Bull Environ Contam Toxicol (2014) 93:53–59 DOI 10.1007/s00128-014-1296-4
Toxicity of Citrate-Coated Silver Nanoparticles Differs According to Method of Suspension Preparation June-Woo Park • Ji-Hyun Oh • Woo-Keun Kim Sung-Kyu Lee
•
Received: 16 May 2013 / Accepted: 10 May 2014 / Published online: 20 May 2014 Ó Springer Science+Business Media New York 2014
Abstract To evaluate substance toxicity, it is critical to maintain specific concentrations of test substances throughout the exposure period. During the last decade, the need to improve methods for nanoparticle (NP) suspension preparations has gained attention because many published results on NPs toxicity have been inconsistent. Here, we compared the toxicity of citrate-coated silver nanoparticles (AgNPs) suspended by two different methods (fractionated vs. colloidal) in freshwater organisms (daphnia and medaka). Analytical methods (ICP-OES, DLS and UV absorbance) were employed to characterize behavior of AgNPs in suspension. Results showed that fractionated (stirred and settled) solution was less toxic to daphnia (13.8 lg/L) than colloidal solution (6.1 lg/L), suggesting that method of preparation was a critical factor that affected toxicity. However, differences in toxicity caused by suspension methods were not observed in medaka. Results indicate that the method used to prepare suspensions of NPs can affect toxicity, and that differences can exist among test organisms. Keywords Daphnia magna Oryzias latipes Acute toxicity Suspension methods Silver nanoparticles
J.-W. Park (&) J.-H. Oh W.-K. Kim S.-K. Lee Global Environmental Regulation and Compliance Center, Korea Institute of Toxicology, 141 Gajeongro Yuseong, Daejeon 305-343, South Korea e-mail: [email protected] J.-W. Park S.-K. Lee Human and Environmental Toxicology Program, University of Science and Technology (UST), Daejoen 305-350, South Korea
Early recognition of potential for environmental contamination by engineered nanoparticles (NPs) (Colvin 2003) and forward thinking by environmental regulatory agencies (e.g., U.S. EPA, OECD) has resulted in research on the ecotoxicity of NPs in aquatic organisms. However, results of NP toxicity test have varied considerably among studies in part because of complicated behaviors of NPs in water. Overall behaviors of NPs (i.e., aggregation (agglomeration), association with other compounds etc.) in water are known to be influenced either (or both) by hydrochemical parameters such as temperature, ionic strength, pH, etc., (Brant et al. 2005; Hu et al. 2005; Dunphy Guzman et al. 2006) or by physicochemical characteristics designated to NPs such as particle hydrophobicity, concentrations or size (Filella and Buffle 1993; Lecoanet et al. 2004; Avdeev et al. 2004). Also the existence or absence of coating materials application to NPs can influence the overall aqueous fate of NPs. Some NPs coated with organic compounds (mainly in metallic NPs) can remain stable in aqueous suspensions (Mafune´ et al. 2000), while some uncoated NPs are highly unstable due to high surface hydrophobicity (for example, 1.3 9 10-11 ng/mL for fullerene). Thus, the NPs in aqueous environment are likely to interact among themselves or other materials, resulting in the formation of agglomerates or hetero-agglomerates, respectively rather than existing individually. Because of antimicrobial capacity, silver nanoparticles (AgNPs) are one of most common NPs to be use in consumer products (313 products) (http://www.nanotechpro ject.org/inventories/consumer/analysis_draft/). This mass production and widespread application of AgNPs could indicate that these novel materials, including those in products currently in use, could be continuously released into the environment. The possible influxes of AgNPs into surface water are diverse, for example unintentional spills,
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wastewater or surface run-off via agricultural land that applied wastewater sewage sludge (Mueller and Nowack 2008). Like other metal NPs, AgNPs have been highly evaluated for toxicity, however there is still a lack of knowledge about toxicity because of inconsistency among toxicological studies. Most NPs including AgNPs have been evaluated using internationally agreed test methods such as OECD test guidelines. However, many toxicologists have introduced the incompatibility of method application for testing toxicity of NPs due to propensity of NPs to aggregate/agglomerate in media, resulting in aqueous instability and proposed some efforts to enhance aqueous stability of NPs. For example, Romer et al. (2011) recommended a dilution of standard OECD daphnia media such that the aqueous stability of AgNP was enhanced, but not affecting the viability of test organism. However, this would make test species to alter their susceptibility to NPs and thus could lead unexpected results. To investigate aquatic toxicity, NPs must be well dispersed and various methods for enhancing NPs water stability have been used including stirring, sonication, ultrafiltration, surface modification, and solvents or surfactants application. However, these methods often caused some unintentional errors in the early studies of AgNPs toxicity due to experimental artifacts (Henry et al. 2007). The toxicity of NPs was also dependent on the types of stabilizers applied such as natural or synthetic polymers. The 96-h LC50 s for daphnia exposed to multi-walled carbon nanotubes were different among three different types of natural organic matters (Suwanee, Black, and Edisto Rivers) that applied as stabilizers (Edgington et al. 2010). This was also observed in mouse and human cells exposed to AgNPs. The AgNPs stabilized with three different polymer surfactants induced different cytotoxicity among cells, suggesting an importance of selecting stabilizers prior to determining toxicity of AgNPs (Lin et al. 2012). More recently, Jo et al. (2012) reported that concentrations of released ionic silver from AgNPs differed by preparation method. Therefore, it is necessary to understand inconsistencies in toxicity of NPs induced by suspension methods and to develop standardized methods that allow NPs well-stable in water for properly evaluating toxicity. Our hypothesis was that the toxicity of AgNPs is changed according to preparation method used to suspend AgNPs in the aqueous phase. Differences in toxicity according to preparation method (fractionated and colloidal) were tested by exposing freshwater test species (Oryzias latipes and Daphnia magna) in accordance with OECD acute toxicity test guidelines. The physicochemical characteristics of AgNPs in exposing solutions were investigated for their behaviors.
