowry et al., 2012; Pokhrel et al., 2012, in review; Tejamaya et al., 2012; Xiu et al., 2012). Citrate, polyvinylpyrrolidone (PVP), and branched polyethyleneimine (BPEI) represent, in part, the most commonly employed coating/ stabilizing agents (Tolaymat et al., 2010) as they enable effective nanoparticle dispersion (El Badawy et al., 2012); whilst citrate has been widely used as a reductant for nanoparticle synthesis (El Badawy et al., 2012; Pokhrel et al., 2012). In this study, not only that these coating materials differentially charge AgNPs surface, they also confer stability via distinctly different mechanisms, namely, electrostatic (for citrate-coated AgNPs), steric (for PVP-coated AgNPs), and electrosteric (for BPEI-coated AgNPs). Different primary particle sizes are obtained

employing different methods of synthesis (El Badawy et al., 2012). We systematically evaluate AgNP toxicity using the three different types of organo-coated AgNPs: Citrate-AgNP, PVP-AgNP, and BPEI-AgNP, and characterize for hydrodynamic diameters (HDD), particle morphology (i.e., diameter and shape using transmission electron microscopy (TEM)), state of aggregation, ion release rate, solution pH, and zeta (ζ) potential including that of the biologic receptor surfaces. Our results demonstrate particle size, surface charge, and concentration dependent toxicity of the three different organo-coated AgNPs against both the prokaryotic (Escherichia coli) and eukaryotic (Daphnia magna) organisms. Using the regression method of general linear model (GLM), herein we demonstrate for the first time a significant interactive effect of primary particle size (TEM diameter) and surface charge to satisfactorily explain acute toxicity of the three different organo-coated AgNPs against both the test organisms. Notably, our GLM shows an association between a minimum set of AgNP properties and the biologic responses in E. coli and D. magna.

2. Materials and methods 2.1. Organo-coated silver nanoparticle synthesis and characterization This study considers three different organo-coated silver nanoparticles (AgNPs): citrate-coated AgNP (Citrate-AgNP), polyvinylpyrrolidone-coated AgNP (PVP-AgNP), and branched polyethyleneiminecoated AgNP (BPEI-AgNP), presenting different surface charge scenarios and particle sizes which enabled us to study the potential main effects of the particle size and surface charge, including their interactions, on AgNP toxicity. These AgNPs were synthesized as described previously by El Badawy et al. (2010) (detailed in Supplementary Information), and purified using a tangential flow filtration (TFF) system equipped with 10 kD hollow fiber polysulfone membranes (www.spectrumlabs. com). The purification protocol was previously described in detail (Pokhrel et al., 2012; Pokhrel and Dubey, 2012). The NPs were wellcharacterized as follows: the hydrodynamic diameter (HDD) and zeta (ζ) potential were measured using the dynamic light scattering (DLS) and phase analysis light scattering (PALS), respectively, by a NICOMP particle sizer/zeta potential unit (PSS NICOMP Particle Sizing Systems, CA); the surface plasmon resonances were recorded as absorbance spectra using an UV/vis spectrophotometer (HACH DR 5000, HACH Company, CO); the particles were imaged using a Transmission electron microscope (TEM, Philips EM 420) and analyzed for particle size and shape; and the suspension pH was determined using a pH meter. Samples were digested using ultrapure HNO3 (Method 3050B) (USEPA 1996), and total Ag concentration was quantified using a Graphite furnace-atomic absorption spectrometry (GF-AAS, Varian Spectra 220Z) following the USEPA method 7010 (USEPA, 2007). For comparison, ion-specific toxicity was conducted using AgNO3 (Fisher Scientific, Cat. # S486-100). For nano-bio interactions to commence, the NPs are expected to have some degree of physical contact with the biologic surface which occurs due to attractive or repulsive forces (Vecitis et al., 2010; El Badawy et al., 2011). Estimating organisms' surface charge (as ζ potential) in tandem with particles' surface charge in the test matrix should offer insight into the strength of interaction at the nano-bio interface (Vecitis et al., 2010; El Badawy et al., 2011; Pasquini et al., 2012). To determine ζ potential of D. magna body surface, ten b 24 h old neonates were cleaned several times with moderately hard water (MHW), carefully removed the gastrointestinal tract under a dissecting microscope, washed three times with MHW, then the integument (skin) was resuspended in MHW and disintegrated using a tissue homogenizer

