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Keywords:

  • Silver nanoparticles;
  • Ecotoxicity effects;
  • Ion-release kinetics

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. References
  10. Supporting Information

The environmental toxicity associated with silver nanoparticles (AgNPs) has been a major focus in nanotoxicology. The Ag+ released from AgNPs may affect ecotoxicity, although whether the major toxic effect is governed by Ag+ ions or by AgNPs themselves is unclear. In the present study, we have examined the ecotoxicity of AgNPs in aquatic organisms, silver ion-release kinetics of AgNPs, and their relationship. The 48-h median effective concentration (EC50) values for Daphnia magna of powder-type AgNP suspensions were 0.75 µg/L (95% confidence interval [CI] = 0.71–0.78) total Ag and 0.37 µg/L (95% CI = 0.36–0.38) dissolved Ag. For sol-type AgNP suspension, the 48-h EC50 values for D. magna were 7.98 µg/L (95% CI = 7.04–9.03) total Ag and 0.88 µg/L (95% CI = 0.80–0.97) dissolved Ag. The EC50 values for the dissolved Ag of powder-type and sol-type AgNPs for D. magna showed similar results (0.37 µg/L and 0.88 µg/L) despite their differences of EC50 values in total Ag. We observed that the first-order rate constant (k) of Ag+ ions released from AgNPs was 0.0734/h at 0.05 mg/L total Ag at 22°C within 6 h. The kinetic experiments and the toxicity test showed that 36% and 11% of sol-type AgNPs were converted to the Ag+ ion form under oxidation conditions, respectively. Powder-type AgNPs showed 49% conversion rate of Ag+ ion from AgNPs. We also confirmed that Ag+ ion concentration in AgNP suspension reaches an equilibrium concentration after 48 h, which is an exposure time of the acute aquatic toxicity test. Environ. Toxicol. Chem. 2012;31:155–159. © 2011 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. References
  10. Supporting Information

Engineered nanoparticles with sizes between 1 and 100 nm in at least one dimension have versatile applications in many commercial products, including tires, stain-resistant clothing, cosmetics, computer chips, drug-delivery systems, medical imaging, and implants 1. However, despite the already widespread use of nanomaterials in modern technology, the lack of information on the human and environmental implications of manufactured nanomaterials is acute 2, 3.

Worldwide, markets for Ag-containing nano-functionalized products are seeing substantial growth 4, 5. According to the nanotechnology consumer products inventory by the Project on Emerging Nanotechnologies, of the 1,317 nanotechnology-based consumer products or product lines available on the market in March 2011, products containing silver nanoparticles (AgNPs) accounted for the largest (24%) and fastest growing category (http://www.nanotechproject.org/inventories/consumer/analysis_draft/). Because of the antibacterial properties of AgNPs, their use in consumer and medical products has become increasingly common, given the biocidal effect of silver ions 6. However, major concerns exist regarding the potential toxic impacts on ecosystems and microorganisms when AgNPs are released into the environment. Silver in its ionic form (Ag+) is well known to be toxic to aquatic organisms at concentrations of a few micrograms per liter 7. For freshwater fish and invertebrates, a key mechanism of acute Ag toxicity consists of reduction in Na+ uptake by the blockade of gill Na+, K+-adenosine triphosphatase 8, 9. The Ag+ ion inhibits enzymes acting in the phosphorus, sulfur, and nitrogen cycles of nitrifying bacteria 10. The biocidal mechanism of silver-containing products results from long-term release of silver ions (Ag+) by oxidation of metallic silver (Ag°) in contact with water 11. The production of Ag+ ions from AgNPs may have toxic effects 12, 13. However, whether the toxicity of AgNPs is indeed only attributable to dissolved Ag, or whether AgNPs themselves show a toxic effect 13, is unclear. Choi et al. 14 showed that released Ag ions react very quickly with common environmental ligands such as sulfide and chloride, which can decrease the toxicity of AgNPs. Many important aspects of nanosilver behavior are influenced by the ionic activity associated with particle suspension, including antibacterial potency, eukaryotic toxicity, environmental release, and particle persistence 15. Although a number of authors have published literature on toxicological information of AgNPs 16, 17, exactly how, at which concentrations, and in what form, AgNPs will be released into the environment is unclear.

The main objectives of the present study were to understand the release kinetics of Ag+ ions from AgNPs into water environment, and to determine to what extent Ag+ ions released from sol-type and powder-type AgNPs contribute to ecotoxicity in Daphnia magna.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. References
  10. Supporting Information

Materials

The following two types of AgNPs were used: sol-type AgNPs (citrate-stabilized AgNPs colloids [20.36% (wt/wt), product name: SARPU 200KW, average particle size: 13.3 nm]); and powder-type AgNPs (product name: SNC-S100, primary particle size: 60–100 nm). Both were purchased from ABC Nanotech. The size and shape of the two types of AgNPs are characterized and summarized in Supplemental Data, Figure S1. The sol-type AgNPs had a negative surface charge (zeta potential −45.2 mV), suggesting stabilization by a surface layer of citrate anions.