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Bull Environ Contam Toxicol (2014) 93:53–59 Table 1 Physicochemical characteristics of citrate-coated AgNPs Average Particle diameter
11.27 nm (smallest 4.52 nm, largest 22.26 nm, CV 31.2 %)
Particle surface area
4.38 9 102 nm2/particle
Particle mass
10.4 9 10-18 g
Particle volume
9.92 9 102 nm3
Materials and Methods Preparation of AgNPs Solutions Citrate-coated AgNPs were obtained from ABC Nanotech Co., (Daejon, South Korea) as a suspension in DI water. Manufacturer-provided physicochemical information on AgNPs are shown in Table 1. Originated from this solution, water accommodated fractionated AgNPs stock was prepared as below. To define optimal stirring and settling times that allow AgNPs stable in daphnia or fish exposing solution, preliminary tests were conducted. In daphnia exposing solution, they were 48 and 16 h, respectively and in fish exposing solution, they were 4 and 48 h, respectively. For daphnia acute toxicity test, 100 mg/L of AgNPs solution were stirred for 48 h and then were kept for 16 h without agitation, followed by taking upper aqueous part of solutions (fractionated stock). The fractionated stock was then diluted with standard daphnia solution (M4 medium) for exposure. As a counterpart of fractionated stock, original AgNPs solution was simply diluted with daphnia solution to be 100 mg/L (AgNP stock) and then further diluted for exposure. To make fractionated stock and exposing solution for fish acute toxicity test, same approach was applied except that dechlorinated and filtered (1 lm) tap water was used. Physicochemical Analysis To investigate changes in physicochemical properties of AgNPs in daphnia or fish exposing solution, samples (10 mg/L) after 5 min stirring were collected at specific times and directly analyzed for the particle size and UV observance using Zetasizer Nano-ZS90 (Malvern, Worcestershire, UK) and Lambda 25 UV/VIS Spectrophotometer (Perkin Elmer, Waltham, MA, USA), respectively. Samples (3 mL) in exposing solutions were collected by pipette from the middle of water column at beginning (before adding organisms) and at the end of exposures, and aqueous concentrations of total Ag or ionic Ag were determined with Inductively Coupled Plasma (ICP)-OES (Optima 4300 DV ICP-OES) (Perkin Elmer, Waltham, MA, USA). The standard used for ICP analysis
Bull Environ Contam Toxicol (2014) 93:53–59
was purchased from the Korea Research Institute of Standards and Science as certified reference materials of silver (CRM No. 10502AG2, Serial No. 050414-44). With this materials, spike test showed 100 % recovery rate of ICPOES analysis. The detection limit of ICP-OES is 0.27 lg/L. For ionic Ag concentration, AmiconÒ Ultra centrifugal filters Ultracel-3K (Millipore, Billerica, MA, USA) was applied to the collected samples and it was considered that any particles do not exist in the filtered samples. Transmission electron microscope analysis (TEM, JEM-1010, Jeol, Tokyo, Japan) was also employed (Center for Research Facilities, Chungnam National University, Daejeon, South Korea) to address the difference in shape of AgNPs. Simply, samples were placed on a slot-copper grid and dried overnight in a sterilized dust box. Samples were then imaged using TEM at 80 kV. Exposure Scenario Daphnia magna Acute Toxicity Test Acute toxicity test followed OECD test guideline (Test No. 202: Daphnia sp. Acute immobilization test). D. magna were obtained from Carolina Biological Supply Company (Burlington, NC USA) and cultured in the Division of NonClinical Studies, Korea Institute of Toxicology. Five daphnids (\24-h old) per test concentration were exposed for 48-h and inspected for immobility. In fractionated AgNP treatment, test concentrations were control, 10.1, 13.1, 17.1, 22.3, and 29.0 lg/L with four replicates. To compare toxicity, daphnids were also exposed to serial dilutions of colloidal (i.e., non-fractionated) AgNPs solution at control, 4.9, 7.4, 11.1, 16.6, and 25.0 lg/L. During exposure, daphnids were kept under following characteristics without feeding: pH 7.6 ± 0.2; dissolved oxygen concentration 8.7 ± 0.1 mg/L; temperature 20.2 ± 0.3°C. Photoperiod was 16:8-h light/dark. Fish Acute Toxicity Test Acute toxic effects of AgNPs to Japanese medaka (O. latipes) were evaluated using OECD test guideline (Test No. 203: Fish, acute toxicity test). O. latipes were obtained from Carolina Biological Supply Company (Burlington, NC, USA) and acclimated under a 16:8-h light/dark photoperiod with a 30-min transition period at 23–25°C prior to exposing. Seven fish (3 months aged) per test concentration were exposed to fractionated solutions at control, 20.6, 41.3, 82.5, 165.0, 330.0, and 660.0 lg/L for 96-h and recorded for mortality. As well, fish were exposed to serial dilutions of colloidal solutions at control, 19, 39, 70, 150, 310, and 620 lg/L. During exposure, fish were kept under following characteristics without feeding: temperature 22.2 ± 0.4°C;
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dissolved oxygen concentration: 8.5 ± 0.1 mg/L; pH 7.9 ± 0.1. Photoperiod was 16:8-h light/dark. Statistical Analysis 48-h median effective concentration (EC50 and LC50) with confidence limits for immobilizing rate (daphnia acute toxicity test) or mortality (fish acute toxicity test) were calculated with probit analysis (ToxCalc 5.0, Tidepool Scientific Software, McKinleyville, CA USA).