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(Cole-Parmer Ultrasonic Homogenizer, 125 w/cm2, 25 kHz, Cat. # R04717-00) before measuring the ζ potential using PALS. For E. coli, the cells (as recommended by the supplier) were reconstituted in MHW for 15 min, following which its surface charge was recorded using PALS. 2.2. Toxicity bioassays Two different toxicity bioassays very well representing aquatic toxicity were performed, namely, E. coli bioassay and D. magna 48 h survival assay. 2.2.1. Escherichia coli bioassay The potential of three different organo-coated AgNPs to inhibit β-galactosidase enzyme activity was evaluated in E. coli. Chlorophenolred β-galactopyranoside (CPRG) used as a chromogenic substrate is catalyzed at the glycosidic bond by β-galactosidase, forming galactopyranose and chlorophenol red as the reaction products (Bitton et al., 1994; Pokhrel et al., 2012). The details of this bioassay have been described in our previous publication (Pokhrel et al., 2012). Briefly, 100 μL of reconstituted bacteria was added to 900 μL of the sample and incubated at 35 °C for 90 min in a glass test tube. Then 200 μL of this suspension (sample with bacteria) was transferred to a 96 well plate and added 100 μL of CPRG to each well, following which the well plate was incubated at 35 °C for another 90 min; thus the total exposure duration was 3 h. The bacteria were exposed to a wide range of concentrations of organocoated AgNPs (0.01–41.2 mg/L as total Ag), following which enzyme activity was recorded at 570 nm using a microplate reader. The MHW (conductance 560 μS cm−1, hardness 280 mg/L as CaCO3) and 1 mg Cu2+/L (as CuSO4) represented negative and positive controls, respectively (Pokhrel et al., 2012). At least triplicate samples were analyzed for each sample dilution, including the controls. All test solutions were maintained in a narrow pH range of 7.0–7.2. AgNO3 was also used to test ion-specific toxicity. To eliminate the potential effect of coating material on AgNP toxicity, tests were performed for each coating material separately (i.e., sodium citrate dihydrate, PVP, and BPEI) at the highest theoretical concentration present in the nanosuspension. The bacterial reagent and CPRG were obtained from M2B Research & Innovative Technologies, LLC, Gainesville, FL. 2.2.2. Daphnia magna mortality bioassay A static, non-renewable D. magna 48 h bioassay was performed following the standard USEPA guidelines, including the culture and maintenance of the daphnids (USEPA, 1987). Briefly, ten b 24 h old neonates were introduced randomly into each 50 mL test beaker containing AgNP treatment (0.01–40.3 μg/L as total Ag) or the control. As the negative and positive controls, MHW and CuSO4 were used, respectively. Animals were maintained at 20 ± 1 °C with a 16 h photoperiod cycle and were unfed during the test. Triplicate test runs were performed for each sample concentration. Total dead daphnids in each test beaker were recorded at the end of the test. 2.3. Analysis of dissolved silver in AgNP samples The dissolved Ag fraction in three different organo-coated AgNP samples was determined by incubating a 50 mL sample at 20 ± 1 °C in a centrifuge tube (Fisher Scientific, Cat. # 06-443-18) and maintaining a 16 h photoperiod cycle for 48 h—the condition similar to D. magna test described above. Soon the samples were centrifuged for 30 min at ~3150 g (4000 rpm, Thermo Electron, IEC Centra CL3 Series Centrifuge), then 5 mL supernatant was removed for digestion with HNO3 (ultrapure grade) following the method 3050B and analyzed in duplicates for the Ag amount released from each AgNP sample using a GF-AAS (Pokhrel and Dubey, 2012). Typical sample analysis consisted of the sample blanks, internal standards, spiked samples, and sample duplicates; the recovery of Ag was in the range 96–101%.