Characterization of AgNPs

The morphology and size of AgNPs were confirmed by transmission electron microscopy using a field emission transmission electron microscope (Tecnai G2 F30 S-Twin) and scanning electron microscopy using a field emission scanning electron microscope (Sirion). The surface charge and size distribution of AgNPs were evaluated by zeta potential and dynamic light scattering, respectively, using a nanoparticle analyzer (Nano ZS 90, Malvern Instruments).

Acute toxicity tests for AgNP suspensions using D. magna

The ecotoxicity tests of two types of AgNPs were performed as the procedures shown in Figure 1. Because powder-type AgNPs are poorly dispersed in water, the test solution is assumed to contain the following three forms of silver 15: Ag0 solids, free Ag+ ions or complexes (dissolved form), and surface-adsorbed Ag+ ions. Mixtures of individual substances with different solubilities and physical-chemical properties are frequently referred to as “complex mixtures” 18. Therefore, AgNP suspensions that are only partially dispersed in water could be considered complex multi-component substances, and their toxicity could be determined by preparing water-accommodated fractions (WAFs). We prepared WAFs as follows: The powders were added directly to the culture water (Elendt's M4 medium), in which AgNPs at concentrations of 100 mg/L were prepared, and stirred for 24 h and settled for 48 h (step 1 in Fig. 1). We assumed that the aggregated AgNPs, which are mostly settled at the bottom of the suspension, would not affect ecotoxicity because the size of aggregated nanoparticles is so great that they cannot penetrate the cell membrane; therefore the top part of the suspension was taken for the ecotoxicity test (step 2 in Fig. 1). Additional filtration was performed to remove any aggregated AgNPs that presented in the top part of the suspension, using 0.45-µm membrane filters (Advantec MFS). Because sol-type AgNPs were well dispersed in a suspension, when AgNPs were much smaller than 0.45 µm, they were simply added to the culture water for the ecotoxicity test without any further filtration. The testing solution for the ecotoxicity test of powder-type AgNP was prepared through steps of 48-h settling and 0.45-µm filtering. Aggregation/agglomeration of nanoparticles occurs during the preparation of test solutions; therefore, maintaining reproducible exposure suspensions of the powder-type of AgNPs within typical aquatic toxicity tests is important. The amount of total Ag is measured during the settling step, and it also fluctuates because of the aggregation/agglomeration of nanoparticles (see Supplemental Data, Fig. S2). Therefore, we arbitrarily set the settling time as 48 h and prepared WAFs for the ecotoxicity test. Nevertheless, the actual amount of Ag for the ecotoxicity test is not affected by the fluctuation because WAFs, not powder-type AgNPs, are applied to the testing solution. The prepared WAFs were diluted with media culture to the desired concentration, and the toxicity tests were conducted.

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Figure 1. Ecotoxicity test scheme for powder-type and sol-type silver nanoparticles (AgNPs).

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An acute immobilization test for D. magna was carried out in accordance with Test Guideline 202 of the Organisation for Economic Co-operation and Development 19. Daphnia magna were purchased from Carolina Biological Supply Company in 2008 and reared in the culture room in the Division of Nonclinical Studies, Korea Institute of Toxicology, under a 16:8-h light:dark photoperiod, with a 30-min transition period at 18 to 22°C. Gravid adults were isolated 24 h before initiation of the test, and young daphnids produced overnight (<24 h of age) were used in toxicity tests. A total of 20 daphnids, divided into four groups of five, were used for each test concentration and the control. Each group was placed in a 120-ml glass vessel filled with 100-ml test solutions prepared from the WAF or AgNP suspensions. The test was conducted on the basis of the static method under the same culture conditions as described.

To determine dissolved Ag containing free Ag ions or its complexes (for example, Ag+ complexed to citrate), AgNPs were removed using Amicon centrifugal ultrafilter devices (Millipore) containing porous cellulose membranes with a nominal particle size limit of 1 to 2 nm. The suspensions were centrifuged for 1 h at 3,220 g (Eppendorf Centrifuge 5810R). Dissolved Ag was tracked by inductively coupled plasma-mass spectrometry (ICP-MS) using a PerkinElmer Elan 6000. The ultrafiltration ICP-MS method did not distinguish between the forms of dissolved Ag (Ag ions and Ag complexes). The total Ag concentration was quantitatively analyzed by ICP-MS after HNO3 digestion.

The EC50 values and 95% confidence intervals (CIs) were calculated using the Probit method 20. Mean ( ± standard deviation [SD]) values of the measured Ag concentrations were calculated from three replicates.