Results and Discussion Physicochemistry of AgNPs The aqueous behaviors of AgNPs were evaluated and compared in different solutions (Fig. 1). The OECD daphnia solution is comprised of diverse inorganic compounds and thus contains high ionic strength in the solution. In contrast, the ionic strength of fish exposure solution was lower than that of daphnia solution due to the absence of inorganic elements. The behavior of AgNPs in water is generally believed to be governed by the kinetics of agglomeration/aggregation in which ionic strength is one of important regulating factors. At high ionic strength, AgNPs are easily aggregated by electrostatic destabilization (Badawy et al. 2010). Although size distribution over 1,000 nm by DLS analysis is not sufficient for polydispersity of NPs in suspension (PDIs [0.7) as shown in Fig. 1, there is no doubt that AgNPs were highly agglomerated and less stable in daphnia solution, likely observed in elsewhere (Romer et al. 2011). However AgNPs in fish exposing solution were aqueous stable (Fig. 1). With high ionic strength in OECD daphnia solution, the presence of diverse divalent cations (especially for Ca2? from 0.01 M CaCl2) in the solution could enhance the aggregation and settlement of citrated coated AgNPs by complexation with carboxyl groups on the coating material (Badawy et al. 2010). Also Huynh and Chen (2011) reported that aggregation of NPs was positively related with salinity in water. The propensity of AgNPs to aggregate in daphnia exposing solution compared to fish exposing solution was further supported by TEM images (image not shown). Interestingly, during fractionation (i.e., stirring and settling), small sized AgNPs (about 10 nm) were rarely observed in daphnia exposing solution, but in fish exposing solution (Fig. 2). Different Toxicity of AgNPs by Suspension Methods Regardless of preparation method, actual concentrations as aqueous total Ag were close to their corresponding nominal
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Fig. 1 Changes in size and stability of AgNPs (10 mg/L) in exposure preparations
Fig. 2 Transmission electron microscope images of AgNPs in colloidal and fractionated daphnia (a, b) or fish exposure preparations (c, d)
concentrations at time 0 in both daphnia and fish acute toxicity test except for highly treated groups in fish acute test. At 48 h, total silver concentrations were significantly decreased in all treatments and the loss of total aqueous Ag concentration in fractionated AgNP groups was greater than that in colloidal counterparts. Both types of AgNPs appeared to be toxic to D. magna and O. latipes, but less toxic compared to ionic Ag as AgNO3 (Table 2). In daphnia, the toxicity of fractionated AgNPs was lower than that of colloidal AgNPs as indicated that EC50s of fractionated and colloidal AgNPs were 13.8 and 6.1 lg/L, respectively, which suggests that preparation method is a considerable determinant to evaluate the toxicity of NPs. Meanwhile, O. latipes was less sensitive to AgNPs than D. magna. The medaka-48h-LC50s of
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Table 2 Different toxicity in D. magna and O. latipes exposed to AgNPs prepared by two different suspension methods Daphnia magna Total Ag EC50 (CI 95 %, lg/L)
Oryzias latipes Total Ag LC50 (CI 95 %, lg/L)
Fractionated AgNPs
13.8 lg/L (13.3–14.0)
64.7 lg/L (–a)
Colloidal AgNPs
6.1 lg/L (5.6–6.5)
76.0 lg/L (55.0–99.0)
AgNO3
2.1 lg/L (2.1–2.1)
49.3 lg/L (43.9–55.3)
a
Not applicable
colloidal and fractionated AgNPs were 76.0 and 64.7 lg/L, respectively (Table 2). Interestingly, distinct differences in toxicity of AgNPs according to different suspension
Bull Environ Contam Toxicol (2014) 93:53–59 Table 3 Actual concentrations (lg/L) of ionic Ag in fractionated and colloidal daphnia and fish exposure solutions at the end of exposure (48 h for D. magna and 96 h for O. latipes)
Daphnia exposing solution
Fish exposing solution
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Treatments
Control
10.1
13.1
17.1
22.3
29.0
Fractionated
–
–
0.2
0.3
0.4
0.6
Treatments
Control
4.9
7.4
11.1
16.6
25.0
Colloidal Treatments
– Control
0.6 20.6
1.3 41.3
1.9 82.5
2.3 165.0
3.1 330.0
660.0
Fractionated
–
0.2
1.5
–
–
–
0.2
Treatments
Control
19.0
39.0
70.0
150.0
310.0
620.0
Colloidal
–
–
1.7
–
–
–
0.2
methods were not observed in fish acute toxicity test (Table 2), which is inconsistent to the suggestion of Kennedy et al. (2010) that fractionated AgNPs was more predictive of acute toxicity than total AgNPs. This discrepancy could be due to different fractionation method applied and/ or different susceptibility of AgNPs to test species. The levels of ionic Ag in solutions were evaluated at the end of exposure (Table 3) and concentration did not decrease during exposure in daphnia exposing solutions, meanwhile most of ionic Ag was disappeared in fish exposure solutions. To evaluate if ionic silver released from AgNPs in solutions causes additive toxic effects to daphnia or fish, preliminary experiments using silver nitrate (AgNO3) were conducted. Higher toxicity of ionic Ag as AgNO3 to test species compared to either fractionated or colloidal solutions was observed (Table 2). Except for colloidal daphnia exposing solutions, the aqueous concentrations of ionic Ag in fractionated daphnia or fish exposing solutions were lower than 1.4 lg AgNO3/L of daphnia 96 h-NOAEL (no observed adverse effective level) or 30.1 lg AgNO3/L of O. latipes 96 h-NOAEL, indicating that harmful effect observed in daphnia or fish was not likely attributable from the released ionic Ag (Table 3). In daphnia test, higher ionic Ag concentrations were observed in colloidal solutions than in fractionated counterpart. This could be explained by the existence of large number of small sized particles (about 10 nm), which was not observed in fractionated solution (Fig. 2a vs. b) (Zhang et al. 2011; Li and Lenhart 2012). In condition of high ionic strength, counter-ion such as chloride (Cl-) could bind with released silver ions (Ag?) from AgNPs, resulting in the formation of aqueous (stable) Ag-chloride complex. Li and Lenhart (2012) investigated that aggregated AgNPs did not easily release ionic silver, and suggested that the smaller size of particles, the more release of ionic silver into water. Therefore the enhanced toxicity of colloidal AgNPs to daphnia may relate to the higher aqueous levels of ionic silver than in fractionated AgNPs. This finding might implicate that high ionic strength could not only affect the kinetics of silver ion release from AgNPs but also the residence of released ionic silver in