2.4. Statistical analysis As data satisfied normal probability distribution (Kolmogorov– Smirnov test, p N 0.1 in all cases), they were used untransformed. The EC50 (i.e., effective concentration for 50% enzyme activity inhibition in E. coli) or LC50 (i.e., lethal concentration that kills 50% of D. magna neonates) values were estimated using the linear regression analysis. Comparison of the means of EC50 or LC50 among different treatments was performed using the ANOVA followed by the Dunnett t test (2-tailed post-hoc) for multiple comparisons. The only association that has been quantitatively described previously in “nano” environmental health and safety (EHS) studies has been the use of univariate correlation method (Vecitis et al., 2010; Diedrich et al., 2012; Ma et al., 2012), explaining whether (or not) a given physico-chemical property (e.g., particle size, electron structure) of the model ENM correlates with the observed toxicity. Not pursued previously, but the important questions addressed in this work are whether interactions exist between the physico-chemical characteristics of the three organo-coated AgNPs and how such interactions (should those occur) could explain AgNP toxicity against both the prokaryotic and eukaryotic organisms. Using the general linear model (GLM), herein we address these important questions by investigating the potential main effects of TEM diameter (TEMdia) and surface charge (measured as ζ potential), and the potential interactive effects of TEMdia and ζ potential on the toxicity of the three different organo-coated AgNPs. TEMdia representing the primary particle size and ζ potential measured as a function of surface charge were used as the covariates in the linear model. The GLM-predicted toxicity (as LC50/EC50) of the three types of AgNPs, which was quantified using the parameters, TEMdia and ζ potential, is presented and compared with their experimentally derived LC50 or EC50 values. Any unexplained variance of each model is described by its respective error term (ε), and the precision of the predictive power of each model was determined by the coefficient of determination (R2). Statistical analyses were performed using the PASW (a.k.a. SPSS) Statistics 18.0 (PASW, 2009). 3. Results and discussion The physico-chemical characteristics of the TFF-purified three different organo-coated AgNP samples used in this study are summarized in Table 1. These NPs had primary particle size (TEMdia) within the 100 nm size range, with variable average TEM diameter enabling us for size-dependent toxicity to be evaluated (Table 1). Not only that the three different, yet commonly used, organo-coatings imparted stability to AgNPs by three distinct stabilization mechanisms as previously stated, they also offered variable surface charge scenarios (Table 1), hence allowing us for assessing the potential interactive roles of surface charge and primary particle size on AgNP toxicity against both the prokaryotic (E. coli) and eukaryotic (D. magna) organisms. All three types of organo-coated AgNPs had similar pH and hydrodynamic diameter (HDD) (Table 1), hence excluding their potential roles on AgNP toxicity in this study. The representative TEM images, particle size distributions, and the characteristic surface Plasmon resonance spectra of the AgNP samples are presented in the Supplementary Information (Fig. S1). To evaluate the potential toxicity in E. coli upon exposure to AgNPs, we measured inhibition of β-galactosidase (β-Gal), an extensively conserved metabolic enzyme found in plants, fungi, and animals, including the humans (Taron et al., 1995). In animals, β-Gal catalyzes the conversion of complex sugars (e.g., lactose) to simple sugars (e.g., glucose and galactose) that are utilized as substrates in cellular respiration (Hansen and Gitzelmann, 1975). While in plants, β-Gal activity has been widely associated with ontogenic changes in the tissues such as seeds, seedlings, pollens, and fruits (Figueredo et al., 2011). A significant concentration-dependent β-Gal activity inhibition was observed in E. coli for all the three types of organo-coated AgNPs, including for the free Ag+ (as AgNO3) (Fig. 1). At comparable concentrations, BPEI-


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Table 1 Characteristics of the evaluated organo-coated AgNPs for the toxicity studies. Material

pH

BPEI-AgNP

7.1

Citrate-AgNP

7.2

PVP-AgNP

7.0

Particle size distribution (nm) Hydrodynamic diametera Mean ± S.D.

TEM diameter Mean ± S.D.

Before:10.9 ± 0.8 After: 10.9 ± 0.8 Before:10.9 ± 0.8 After: 11.0 ± 0.7 Before:11.0 ± 0.7 After: 11.0 ± 0.9

10.0 ± 4.6 (n = 63) 56.0 ± 14.0 (n = 98) 72.0 ± 24.0 (n = 57)

Particle circularity

Average zeta potential (mV)

Mechanism of stabilization

0.87

+28.8

Electrosteric

0.88

−20.08

Electrostatic

0.87

−7.49

Steric

a Volume-weighted hydrodynamic diameter measured in the test matrix (moderately hard water) using the DLS method before and after the toxicity tests were conducted; particle circularity of 1 indicates that the particle is a perfect circle in a 2D TEM imagery; PVP-AgNP, polyvinylpyrrolidone-coated AgNPs; Citrate-AgNP, citrate-coated AgNP; BPEI-AgNP, branched polyethyleneimine-coated AgNPs. ImageJ 1.44 program was used to analyze particle size distributions and circularity of the AgNPs from the representative TEM imageries.