Kinetic experiments

Silver nanoparticle dissolution kinetics in air-saturated deionized water (8.5 mg/L dissolved O2, initial pH 5.5–5.8) was investigated. The AgNP stock suspension was diluted with deionized water to three desired starting concentrations (0.05, 0.1, and 1.0 mg/L). Ion-release experiments were conducted at 22°C in 15-ml plastic tubes protected from light unless noted. Dissolved Ag was tracked by ultrafiltration ICP-MS method. We examined the Ag-ion concentration using an Ag+-selective electrode (Orion94-16, Thermo Scientific). A calibration curve between 0.01 mg/L and 0.1 mg/L was made with Ag+ (ICP Standard, KRIAT). Measured values were read against a calibration curve.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. References
  10. Supporting Information

Ecotoxicity effects of AgNPs

Figure 2 represents acute toxicity test results for powder-type and sol-type AgNPs using the testing solution prepared from the WAFs. We found that powder-type AgNPs were highly toxic to D. magna, with a 48-h static EC50 of 0.95 µg/L (95% CI = 0.93–0.99) nominal total Ag, 0.75 µg/L (95% CI = 0.71–0.78) measured total Ag, and 0.37 µg/L (95% CI = 0.36–0.38) dissolved Ag. Approximately 49% of the total Ag was present in the dissolved form. Figure 2B represents acute toxicity test results for sol-type AgNPs. The 48-h EC50 value for D. magna was 10.30 µg/L (95% CI = 8.83–12.02) nominal total Ag, 7.98 µg/L (95% CI = 7.04–9.03) measured total Ag, and 0.88 µg/L (95% CI = 0.80–0.97) dissolved Ag. Approximately 11% of total Ag was present in dissolved form. Kim et al. 21 showed that AgNO3 was highly toxic to D. magna, with a 48-h static EC50 of 0.5 µg Ag/L (95% CI = 0.4–0.6), which is in accordance with the 48-h static EC50 of 0.37 to 0.88 µg dissolved Ag/L obtained in the present study. Surprisingly, EC50 values of dissolved Ag were very similar in both powder-type and sol-type AgNPs (0.37 µg/L and 0.88 µg/L dissolved Ag, respectively), despite the fact that EC50 values differed significantly (0.75 µg/L and 7.98 µg/L measured total Ag, respectively). From our experimental results and previous AgNO3 results, we can conclude that the amount of dissolved Ag showing toxicity in the testing solution was almost the same. Therefore, AgNPs were shown to be acutely toxic to D. magna because of Ag ions released from AgNPs.

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Figure 2. Toxicity of silver nanoparticle (AgNP) suspension to Daphnia magna after 48-h exposure. (A) Powder-type AgNPs. (B) Sol-type AgNPs.

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Kinetic study of Ag ion release in AgNP suspension

Because dissolved Ag is responsible for ecotoxicity to D. magna, one must understand the release kinetics of Ag+ ions from AgNPs into the water environment. Powder-type AgNPs were considered an inadequate sample for conducting the kinetic study because they are not easily dispersed and are likely to form precipitations quickly through interactions between particles. On the contrary, the ion-release kinetic study should be performed with sol-type AgNPs because the nanoparticles are well dispersed in a solution and should maintain a reliable kinetic sample during the test. The surfactants (citrate) coating the surface of sol-type AgNPs might affect the release kinetics, but that effect is not in the scope of the present study.

As AgNPs are introduced to water solution, the nanoparticles undergo the following redox reaction and release Ag+ ions 15.

  • equation image

However, as the released Ag+ ion reaches a certain concentration, the solution is believed to be under equilibrium, where released Ag+ ions can either form aggregates or rejoin existing nanoparticles. The reaction should become an equilibrium reaction occurring through cooperative oxidation involving protons and dissolved oxygen. This argument is well observed in our kinetic experiment and summarized in Figure 3. Within 6 h, the concentrations of Ag+ ions increased exponentially at each of the three concentrations. The concentrations of Ag+ ions fluctuated thereafter because of an unstable aggregation. A previous study on the release kinetics of Ag+ ions claimed that the release reaction follows first-order kinetics 15. In our experiment, we also checked the release kinetics for the first 6 h, where the release of Ag+ ions from AgNPs is the dominant reaction (see Fig. 3B–D). Indeed, our experiments agreed with the previous study, and the rate constants were calculated to 0.0734/h, 0.0709/h, and 0.0278/h for initial AgNPs concentrations of 0.05 mg/L, 0.1 mg/L, and 1 mg/L, respectively. For 0.05-mg/L and 1-mg/L concentrations of samples, the rate constants are very similar (0.0734/h and 0.0709/h, respectively); however, the rate constant in a high-concentration sample (1.0 mg/L AgNPs) was quite different (k = 0.0278/h). Silver nanoparticles with a higher concentration have a smaller rate constant. Ag+ ion-release rate in a high concentration of AgNPs is retarded because the amount of Ag+ ion rapidly reaches a certain critical concentration. The amount of Ag+ ion and dissolved Ag released from 1 mg/L AgNPs are represented distinctively in Figure 4. As expected, Ag+ ions were released abruptly within 6 h from AgNPs and showed a lower concentration than dissolved Ag. Dissolved Ag contains Ag+ and Ag+ complexes, and Ag+ ion-release reaction was almost completed during the first 6 h.