solution, which should be further investigated for better understanding and prediction of AgNPs toxicity. Although a large number of small sized NPs in both colloidal and fractionated solution in fish acute test was observed as well (Fig. 2.c vs. d), measured actual ionic Ag concentrations were lower than those in daphnia exposing solutions, which might be due to different chemistry between solutions such as strong salification rather than fractionation processes. The Importance of AgNP Suspension Methods for Toxicity Recent studies suggested that AgNPs toxicity is mediated by released silver ions (Zhao and Wang 2012), although other toxic mechanisms of AgNPs such as unique physiocochemical properties of AgNP such as size or shape (Scown et al. 2010) and oxidative stress produced by the AgNPs in aqueous phase (Choi and Hu 2008) are still of concern. The present study applied two types of AgNPs suspension methods and compared their toxicities to daphnia or fish and demonstrated that they were toxic to test organisms, but the level of toxicity was different according to the method applied for suspension. Our results indicated that fractionated solution was less acutely toxic to D. magna than the non-fractionated solution. The daphnia48h-EC50 of colloidal AgNPs was 6.1 lg/L, which is close to the results of Asghari et al. (2012) (2–4 lg/L) and Lee et al. (2012) (7.98 lg/L) that applied similar exposing processes to ours. On the other hand, fractionated solution to daphnia (48h-EC50 13.8 lg/L) were about 2 times less toxic than colloidal solution (48h-EC50 6.1 lg/L). It is expected that the toxicity of colloidal solution was added by ionic Ag released as observed high ionic silver concentrations (Table 3). Although many studies have investigated the origins of AgNPs toxicity such as their unique attributes such as size, shape, or coating materials (Scown et al. 2010; Kwok et al. 2012; Zhao and Wang 2012), only a few studies addressed the importance of test preparations for evaluating toxicity of AgNPs (Kennedy et al. 2010; Asghari et al. 2012). Kennedy et al. (2010) compared the toxicity of nanosilver with fractionated nanosilver and
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emphasized the importance of characterizing ionic sliver in nanomaterial suspensions, and Asghari et al. (2012) compared the toxicity of various types (colloids vs. suspension) of AgNPs. However, these studies applied ultracentrifugation and acidification with nitric acid to prepare fractionated suspension (Kennedy et al. 2010), which might cause unexpected toxicity due to the applied suspension method or compared the toxicity of AgNPs suspension to that of AgNPs colloids obtained from different sources (Asghari et al. 2012), which might lead inaccuracy to test whether suspension method is the only factor that affected toxicity of NPs. Current study applied environmentally relevant suspension method, i.e., simple combination of stirring and settlement, and compared toxicity between fractionated and colloidal AgNP solutions coming from same AgNP stocks.
Conclusion The toxicity of AgNPs has already been reported in many aquatic organisms and the number of these studies would be continuously increased. However investigations of the distinct characteristics of AgNPs (such as preparation methods) that might affect their toxicity to organisms and mislead the hazards of AgNPs to human and ecosystem are currently lacking. The present study clearly indicated that methods for preparing AgNP suspensions changed the toxicity to organisms, but these would be only effective on a case by case basis, for example in some toxicity tests that contain high electrolyte concentrations in their test media. These findings will help to draw a toxicological agreement about how to expose and evaluate NPs toxicity in aquatic toxicity tests, and suggest a universal agreement in standardized suspension method to test NP toxicity. Acknowledgments The authors are pleased to acknowledge Prof. Theodore B. Henry (Heriot-Watt University) who provided critical review for this manuscript. This research was funded by the project for Environmental Risk Assessment of Manufactured Nanomaterials (KK-1303-03), the Korea Institute of Toxicology (KIT) and the Industrial Core Technology Program (10034759, Risk Management Technology for Nano Products), the Ministry of Trade, Industry and Energy (MOTIE).
References Asghari S, Johari S, Lee J, Kim Y, Jeon Y, Choi H, Moon M, Yu I (2012) Toxicity of various silver nanoparticles compared to silver ions in Daphnia magna. J Nanobiotechnol 10:14 Avdeev MV, Khokhryakov AA, Tropin TV, Andrievsky GV, Klochkov VK, Derevyanchenko LI, Rosta L, Garamus VM, Priezzhev VB, Korobov MV, Aksenov VL (2004) Structural features of molecular-colloidal solutions of C60 fullerenes in water by small-angle neutron scattering. Langmuir 20:4363–4368
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Bull Environ Contam Toxicol (2014) 93:53–59 Badawy AME, Luxton TP, Silva RG, Scheckel KG, Suidan MT, Tolaymat TM (2010) Impact of environmental conditions (pH, ionic strength, and electrolyte type) on the surface charge and aggregation of silver nanoparticles suspensions. Environ Sci Technol 44:1260–1266 Brant J, Lecoanet H, Wiesner MR (2005) Aggregation and deposition characteristics of fullerene nanoparticles in aqueous systems. J Nanopart Res 7:545–553 Choi O, Hu Z (2008) Size dependent and reactive oxygen species related nanosilver toxicity to nitrifying bacteria. Environ Sci Technol 42:4583–4588 Colvin VL (2003) The potential environmental impact of engineered nanomaterials. Nat Biotechnol 21:1166–1170 Dunphy Guzman KA, Finnegan MP, Banfield JF (2006) Influence of surface potential on aggregation and transport of titania nanoparticles. Environ Sci Technol 40:7688–7693 Edgington AJ, Roberts AP, Taylor LM, Alloy MM, Reppert J, Rao AM, Mao J, Klaine SJ (2010) The influence of natural organic matter on the toxicity of multiwalled carbon nanotubes. Environ Toxicol Chem 29:2511–2518 Filella M, Buffle J (1993) Factors controlling the stability of submicron colloids in natural waters. Colloids Surf A Physicochem Eng Asp 73:255–273 Henry TB, Menn FM, Fleming JT, Wilgus J, Compton RN, Sayler GS (2007) Attributing effects of aqueous C60 nano-aggregates to tetrahydrofuran decomposition products in larval zebrafish by assessment of gene expression. Environ Health Perspect 115:1059–1065 Hu H, Yu A, Kim E, Zhao B, Itkis ME, Bekyarova E, Haddon RC (2005) Influence of the zeta potential on the dispersability and purification of single-walled carbon nanotubes. J Phys Chem B 109:11520–11524 Huynh KA, Chen KL (2011) Aggregation kinetics of citrate and polyvinylpyrrolidone coated silver nanoparticles in monovalent and divalent electrolyte solutions. Environ Sci Technol 45:5564–5571 Jo HJ, Choi JW, Lee SH, Hong SW (2012) Acute toxicity of Ag and CuO nanoparticle suspensions against Daphnia magna: the importance of their dissolved fraction varying with preparation methods. J Hazard Mater 227–228:301–308 Kennedy AJ, Huli MS, Bednar AJ, Goss JD, Gunter JC, Bouldin JL, Vikesland PJ, Steevens JA (2010) Fractionating