AgNPs exhibited significantly greater enzyme activity inhibition among the tested AgNP types, while free Ag+ (as AgNO3) showed significantly greater toxicity than all types of AgNPs evaluated (Fig. 1). In fact, Ag+ (as AgNO3) was, on average, 2.5, 6.4, and 16.4 times acutely inhibitory than BPEI-AgNPs, Citrate-AgNPs, and PVP-AgNPs, respectively; the β-Gal inhibition followed the trend: AgNO3 N BPEI-AgNP N Citrate-AgNP N PVP-AgNP. Similar to E. coli bioassay, a concentration-dependent mortality of D. magna neonates was observed in the 48 h bioassay for all types of AgNPs evaluated, including for the free Ag+ (as AgNO3). The neonatal mortality also followed the same toxicity trend as observed for E. coli assay presented above. Among the three organo-coated AgNP types, BPEI-AgNPs caused significantly higher mortality of the daphnids, while PVP-AgNPs resulted in least toxicity at the comparable concentrations (Fig. 2). It was the free Ag+ (as added AgNO3) that led to the highest mortality of daphnids compared to the AgNPs tested; on average, Ag+ was 1.1, 8.0, and 13.3 times more lethal than BPEI-AgNPs,

Citrate-AgNPs, and PVP-AgNPs, respectively. The respective EC50 or LC50 values of the organo-coated AgNPs for both the test organisms are presented in Table 2. Because the TEMdia, not the HDD (Pearson r = −0.27, p = 0.483), correlated significantly with ζ potential (Pearson r = −0.877, p = 0.002), and to avoid any redundancy in the GLM, TEMdia and ζ potential were used as the covariates in the linear models. Ma et al. (2012) have recently shown TEMdia, not the HDD, as a good predictor of organocoated AgNP dissolution into Ag ions, a premise consistent to our analysis. In the current work, we documented for the first time a significant main effect of primary particle size (TEMdia) and surface charge, and a significant interaction effect of primary particle size and surface charge, explaining empirically-derived acute toxicity correctly using the GLM (Tables 2 and 3). As the intercept term is not included in the model by the model selection procedure, this means that if the particle size and surface charge are zero, the toxicity in E. coli and D. magna would be securely anchored at zero and other factors would not significantly impact

Fig. 1. Concentration-dependent inhibition of β-galactosidase activity, compared to negative control, in Escherichia coli exposed to the three types of organo-coated AgNPs, including the Ag+ ions (as AgNO3). Error bars represent ±1 standard deviation of the triplicate runs. PVP-AgNP, polyvinylpyrrolidone-coated AgNPs; Citrate-AgNP, citrate-coated AgNPs; BPEI-AgNP, branched polyethyleneimine-coated AgNPs.

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Fig. 2. Concentration-dependent mortality of Daphnia magna exposed to the three types of organo-coated AgNPs, including the Ag+ ions (as AgNO3), as shown by 48 h test bioassay. Error bars represent ±1 standard deviation of the triplicate samples. PVP-AgNP, polyvinylpyrrolidone-coated AgNPs; Citrate-AgNP, citrate-coated AgNPs; BPEI-AgNP, branched polyethyleneimine-coated AgNPs.

where TEMdia denotes TEM diameter, ζ represents zeta potential, and εi and εii are the respective error terms of the models representing any

variance unaccounted for by each model. The parameter estimates of the models are presented in Tables 2 and 3. The coefficients of determinations (R2) of the linear models explaining the toxicity in E. coli and D. magna are 0.999 and 0.998, respectively, suggestive of good model fit with sufficient predictability of the models. A schematic summarizes these results in Fig. 3. We calculated the percentage precision of our GLM to predict nanotoxicity (as LC50 or EC50) using the covariates, TEMdia and zeta potential, for all the three types of AgNPs (Table 4). Comparison of GLM-predicted toxicity to that of experimentally-derived toxicity for both the model organisms is presented in Fig. 3 and Table 4. Results showed that the precision of the linear model developed to predict

Table 2 General linear model and parameter estimates showing the main and interactive effects of TEM diameter (TEMdia) and zeta potential on the toxicity of AgNPs (used as EC50 values, a dependent variable in the model) against Escherichia colia.

Table 3 General linear model and parameter estimates showing the main and interactive effects of TEM diameter (TEMdia) and zeta potential on the toxicity of AgNPs (used as LC50 values, a dependent variable in the model) against Daphnia magna.a

the toxicity. Our models explaining the toxicity of AgNPs in E. coli and D. magna are presented in Eqs. (1) and (2), respectively.