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Figure 3. Kinetic experimental results of Ag+ ion release from sol-type silver nanoparticles (AgNPs). (a) Extent of dissolved silver release from AgNPs as a function of time in air-saturated deionized water at 22°C. (b) First-order Ag+ ion-release rate in 0.05 mg/L AgNPs for the first 6 h. (c) First-order Ag+ ion-release rate in 0.1 mg/L AgNPs for the first 6 h. (d) First-order Ag+ ion-release rate in 1.0 mg/L AgNPs for the first 6 h.

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Figure 4. 1.0 mg/L silver nanoparticle (AgNP) dissolution (Ag+ ion and dissolved Ag) kinetics in air-saturated deionized water at 22°C.

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We estimated the concentration of dissolved Ag in the AgNP suspension using the obtained first-order rate constant (k) and compared it with the result of the toxicity test. The reason for selecting the k at the lowest level of AgNPs is that AgNPs are very toxic to D. magna and can exist at a very low level (ppb) in real environments 6. By substituting [Ag]o in [Ag] = [Ag]oe-kt with the measured total Ag of 7.98 µg/L obtained in the toxicity test of sol-type AgNPs, total [Ag] would be 5.14 µg/L, and [Ag+] would be 2.84 µg/L at 6 h. Approximately 36% of AgNPs were converted into the dissolved Ag form. Liu et al. 15 investigated long-term AgNP dissolution kinetics, and complete reactive dissolution appeared after 6 to 125 d. However, in the present study, complete reactive dissolution was not observed until 7 d. Despite the small fluctuation of Ag+ ion concentration after 6 h, we assumed that it should reach equilibrium after 48 h, which is an exposure time during the acute ecotoxicity test (Fig. 3A). Therefore, we can assume that the EC50 value (in dissolved form) should be the concentration of Ag+ ion in AgNP suspension at equilibrium. An aquatic organism is a very important kinetic compartment that will rapidly uptake Ag+ ions from the system. However, the conversion ratio in the toxicity test was 11%, which is quite different from the conversion ratio, 36% in the release kinetic experiment. Because the ion-release kinetic experiments were not performed in the same medium as in the toxicity test but in a very simple solution (deionized water medium), Ag+ release kinetics should be slowed in a more complex environment. For powder-type AgNPs, the toxicity test showed that 49% of total Ag was present in the dissolved form and showed a higher conversion rate than the calculation (36%). Considering that the two types of AgNPs (powder-type and sol-type) are different in their characteristics, we could expect the conversion rate of Ag ions to be higher in noncapped powder-type AgNPs than in capped sol-type AgNPs.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. References
  10. Supporting Information

We evaluated the ecotoxicity of AgNPs represented in total Ag concentration and dissolved Ag (Ag+ ion) concentration, respectively, recognizing that the preparation method for obtaining WAFs was very useful in obtaining a reproducible testing solution of powder-type AgNPs. Silver nanoparticles were acutely toxic to D. magna, the result of Ag+ ions released from AgNPs. Despite some differences between EC50 values in total Ag in powder-type and sol-type AgNPs, the EC50 values in dissolved Ag form showed little difference. The conversion ratio of sol-type AgNP suspension into Ag ions is higher in the kinetic experiment performed in deionized water (36%) than in the toxicity test performed in Elendt's M4 medium (11%). In a more complex environment, the Ag+ release rate should be slowed. In the wild, the Ag ion-release rate might be slower than in the laboratory because of the presence of nutrients, salts, and natural organic matter. Thus, AgNPs may not persist long enough to be converted into Ag ions in nature. However, given that free Ag ions are highly toxic to aquatic organisms, a low conversion rate of AgNPs to Ag+ ions would be fatal.

SUPPLEMENTAL DATA

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. References
  10. Supporting Information

Figure S1. Morphology and size distribution of silver nanoparticle (AgNP) samples used in this study.

Figure S2. Changes in total Ag concentrations in powder-type silver nanoparticle (AgNP) suspension according to settling time in Figure 1. (142 KB DOC)

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. References
  10. Supporting Information

We acknowledge financial support from the Ministry of Knowledge Economy, Republic of Korea, for the National Platform Technology Project, and SHK acknowledges the support by the National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF-2010-M2-20100028702).

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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