nanosilver: importance for determining toxicity to aquatic test organisms. Environ Sci Technol 44:9571–9577 Kwok KW, Auffan M, Badireddy AR, Nelson CM, Wiesner MR, Chilkoti A, Liu J, Marinakos SM, Hinton DE (2012) Uptake of silver nanoparticles and toxicity to early life stages of Japanese medaka (Oryzias latipes): effect of coating materials. Aquat Toxicol 120–121:59–66 Lecoanet HF, Bottero JY, Wiesner MR (2004) Laboratory assessment of the mobility of nanomaterials in porous media. Environ Sci Technol 38:5164–5169 Lee YJ, Kim J, Oh J, Bae S, Lee S, Hong IS, Kim SH (2012) Ionrelease kinetics and ecotoxicity effects of silver nanoparticles. Environ Toxicol Chem 31:155–159 Li X, Lenhart JJ (2012) Aggregation and dissolution of silver nanoparticles in natural surface water. Environ Sci Technol 46:5378–5386 Lin JJ, Lin WC, Dong RX, Hsu SH (2012) The cellular responses and antibacterial activities of silver nanoparticles stabilized by different polymers. Nanotechnology 23:065102 Mafune´ F, Kohno JY, Takeda Y, Kondow T, Sawabe H (2000) Structure and stability of silver nanoparticles in aqueous solution produced by laser ablation. J Phys Chem B 104:8333–8337 Mueller NC, Nowack B (2008) Exposure modelling of engineered nanoparticles in the Environment. Environ Sci Technol 42:4447–4453
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59 Zhang W, Yao Y, Sullivan N, Chen Y (2011) Modeling the primary size effects of citrate-coated silver nanoparticles on their ion release kinetics. Environ Sci Technol 45:4422–4428 Zhao CM, Wang WX (2012) Importance of surface coatings and soluble silver in silver nanoparticles toxicity to Daphnia magna. Nanotoxicology 6:361–370
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Toxicity of Citrate-Coated Silver Nanoparticles Differs According to Method of Suspension Preparation June-Woo Park • Ji-Hyun Oh • Woo-Keun Kim Sung-Kyu Lee
•
Received: 16 May 2013 / Accepted: 10 May 2014 / Published online: 20 May 2014 Ó Springer Science+Business Media New York 2014
Abstract To evaluate substance toxicity, it is critical to maintain specific concentrations of test substances throughout the exposure period. During the last decade, the need to improve methods for nanoparticle (NP) suspension preparations has gained attention because many published results on NPs toxicity have been inconsistent. Here, we compared the toxicity of citrate-coated silver nanoparticles (AgNPs) suspended by two different methods (fractionated vs. colloidal) in freshwater organisms (daphnia and medaka). Analytical methods (ICP-OES, DLS and UV absorbance) were employed to characterize behavior of AgNPs in suspension. Results showed that fractionated (stirred and settled) solution was less toxic to daphnia (13.8 lg/L) than colloidal solution (6.1 lg/L), suggesting that method of preparation was a critical factor that affected toxicity. However, differences in toxicity caused by suspension methods were not observed in medaka. Results indicate that the method used to prepare suspensions of NPs can affect toxicity, and that differences can exist among test organisms. Keywords Daphnia magna Oryzias latipes Acute toxicity Suspension methods Silver nanoparticles
J.-W. Park (&) J.-H. Oh W.-K. Kim S.-K. Lee Global Environmental Regulation and Compliance Center, Korea Institute of Toxicology, 141 Gajeongro Yuseong, Daejeon 305-343, South Korea e-mail: [email protected] J.-W. Park S.-K. Lee Human and Environmental Toxicology Program, University of Science and Technology (UST), Daejoen 305-350, South Korea
Early recognition of potential for environmental contamination by engineered nanoparticles (NPs) (Colvin 2003) and forward thinking by environmental regulatory agencies (e.g., U.S. EPA, OECD) has resulted in research on the ecotoxicity of NPs in aquatic organisms. However, results of NP toxicity test have varied considerably among studies in part because of complicated behaviors of NPs in water. Overall behaviors of NPs (i.e., aggregation (agglomeration), association with other compounds etc.) in water are known to be influenced either (or both) by hydrochemical parameters such as temperature, ionic strength, pH, etc., (Brant et al. 2005; Hu et al. 2005; Dunphy Guzman et al. 2006) or by physicochemical characteristics designated to NPs such as particle hydrophobicity, concentrations or size (Filella and Buffle 1993; Lecoanet et al. 2004; Avdeev et al. 2004). Also the existence or absence of coating materials application to NPs can influence the overall aqueous fate of NPs. Some NPs coated with organic compounds (mainly in metallic NPs) can remain stable in aqueous suspensions (Mafune´ et al. 2000), while some uncoated NPs are highly unstable due to high surface hydrophobicity (for example, 1.3 9 10-11 ng/mL for fullerene). Thus, the NPs in aqueous environment are likely to interact among themselves or other materials, resulting in the formation of agglomerates or hetero-agglomerates, respectively rather than existing individually. Because of antimicrobial capacity, silver nanoparticles (AgNPs) are one of most common NPs to be use in consumer products (313 products) (http://www.nanotechpro ject.org/inventories/consumer/analysis_draft/). This mass production and widespread application of AgNPs could indicate that these novel materials, including those in products currently in use, could be continuously released into the environment. The possible influxes of AgNPs into surface water are diverse, for example unintentional spills,
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wastewater or surface run-off via agricultural land that applied wastewater sewage sludge (Mueller and Nowack 2008). Like other metal NPs, AgNPs have been highly evaluated for toxicity, however there is still a lack of knowledge about toxicity because of inconsistency among toxicological studies. Most NPs including AgNPs have been evaluated using internationally agreed test methods such as OECD test guidelines. However, many toxicologists have introduced the incompatibility of method application for testing toxicity of NPs due to propensity of NPs to aggregate/agglomerate in media, resulting in aqueous instability and proposed some efforts to enhance aqueous stability of NPs. For example, Romer et al. (2011) recommended a dilution of standard OECD daphnia media such that the aqueous stability of AgNP was enhanced, but not affecting the viability of test organism. However, this would make test species to alter their susceptibility to NPs and thus could lead unexpected results. To investigate aquatic toxicity, NPs must be well dispersed and various methods for enhancing NPs water stability have been used including stirring, sonication, ultrafiltration, surface modification, and solvents or surfactants application. However, these methods often caused some unintentional errors in the early studies of AgNPs toxicity due to