Escherichia coli EC50 ¼ 37:599 TEMdia –17:222 ζ þ 1:476ðTEMdia ζÞ þ εi

ð1Þ

Daphnia magna LC50 ¼ 0:077 TEMdia –0:031 ζ þ 0:002ðTEMdia ζÞ þ εii

ð2Þ

Dependent variable: EC50

Type III sum of squares

Mean squares

F

p

Source

Dependent variable: LC50

Type III sum of squares

Mean squares

F

p

Source

Model TEMdia Zeta potential TEMdia × Zeta potential Error

1.465E7 6.202E6 2.896E5 9.515E5 1.234E4

4.883E6 6.202E6 2.896E5 9.515E5 2.057E3

2373.454 3014.463 140.771 462.510

b0.0001 b0.0001 b0.0001 b0.0001

Model TEMdia Zeta potential TEMdia × Zeta potential Error

94.373 26.014 0.920 1.445 0.156

31.458 26.014 0.920 1.445 0.026

1207.334 998.396 35.296 55.474

b0.0001 b0.0001 0.001 b0.001

Predictor

Coefficient B

Std. error

t

p

Predictor

Coefficient B

Std. error

t

p

TEMdia Zeta potential TEMdia x Zeta potential

37.599 −17.222 1.476

0.685 1.452 0.069

54.904 −11.865 21.506

b0.0001 b0.0001 b0.0001

TEMdia Zeta potential TEMdia × Zeta potential

0.077 −0.031 0.002

0.002 0.005 0

31.597 −5.941 7.448

b0.0001 0.001 b0.001

a The coefficient of determination (R2) of the model was 0.999, suggestive of good model fit with sufficient predictive power.

a The coefficient of determination (R2) of the model was 0.998, suggestive of good model fit with sufficient predictive power.


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Fig. 3. Schematic showing the significant main effects and the significant interactive effects of primary particle size (TEM diameter) and surface charge explaining empirically-derived acute toxicity in E. coli and D. magna using the general linear model (GLM). Bar graph to the right shows concentration-, particle size-, and surface charge-dependent toxicity of the three different organo-coated AgNPs. PVP-AgNP, polyvinylpyrrolidone-coated AgNPs; Citrate-AgNP, citrate-coated AgNPs; BPEI-AgNP, branched polyethyleneimine-coated AgNPs.

nanotoxicity of the three different organo-coated AgNPs was in the range 99.8–100% for E. coli. For D. magna survival assay, some degree of deviation recorded in % precision of predicting toxicity by the GLM can be attributed to the higher sensitivity of zooplanktons to AgNPs, resulting in narrow differences in the experimentally-derived LC50 values (Table 4), which is in good agreement with the smaller coefficient values of the predictors (i.e., TEMdia, zeta potential) including of the interaction term (i.e., TEMdia × zeta potential) as noted in Table 3. Potential energy barrier, which has been hypothesized to act as a limiting factor (Vecitis et al., 2010; El Badawy et al., 2011), between the biologic surface and AgNP needs to be removed before the particles could chemically interact with the receptor molecules within the cell envelope (cell wall and/or cell membrane) and/or with the cellular content following permeation of nanoparticle into the cell (El Badawy et al., 2011), which may likely be governed by its nano-size or materialspecific characteristics (Pokhrel et al., 2012). Applying the single-chain mean field (SCMF) theory, studies have modeled the potential interaction of rod shaped biomolecules, including the carbon nanotubes, with the phospholipid bilayer and suggested that the change in surface patterning, and therefore the energy associated with the surface, of the rod shaped objects may influence, or rather enhance, object-cell membrane association, and perhaps cell entry (Pogodin et al., 2011, 2012), and subsequently the toxicity. Fig. 4 strongly supports primary particle size and surface charge dependent toxicity as predicted by our models for the three types of AgNPs against both the test organisms. Our analysis of the magnitude of charge difference between the nano-

bio interfaces, revealing higher attraction forces between (each of) the biologic receptors’ surface and the BPEI–AgNP surface, might have enabled potential physical contact between BPEI–AgNP and the receptor organism, leading to higher toxicity (Tables 5). Likewise, the lowest charge difference as observed for PVP-AgNPs suggests that the dominant repulsive forces (lower attraction) might have played a key role in keeping PVP-AgNPs away from the biologic surfaces and this may explain the lowest observed toxicity (Table 5). A previous study has shown surface charge-dependent toxicity of organo-coated AgNPs on the physiological activity and mortality in Gram positive Bacillus species, demonstrating BPEI-AgNPs as the most toxic among the types of AgNPs evaluated (El Badawy et al., 2011). The authors attributed higher toxicity of BPEI-AgNPs to the greater charge difference between the surface of Bacillus cell wall and the AgNPs, which was large enough to overcome the energy (electrostatic) barrier at the nano-bio interface, therefore leading to higher toxicity. This finding is consistent with our results (Fig. 4A,B). However, the toxicity trend for other two types of AgNPs (i.e., Citrate-AgNP and PVP-AgNP) observed in the previous study (El Badawy et al., 2011) was modified in the present study; this suggests that the disparity in toxicity observed could be largely due to the differences in the sensitivity and/or surface charge of the types of test organisms used including that of the Citrate-AgNPs in these two studies (El Badawy et al., 2011). In good agreement with our findings for Citrate-AgNP and PVP-AgNP, Kennedy et al. (2010) had observed similar toxicity trend for these two organo-coated

