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Keywords:

  • Silver nanoparticles;
  • Total surface area;
  • Zebrafish;
  • Microbial toxicology;
  • Nanotoxicology

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Silver nanoparticles (Ag NPs) have been classified as the most abundant NP found in commercial products. In the present study, zebrafish (Danio rerio) and bacteria (Escherichia coli; ATCC 25922) were used to test the size-dependent toxicological effects of Ag NPs, the effects of ionic silver versus Ag NPs, and Ag NP effects on mortality using mass concentration (mg/L) compared with total surface area (nm2/L). Several diameters of Ag NPs (20, 50, 110 nm) as well as AgNO3 were chosen as experimental treatments. Treated zebrafish embryos exhibited anomalies of the heart, namely, slower heart rates and pericardial edema. A size-dependent response was not observed in zebrafish when viewing mortality across all Ag NP treatments, although 20 nm elicited the highest incidence of abnormal motility and induced slower development. An Ag NP dose- and size-dependent response was observed in treated bacteria using mass concentration, with 20-nm Ag NP producing the highest mortality rate. In both zebrafish and bacteria, AgNO3 was shown to be more toxic than Ag NPs at equivalent concentrations. When total surface area of Ag NPs was used to gauge bacterial mortality, a total surface area-dependent, but not size-dependent, response was observed for all three Ag NPs used in the present study, with nearly 100% mortality observed once a total surface area of approximately 1E + 18 nm2/L was reached. This trend was not apparent, however, when measuring total surface area for zebrafish mortality. Environ. Toxicol. Chem. 2012; 31: 1793–1800. © 2012 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Nanoparticles (NPs) are identified as particles with dimensions of 1 to 100 nm 1. Nanoparticles have been shown to exhibit highly reactive behavior in biological systems because of a relatively large surface-to-volume ratio, resulting in a high percentage of atoms on the surface of the NPs compared with larger structures 2. The dynamic nature of these particles has proved most useful in many technological applications, including delivering drugs to the cells of cancer patients 3, increasing the efficiency of solar panels 4, and even lowering the emissions and improving the performance of biodiesel engines 5.

It is estimated that by 2015 the nanotechnology economy will be valued at more than $1 trillion 6, but very little is known about the toxicological effects in biological systems. Previous research indicates that NPs may pose a serious risk to people and to the environment 7. Studies performed in vivo show that NPs can be deleterious to model organisms (e.g., bacteria, algae, invertebrates, and fish) in aquatic systems 8.

Traditionally, researchers have gauged organismal toxicity by evaluating the mass concentration (e.g., mg/L) of various chemicals and compounds, although it has been suggested that mass concentration may not be the most appropriate dose metric for NPs 9. Given that surface area may play an integral role in NP toxicity, perhaps there is more relevance in quantifying total surface area of NPs in suspension (e.g., nm2/L), instead of using only mass concentration. Indeed, in one study investigating the inhalation of ultrafine and fine carbon NPs in rats, researchers found significantly different results when gauging toxicity on a mass concentration basis compared with total surface area 10. In addition, Oberdorster 11 treated rats with ultrafine and fine TiO2 NPs and discovered that ultrafine NPs induced a greater neutrophil response when using equivalent mass concentrations. However, when toxicity was gauged using total surface area, the toxic response for both ultrafine and fine TiO2 fit the same dose–response curve. The author of the latter study surmised that total surface area might be a more valid dose metric for assessing toxicity when testing different-sized NPs with the same surface chemistry. Furthermore, researchers studying equivalent mass concentrations of different NPs (e.g., Au, Ag, TiO2) found the corresponding total surface area of these NPs to differ by orders of magnitude 12.

Silver nanoparticles (Ag NPs) have been classified as being the most common nanomaterials to be engineered for commercial use 13. Because of their antimicrobial effects, Ag NPs are frequently used in medicine, water-purification systems, cosmetics, and many consumer products, including washing machines, food containers, and clothing 14. One study has shown that Ag NPs can be leached into wastewater during clothes washing 15, possibly contaminating bacteria in wastewater treatment facilities. Such leaching could consequently place a risk on aquatic organisms in receiving streams and lakes.

Research performed on model organisms, such as zebrafish and Japanese medaka embryos, have shown Ag NPs to induce mortality in a size-dependent manner and to delay hatching 16, 17. Silver nanoparticles have been observed in the brain, testis, liver, and blood throughout embryogenesis of zebrafish 18. Researchers have also shown that Ag NPs penetrate the chorion of zebrafish embryos, leading to circulatory and morphological abnormalities 19. In adult zebrafish, Ag NPs (20–30 nm and spherical) were acutely lethal, with an LC50 of 7.07 mg/L 16. Furthermore, molecular studies of mammalian cell lines have shown Ag NPs to elicit toxicity in a size-dependent manner, with smaller NPs (<20 nm) producing a more toxic effect at concentrations equivalent to larger Ag NP treatments 20.

The exact mechanism of Ag NP toxicity has not been definitively shown, although some researchers have provided evidence that toxicity might result not directly from the NPs themselves but rather from Ag NPs acting as a delivery system for silver ions (Ag+) 8, 21. Other research 22, however, shows Japanese medaka having a greater stress response to Ag NPs compared with Ag+, leading the investigators to conclude that Ag+ dissolution is not solely responsible for toxicity. Similar conclusions have been reached when bacteria are exposed to Ag NPs. For example, Lok et al. 23 showed the dissolution of Ag+ to compromise cell integrity in bacteria. However, other research has demonstrated that the size and shape of Ag NPs play a more significant role in bacterial toxicity, with Ag NPs creating disruptions in cell membranes and inhibiting cellular respiration nearly twice as much as Ag+ 21, 24, 25.