experimental artifacts (Henry et al. 2007). The toxicity of NPs was also dependent on the types of stabilizers applied such as natural or synthetic polymers. The 96-h LC50 s for daphnia exposed to multi-walled carbon nanotubes were different among three different types of natural organic matters (Suwanee, Black, and Edisto Rivers) that applied as stabilizers (Edgington et al. 2010). This was also observed in mouse and human cells exposed to AgNPs. The AgNPs stabilized with three different polymer surfactants induced different cytotoxicity among cells, suggesting an importance of selecting stabilizers prior to determining toxicity of AgNPs (Lin et al. 2012). More recently, Jo et al. (2012) reported that concentrations of released ionic silver from AgNPs differed by preparation method. Therefore, it is necessary to understand inconsistencies in toxicity of NPs induced by suspension methods and to develop standardized methods that allow NPs well-stable in water for properly evaluating toxicity. Our hypothesis was that the toxicity of AgNPs is changed according to preparation method used to suspend AgNPs in the aqueous phase. Differences in toxicity according to preparation method (fractionated and colloidal) were tested by exposing freshwater test species (Oryzias latipes and Daphnia magna) in accordance with OECD acute toxicity test guidelines. The physicochemical characteristics of AgNPs in exposing solutions were investigated for their behaviors.
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Bull Environ Contam Toxicol (2014) 93:53–59 Table 1 Physicochemical characteristics of citrate-coated AgNPs Average Particle diameter
11.27 nm (smallest 4.52 nm, largest 22.26 nm, CV 31.2 %)
Particle surface area
4.38 9 102 nm2/particle
Particle mass
10.4 9 10-18 g
Particle volume
9.92 9 102 nm3
Materials and Methods Preparation of AgNPs Solutions Citrate-coated AgNPs were obtained from ABC Nanotech Co., (Daejon, South Korea) as a suspension in DI water. Manufacturer-provided physicochemical information on AgNPs are shown in Table 1. Originated from this solution, water accommodated fractionated AgNPs stock was prepared as below. To define optimal stirring and settling times that allow AgNPs stable in daphnia or fish exposing solution, preliminary tests were conducted. In daphnia exposing solution, they were 48 and 16 h, respectively and in fish exposing solution, they were 4 and 48 h, respectively. For daphnia acute toxicity test, 100 mg/L of AgNPs solution were stirred for 48 h and then were kept for 16 h without agitation, followed by taking upper aqueous part of solutions (fractionated stock). The fractionated stock was then diluted with standard daphnia solution (M4 medium) for exposure. As a counterpart of fractionated stock, original AgNPs solution was simply diluted with daphnia solution to be 100 mg/L (AgNP stock) and then further diluted for exposure. To make fractionated stock and exposing solution for fish acute toxicity test, same approach was applied except that dechlorinated and filtered (1 lm) tap water was used. Physicochemical Analysis To investigate changes in physicochemical properties of AgNPs in daphnia or fish exposing solution, samples (10 mg/L) after 5 min stirring were collected at specific times and directly analyzed for the particle size and UV observance using Zetasizer Nano-ZS90 (Malvern, Worcestershire, UK) and Lambda 25 UV/VIS Spectrophotometer (Perkin Elmer, Waltham, MA, USA), respectively. Samples (3 mL) in exposing solutions were collected by pipette from the middle of water column at beginning (before adding organisms) and at the end of exposures, and aqueous concentrations of total Ag or ionic Ag were determined with Inductively Coupled Plasma (ICP)-OES (Optima 4300 DV ICP-OES) (Perkin Elmer, Waltham, MA, USA). The standard used for ICP analysis
Bull Environ Contam Toxicol (2014) 93:53–59
was purchased from the Korea Research Institute of Standards and Science as certified reference materials of silver (CRM No. 10502AG2, Serial No. 050414-44). With this materials, spike test showed 100 % recovery rate of ICPOES analysis. The detection limit of ICP-OES is 0.27 lg/L. For ionic Ag concentration, AmiconÒ Ultra centrifugal filters Ultracel-3K (Millipore, Billerica, MA, USA) was applied to the collected samples and it was considered that any particles do not exist in the filtered samples. Transmission electron microscope analysis (TEM, JEM-1010, Jeol, Tokyo, Japan) was also employed (Center for Research Facilities, Chungnam National University, Daejeon, South Korea) to address the difference in shape of AgNPs. Simply, samples were placed on a slot-copper grid and dried overnight in a sterilized dust box. Samples were then imaged using TEM at 80 kV. Exposure Scenario Daphnia magna Acute Toxicity Test Acute toxicity test followed OECD test guideline (Test No. 202: Daphnia sp. Acute immobilization test). D. magna were obtained from Carolina Biological Supply Company (Burlington, NC USA) and cultured in the Division of NonClinical Studies, Korea Institute of Toxicology. Five daphnids (\24-h old) per test concentration were exposed for 48-h and inspected for immobility. In fractionated AgNP treatment, test concentrations were control, 10.1, 13.1, 17.1, 22.3, and 29.0 lg/L with four replicates. To compare toxicity, daphnids were also exposed to serial dilutions of colloidal (i.e., non-fractionated) AgNPs solution at control, 4.9, 7.4, 11.1, 16.6, and 25.0 lg/L. During exposure, daphnids were kept under following characteristics without feeding: pH 7.6 ± 0.2; dissolved oxygen concentration 8.7 ± 0.1 mg/L; temperature 20.2 ± 0.3°C. Photoperiod was 16:8-h light/dark. Fish Acute Toxicity Test Acute toxic effects of AgNPs to Japanese medaka (O. latipes) were evaluated using OECD test guideline (Test No. 203: Fish, acute toxicity test). O. latipes were obtained from Carolina Biological Supply Company (Burlington, NC, USA) and acclimated under a 16:8-h light/dark photoperiod with a 30-min transition period at 23–25°C prior to exposing. Seven fish (3 months aged) per test concentration were exposed to fractionated solutions at control, 20.6, 41.3, 82.5, 165.0, 330.0, and 660.0 lg/L for 96-h and recorded for mortality. As well, fish were exposed to serial dilutions of colloidal solutions at control, 19, 39, 70, 150, 310, and 620 lg/L. During exposure, fish were kept under following characteristics without feeding: temperature 22.2 ± 0.4°C;
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dissolved oxygen concentration: 8.5 ± 0.1 mg/L; pH 7.9 ± 0.1. Photoperiod was 16:8-h light/dark. Statistical Analysis 48-h median effective concentration (EC50 and LC50) with confidence limits for immobilizing rate (daphnia acute toxicity test) or mortality (fish acute toxicity test) were calculated with probit analysis (ToxCalc 5.0, Tidepool Scientific Software, McKinleyville, CA USA).