Table 4 Impact of surface charge on the toxicity of organo-coated AgNPs to Escherichia coli and Daphnia magna. Nanoparticle

BPEI-AgNP Citrate-AgNP PVP-AgNP

Zeta potentiala (mV)

Magnitude of charge differenceb c

AgNP (A)

E. coli (B)

D. magna (C)

E. coli |A–B|

D. magna |A–C|

+28.8 −20.08 −7.49

−11.0

−2.55

39.8 9.08 3.51

31.35 17.53 4.94

Strength of interaction

Expected toxicity

Observed toxicity

Higher attraction Lower attraction Higher repulsion

Higher Medium Lower

Higher Medium Lower

Note greater difference in charge indicates higher attraction (or lower repulsion) whereas lower difference in charge indicates higher repulsion (or lower attraction). PVP-AgNP, polyvinylpyrrolidone-coated AgNPs; Citrate-AgNP, citrate-coated AgNP; BPEI-AgNP, branched polyethyleneimine-coated AgNPs. a Zeta potential measured in the test matrix. b Magnitude of charge difference is taken as an absolute value. c Daphnia integument suspended in MHW (detailed in Materials and methods section, see Supporting Information).

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Fig. 4. Primary particle size (TEM diameter)- and surface charge (as ζ potential)-dependent toxicity of organo-coated AgNPs to (A) E. coli and (B) D. magna. Two bars with different letters indicate significant difference between the two treatments at the p = 0.01 level. Comparison of the means for E. coli EC50 and D. magna LC50 among different treatments was performed using the one-way ANOVA followed by Dunnett t test (2-tailed posthoc) for multiple comparisons. E. coli ζ potential was −11 mV and that of D. magna was −2.5 mV; details of surface charge are presented in Table 4. PVP-AgNP, polyvinylpyrrolidone-coated AgNPs; Citrate-AgNP, citrate-coated AgNPs; BPEI-AgNP, branched polyethyleneimine-coated AgNPs. NRWQC denotes USEPA national recommended water quality criterion for fresh water system (USEPA, 2009). The lines are meant to guide the eyes.

AgNPs against D. magna and Pimephales promelas. Results of the other studies also indicate a potential for direct physical interaction between the nanoparticles (e.g., various fullerene derivatives) and biologic receptors (but not the effect of reactive oxygen species being formed), which could likely be an important mechanism of nanotoxicity (Ali et al., 2004; Tang et al., 2007). Potential oxidation of AgNPs under aerobic experimental conditions, as in this study, has been shown to promote AgNP dissolution into Ag ions (Xiu et al., 2012). Our analysis showed more dissolved Ag ions released from PVP-AgNP suspension (3.74 μg/L), rather than from BPEIAgNP (3.17 μg/L), or Citrate-AgNP (2.39 μg/L) suspension. Because the concentrations of dissolved Ag were in the range 33–52 times lower than the EC50 value for Ag+ (as AgNO3) in E. coli, the observed toxicity of the types of AgNPs may not be associated with the aerobically

released dissolved Ag ion emanating from the AgNP suspensions; this suggests negligible contribution of dissolved Ag ion to the observed toxicity of AgNPs. Our analysis of HDD of the three types of AgNPs evaluated revealed higher stability of the particles in the test matrix (i.e., moderately hard water) during the test periods as no change in HDD occurred (Table 1), confirming an absence of aggregation or settling of particles in both the test media; thus particle aggregation was not a factor accounting for the observed differences in toxicity among the types of AgNPs evaluated against both the test organisms. Employing chemically different, yet commonly used, coating agents during synthesis of the three types of AgNPs contributed to the colloidal particle stability via different stabilization mechanisms (see Table 1) by hindering particle aggregation, and enabled us to acquire desired particle properties such as size, surface charge, and shape of the synthesized AgNPs (El

Table 5 Comparison of the general linear model (GLM)-predicted toxicity (LC50/EC50) versus experimentally-derived toxicity of the three types of organo-coated AgNPs against Escherichia coli and Daphnia magna. Nanoparticle

Daphnia magna Experimental LC50 ± S.D. (μg/L)

BPEI-AgNP Citrate-AgNP PVP-AgNP

0.41 ± 0.1 2.88 ± 0.08 4.79 ± 0.25

Escherichia coli GLM-predicted

Experimental EC50 ± S.D. (μg/L)

LC50 (μg/L)

% Precision

0.45 2.69 4.71

109.7 93.4 98.3

305 ± 33 793 ± 71 2041 ± 5

GLM-predicted EC50 (μg/L)

% Precision

305.08 791.63 2040.14

100 99.8 99.9

GLM, general linear model; % Precision = (GLM predicted LC50 or EC50 / Experimental LC50 or EC50) × 100. PVP-AgNP, polyvinylpyrrolidone-coated AgNPs; Citrate-AgNP, citrate-coated AgNP; BPEI-AgNP, branched polyethyleneimine-coated AgNPs.


Badawy et al., 2010). Because all the three types of organo-coated AgNPs evaluated in this study did not appear morphologically distinct from each other in shape, as they were roughly oval or spherical with narrow average circularity (Table 1; Supplementary Fig. S1), particle shape may not explain the observed differences in acute toxicity among the types of organo-coated AgNPs. Because the potential toxicity of each coating material used in this study was negligible at the highest theoretically relevant concentration (tri sodium citrate = 10 mM, PVP = 0.25%, and BPEI = 0.5 mM) against both the test organisms, it negates the possibility that the coating material alone could have any contribution to the observed AgNP toxicity. This is in good agreement with the published literature using the same coating materials and similar model species (El Badawy et al., 2011; Pokhrel et al., 2012). Under section 304 of the Clean Water Act (CWA), water quality criteria are established with an aim to protect human health and aquatic life from the contaminants of concerns. Recently revised USEPA national recommended water quality criterion (NRWQC) for fresh water system is 3.2 μg Ag/L (USEPA, 2009). As our experimentally obtained or the GLM-predicted LC50 values against D. magna for BPEI-AgNP and Citrate-AgNP fall below the NRWQC threshold, this suggests that the daphnid population might be vulnerable to AgNPs in the fresh water systems. Recently, a study has shown that a low, environmentally relevant concentration of Citrate-AgNPs (2 μg Ag/L) could influence daphnid migratory behaviors (i.e., horizontal and vertical migration patterns), survival, and reproductive potential in the presence of its common Odonate predator, Anax junius nymphs (Pokhrel and Dubey, 2012). Growing demand for AgNPs as an antimicrobial agent, including of other various kinds of nanomaterials (e.g., ZnO NP, TiO2 NP, CdSe quantum dots, C60/C70 fullerenes), in hundreds of consumer products will inevitably contaminate the aquatic systems, potentially affecting the biota, and perhaps the ecosystems, therein (Lovern et al., 2007; Brausch et al., 2011; El Badawy et al., 2011; Pokhrel et al., 2012; Pokhrel and Dubey, 2012). Overall, among the three types of organo-coated AgNPs evaluated in this study, BPEI-AgNP was the most biocidal due to its smaller primary particle size, and greater charge differences between the NP surface and the biologic surface leading to higher attractive forces, potentially promoting the toxicity. Notably, however, Ag+ (as added AgNO3) was the most toxic of all the chemicals tested against E. coli, while the toxicity of free Ag+ (as AgNO3) and BPEI-AgNP did not differ significantly against D. magna (p N 0.5; Fig. 3). Potential release of dissolved Ag ions from the evaluated AgNPs in our experimental scenarios was inadequate to explain the observed toxicity. Likewise, particle state of aggregation, suspension pH, coating material alone, and particle shape did not contribute to the toxicity of the AgNPs evaluated. This study, therefore, highlights the significance of considering various measureable physicochemical characteristics of nanoparticles including the particle size, surface charge of nanoparticles including that of the receptors' surface, and a proper evaluation of the potential main and interaction effects of the fundamental characteristics of nanoparticles using the quantitative modeling, such as the GLM applied in this work, could significantly contribute to our understanding of nanotoxicology. Our choice of different non-toxic surface coatings, which rendered different charge scenarios, on AgNPs surface also enabled the assessment of surface charge-dependent toxicity of AgNPs against both the prokaryotic and eukaryotic model organisms. The toxicity profiles developed here for the three organo-coated AgNPs demonstrate the importance of selecting coating materials in tandem with particle size and surface charge while considering safer AgNPs for environmental purposes, or alternately antimicrobial AgNPs for biomedical applications or for delaying membrane biofouling in water purification systems. Current paradigm that the particle size and surface charge matter still prevails. Notably, however, the results of this study suggest probing for the potential interaction effects amongst the different physico-chemical properties of the nanomaterials by deploying appropriate quantitative modeling, thus offering a new perspective on the