Results and Discussion Physicochemistry of AgNPs The aqueous behaviors of AgNPs were evaluated and compared in different solutions (Fig. 1). The OECD daphnia solution is comprised of diverse inorganic compounds and thus contains high ionic strength in the solution. In contrast, the ionic strength of fish exposure solution was lower than that of daphnia solution due to the absence of inorganic elements. The behavior of AgNPs in water is generally believed to be governed by the kinetics of agglomeration/aggregation in which ionic strength is one of important regulating factors. At high ionic strength, AgNPs are easily aggregated by electrostatic destabilization (Badawy et al. 2010). Although size distribution over 1,000 nm by DLS analysis is not sufficient for polydispersity of NPs in suspension (PDIs [0.7) as shown in Fig. 1, there is no doubt that AgNPs were highly agglomerated and less stable in daphnia solution, likely observed in elsewhere (Romer et al. 2011). However AgNPs in fish exposing solution were aqueous stable (Fig. 1). With high ionic strength in OECD daphnia solution, the presence of diverse divalent cations (especially for Ca2? from 0.01 M CaCl2) in the solution could enhance the aggregation and settlement of citrated coated AgNPs by complexation with carboxyl groups on the coating material (Badawy et al. 2010). Also Huynh and Chen (2011) reported that aggregation of NPs was positively related with salinity in water. The propensity of AgNPs to aggregate in daphnia exposing solution compared to fish exposing solution was further supported by TEM images (image not shown). Interestingly, during fractionation (i.e., stirring and settling), small sized AgNPs (about 10 nm) were rarely observed in daphnia exposing solution, but in fish exposing solution (Fig. 2). Different Toxicity of AgNPs by Suspension Methods Regardless of preparation method, actual concentrations as aqueous total Ag were close to their corresponding nominal
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Fig. 1 Changes in size and stability of AgNPs (10 mg/L) in exposure preparations
Fig. 2 Transmission electron microscope images of AgNPs in colloidal and fractionated daphnia (a, b) or fish exposure preparations (c, d)
concentrations at time 0 in both daphnia and fish acute toxicity test except for highly treated groups in fish acute test. At 48 h, total silver concentrations were significantly decreased in all treatments and the loss of total aqueous Ag concentration in fractionated AgNP groups was greater than that in colloidal counterparts. Both types of AgNPs appeared to be toxic to D. magna and O. latipes, but less toxic compared to ionic Ag as AgNO3 (Table 2). In daphnia, the toxicity of fractionated AgNPs was lower than that of colloidal AgNPs as indicated that EC50s of fractionated and colloidal AgNPs were 13.8 and 6.1 lg/L, respectively, which suggests that preparation method is a considerable determinant to evaluate the toxicity of NPs. Meanwhile, O. latipes was less sensitive to AgNPs than D. magna. The medaka-48h-LC50s of
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Table 2 Different toxicity in D. magna and O. latipes exposed to AgNPs prepared by two different suspension methods Daphnia magna Total Ag EC50 (CI 95 %, lg/L)
Oryzias latipes Total Ag LC50 (CI 95 %, lg/L)
Fractionated AgNPs
13.8 lg/L (13.3–14.0)
64.7 lg/L (–a)
Colloidal AgNPs
6.1 lg/L (5.6–6.5)
76.0 lg/L (55.0–99.0)
AgNO3
2.1 lg/L (2.1–2.1)
49.3 lg/L (43.9–55.3)
a
Not applicable
colloidal and fractionated AgNPs were 76.0 and 64.7 lg/L, respectively (Table 2). Interestingly, distinct differences in toxicity of AgNPs according to different suspension
Bull Environ Contam Toxicol (2014) 93:53–59 Table 3 Actual concentrations (lg/L) of ionic Ag in fractionated and colloidal daphnia and fish exposure solutions at the end of exposure (48 h for D. magna and 96 h for O. latipes)
Daphnia exposing solution
Fish exposing solution
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Treatments
Control
10.1
13.1
17.1
22.3
29.0
Fractionated
–
–
0.2
0.3
0.4
0.6
Treatments
Control
4.9
7.4
11.1
16.6
25.0
Colloidal Treatments
– Control
0.6 20.6
1.3 41.3
1.9 82.5
2.3 165.0
3.1 330.0
660.0
Fractionated
–
0.2
1.5
–
–
–
0.2
Treatments
Control
19.0
39.0
70.0
150.0
310.0
620.0
Colloidal
–
–
1.7
–
–
–
0.2
methods were not observed in fish acute toxicity test (Table 2), which is inconsistent to the suggestion of Kennedy et al. (2010) that fractionated AgNPs was more predictive of acute toxicity than total AgNPs. This discrepancy could be due to different fractionation method applied and/ or different susceptibility of AgNPs to test species. The levels of ionic Ag in solutions were evaluated at the end of exposure (Table 3) and concentration did not decrease during exposure in daphnia exposing solutions, meanwhile most of ionic Ag was disappeared in fish exposure solutions. To evaluate if ionic silver released from AgNPs in solutions causes additive toxic effects to daphnia or fish, preliminary experiments using silver nitrate (AgNO3) were conducted. Higher toxicity of ionic Ag as AgNO3 to test species compared to either fractionated or colloidal solutions was observed (Table 2). Except for colloidal daphnia exposing solutions, the aqueous concentrations of ionic Ag in fractionated daphnia or fish exposing solutions were lower than 1.4 lg AgNO3/L of daphnia 96 h-NOAEL (no observed adverse effective level) or 30.1 lg AgNO3/L of O. latipes 96 h-NOAEL, indicating that harmful effect observed in daphnia or fish was