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way that the nanomaterial toxicity can be better explained or predicted when empirical toxicity data are lacking (Pokhrel et al. in review). To test an assertion that a minimum set of properties of the wellcharacterized nanomaterials, as was previously proposed (Pasquini et al., 2012) and clearly demonstrated in this study, could adequately explain the potential toxicity of other types of nanomaterials and against other test organisms will require additional systematic and focused studies. Acknowledgments This study was supported in part by the East Tennessee State University (ETSU) Research Development Council Grant# 82064 and the ETSU Office of Research and Sponsored Programs Grant# 83003. The authors thank TEM Analysis Services Lab, TX for support with TEM characterization of NPs. This study has not been subjected to the US EPA internal review, and the opinions expressed are those of the authors and do not reflect that of the associated institutions. Any mention of the trade names does not imply their endorsements or recommendations for use. Appendix A. Supplementary data Methods for organo-coated AgNP synthesis, TEM characterization, particle size distribution, and UV–vis spectra; plot showing particle sizedependent toxicity; and correlation between parameters of AgNPs. Supplementary data associated with this article can be found online at doi: http://dx.doi.org/10.1016/j.scitotenv.2013.09.006. References Ahamed M, Karns M, Goodson M, Rowe J, Hussain SM, Schlager JJ, et al. DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells. Toxicol Appl Pharmacol 2008;233:404–10. Ali SS, Hardt JI, Quick KL, Kim-Han JS, Erlanger BF, Huang TT, et al. A biologically effective fullerene (C60) derivative with superoxide dismutase mimetic properties. Free Radic Biol Med 2004;37(8):1191–202. Barceló D, Farré M, Bennett J, Hanson M. Nanomaterials in the environment. Sci Total Environ 2013. [http://www.journals.elsevier.com/science-of-the-total-environment/ virtual-special-issues/nanomaterials-in-the-environment/]. Benn T, Cavanagh B, Hristovski K, Posner JD, Westerhoff P. The release of nanosilver from consumer products used in the home. J Environ Qual 2010;39:1875–82. Bitton G, Jung K, Koopman B. Evaluation of a microplate assay specific for heavy metal toxicity. Arch Environ Contam Toxicol 1994;27:25–8. Brausch K, Anderson TA, Smith PN, Maul JD. The effect of fullerenes and functionalized fullerenes on Daphnia magna phototaxis and swimming behavior. Environ Toxicol Chem 2011;30:878–84. Choi O, Hu ZQ. Size dependent and reactive oxygen species related nanosilver toxicity to nitrifying bacteria. Environ Sci Technol 2008;42:4583–8. Costanza J, El Badawy AM, Tolaymat TM. Comment on “120 years of nanosilver history: implications for policy makers”. Environ Sci Technol 2011;45:7591–2. Diedrich T, Dybowska A, Schoot J, Valsami-Jones E, Oelkers EH. The dissolution rates of SiO2 nanoparticles as a function of particle size. Environ Sci Technol 2012;46(9): 4909–15. El Badawy AM, Luxton TP, Silva RG, Scheckel KG, Suidan MT, Tolaymat TM. Impact of environmental conditions (pH, ionic strength, and electrolyte type) on the surface charge and aggregation of silver NPs suspensions. Environ Sci Technol 2010;44(4): 1260–6. El Badawy AM, Silva RG, Morris B, Scheckel KG, Suidan MT, Tolaymat TM. Surface charge-dependent toxicity of silver nanoparticles. Environ Sci Technol 2011;45(1): 283–7. El Badawy AM, Scheckel KG, Suidan MT, Tolaymat TM. The impact of stabilization mechanism on the aggregation kinetics of silver nanoparticles. Sci Total Environ 2012;429: 325–31. Fabrega J, Renshaw JC, Lead JR. Interactions of silver nanoparticles with Pseudomonas putida biofilms. Environ Sci Technol 2009;43:9004–9. Figueredo SA, Lashermes P, Aragao FJL. Molecular characterization and functional analysis of the β-galactosidase gene during Coffea arabica (L.) fruit development. J Exp Bot 2011;62(8):2691–703. Hansen RG, Gitzelmann R. The metabolism of lactose and galactose. In: Jeanes A, Hodge J, editors. Physiological effects of food carbohydratesACS Symposium Series; 1975. p. 100–22. Impellitteri CA, Tolaymat TM, Scheckel KG. The speciation of silver nanoparticles in antimicrobial fabric before and after exposure to a hypochlorite/detergent solution. J Environ Qual 2009;38:1528–30. Jiang W, Kim BYS, Rutka JT, Chan WCW. Nanoparticle-mediated cellular response is size-dependent. Nat Nanotechnol 2008;3:145–50.

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