not likely attributable from the released ionic Ag (Table 3). In daphnia test, higher ionic Ag concentrations were observed in colloidal solutions than in fractionated counterpart. This could be explained by the existence of large number of small sized particles (about 10 nm), which was not observed in fractionated solution (Fig. 2a vs. b) (Zhang et al. 2011; Li and Lenhart 2012). In condition of high ionic strength, counter-ion such as chloride (Cl-) could bind with released silver ions (Ag?) from AgNPs, resulting in the formation of aqueous (stable) Ag-chloride complex. Li and Lenhart (2012) investigated that aggregated AgNPs did not easily release ionic silver, and suggested that the smaller size of particles, the more release of ionic silver into water. Therefore the enhanced toxicity of colloidal AgNPs to daphnia may relate to the higher aqueous levels of ionic silver than in fractionated AgNPs. This finding might implicate that high ionic strength could not only affect the kinetics of silver ion release from AgNPs but also the residence of released ionic silver in
solution, which should be further investigated for better understanding and prediction of AgNPs toxicity. Although a large number of small sized NPs in both colloidal and fractionated solution in fish acute test was observed as well (Fig. 2.c vs. d), measured actual ionic Ag concentrations were lower than those in daphnia exposing solutions, which might be due to different chemistry between solutions such as strong salification rather than fractionation processes. The Importance of AgNP Suspension Methods for Toxicity Recent studies suggested that AgNPs toxicity is mediated by released silver ions (Zhao and Wang 2012), although other toxic mechanisms of AgNPs such as unique physiocochemical properties of AgNP such as size or shape (Scown et al. 2010) and oxidative stress produced by the AgNPs in aqueous phase (Choi and Hu 2008) are still of concern. The present study applied two types of AgNPs suspension methods and compared their toxicities to daphnia or fish and demonstrated that they were toxic to test organisms, but the level of toxicity was different according to the method applied for suspension. Our results indicated that fractionated solution was less acutely toxic to D. magna than the non-fractionated solution. The daphnia48h-EC50 of colloidal AgNPs was 6.1 lg/L, which is close to the results of Asghari et al. (2012) (2–4 lg/L) and Lee et al. (2012) (7.98 lg/L) that applied similar exposing processes to ours. On the other hand, fractionated solution to daphnia (48h-EC50 13.8 lg/L) were about 2 times less toxic than colloidal solution (48h-EC50 6.1 lg/L). It is expected that the toxicity of colloidal solution was added by ionic Ag released as observed high ionic silver concentrations (Table 3). Although many studies have investigated the origins of AgNPs toxicity such as their unique attributes such as size, shape, or coating materials (Scown et al. 2010; Kwok et al. 2012; Zhao and Wang 2012), only a few studies addressed the importance of test preparations for evaluating toxicity of AgNPs (Kennedy et al. 2010; Asghari et al. 2012). Kennedy et al. (2010) compared the toxicity of nanosilver with fractionated nanosilver and
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emphasized the importance of characterizing ionic sliver in nanomaterial suspensions, and Asghari et al. (2012) compared the toxicity of various types (colloids vs. suspension) of AgNPs. However, these studies applied ultracentrifugation and acidification with nitric acid to prepare fractionated suspension (Kennedy et al. 2010), which might cause unexpected toxicity due to the applied suspension method or compared the toxicity of AgNPs suspension to that of AgNPs colloids obtained from different sources (Asghari et al. 2012), which might lead inaccuracy to test whether suspension method is the only factor that affected toxicity of NPs. Current study applied environmentally relevant suspension method, i.e., simple combination of stirring and settlement, and compared toxicity between fractionated and colloidal AgNP solutions coming from same AgNP stocks.
Conclusion The toxicity of AgNPs has already been reported in many aquatic organisms and the number of these studies would be continuously increased. However investigations of the distinct characteristics of AgNPs (such as preparation methods) that might affect their toxicity to organisms and mislead the hazards of AgNPs to human and ecosystem are currently lacking. The present study clearly indicated that methods for preparing AgNP suspensions changed the toxicity to organisms, but these would be only effective on a case by case basis, for example in some toxicity tests that contain high electrolyte concentrations in their test media. These findings will help to draw a toxicological agreement about how to expose and evaluate NPs toxicity in aquatic toxicity tests, and suggest a universal agreement in standardized suspension method to test NP toxicity. Acknowledgments The authors are pleased to acknowledge Prof. Theodore B. Henry (Heriot-Watt University) who provided critical review for this manuscript. This research was funded by the project for Environmental Risk Assessment of Manufactured Nanomaterials (KK-1303-03), the Korea Institute of Toxicology (KIT) and the Industrial Core Technology Program (10034759, Risk Management Technology for Nano Products), the Ministry of Trade, Industry and Energy (MOTIE).
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