The purpose of the present study was to (1) characterize the Ag NPs (20, 50, 110 nm) employed in the study using transmission electron microscopy (TEM), ultraviolet-visible spectroscopy, and dynamic light scattering (DLS) techniques; (2) determine whether developing zebrafish (Danio rerio) embryos and bacteria (Escherichia coli; ATCC 25922) elicit a size-dependent toxicological response to three different sizes of Ag NPs (20, 50, 110 nm); (3) determine whether ionic silver (AgNO3) produces a more toxic effect in developing zebrafish embryos and E. coli bacteria compared with various Ag NP sizes; and (4) gauge the toxicity of Ag NPs to zebrafish and E. coli bacteria based on a comparison of total surface area (nm2/L) versus mass concentration (mg/L).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Silver nanoparticles characterization methods

Silver nanoparticles with nominal diameters of 20, 50, and 110 nm were provided by nanoComposix and synthesized via methods explained by Park et al. 9. Briefly, Ag NPs were synthesized using an aqueous reduction technique from silver salts to a final concentration of 2 mM in phosphate buffer. After extensive washing, the surface of Ag NPs consisted of a phosphate monolayer. Particle size distribution and particle shape were determined by using TEM, dynamic light scattering techniques, and ultraviolet-visible spectroscopy. All NPs used in the present study were approximately spherical based on TEM images. Therefore, based on the diameter of >100 particles selected at random, surface area calculations for each size class of NPs (20, 50, 110 nm) were performed by the following: (1) multiplying the density of silver (10.49 g/cm3) by the average volume of an NP to determine the mass of an individual NP in each size class; (2) measuring mass concentration of each solution by inductively coupled plasma–mass spectroscopy (ICP-MS); (3) dividing the mass concentration of each dose (mg/L) by the mass of a particle to calculate the number of particles in each dose (particles/L); and (4) multiplying particles per liter by the surface area of an individual particle to determine the amount of total surface area per liter in each dose (nm2/L). Calculations to determine surface area per liter were performed on the actual average particle size measured via TEM imaging for each size class. Standard error for the surface area per liter of each size class was calculated by using standard errors of the particle diameter measurements. Nominal particle size classes were used to report E. coli and zebrafish results. Silver nitrate (AgNO3) was purchased from Sigma-Aldrich.

Zebrafish culturing and assay

Wild-type, adult zebrafish (Danio rerio) were kept in aquaria (30 L) at 28.5 ± 2.0°C with a 14:10 h light:dark cycle. Aquarium water consisted of 60 mg/L Instant Ocean in deionized water at a pH of 7.0 ± 0.2. Zebrafish were fed twice daily with Omega One dry flakes and were intermittently given brine shrimp for supplementation. Mating protocols consisted of four 0.8-L breeding tanks, each containing two males and two females. Adult zebrafish were introduced to the breeding tanks 1 h before the beginning of the dark cycle and occasionally fed brine shrimp to reduce stress. Embryos were randomly collected 1 h postfertilization (hpf) and washed briefly (∼1 min) in a 0.05% bleach solution. To ensure the removal of bleach, embryos were washed three times with fresh aquarium water.

Each replicate consisted of five embryos placed in a 30-mm petri dish containing 10 ml of the appropriate dosing suspension. Silver nanoparticle treatments (20, 50, 110 nm) were prepared at 0.1, 0.5, 1, and 5 mg/L. Silver nitrate treatments were prepared at 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 mg/L. All treatments were performed in triplicate, resulting in 15 embryos per treatment. All dilutions were made using aquarium water. Controls were treated identically to the treatments with the exception of Ag NPs or AgNO3 being absent from solution. All Ag treatments were prepared and probe sonicated (Fisher Scientific 4C15) at 60 Hz for approximately 30 s prior to dosing to ensure even dispersal of Ag NPs.

Experiments were performed in environmental chambers (Fisher Scientific 100FS) at 28.5 ± 1.0°C. Exposed embryos were observed at 24, 72, and 120 hpf using an inverted microscope (Nikon Eclipse TS100). Embryological and larval toxicity was determined by using the following endpoints: mortality, heart rate, pericardial edema, chorion turbidity, motility, and otic vesicle-to-optic cup distance (OV-OP; Fig. 1). Table 1 defines each endpoint, the time points at which each endpoint was observed, and the method of data collection. Data for pericardial edema, chorion turbidity, motility, and OV-OP were calculated as percentage of difference relative to control.

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Figure 1. Demonstration images of selected zebrafish (Danio rerio) endpoints. (A) Control embryo at 24 h postfertilization, (B) arrow indicates pericardial edema at 72 h postfertilization, (C) arrow indicates chorion turbidity at 24 h postfertilization. (D) bar spans the length (µm) of otic vesicle-to-optic cup distance (OV-OP), which defines OV-OP, in an embryo at 24 h postfertilization. A,C,D: ×100; B: ×40.

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Table 1. List of endpoints used to assess zebrafish response to silver treatments
EndpointObservation time (hours postfertilization)Endpoint descriptor
Mortality24, 72, and 120Lack of heart beat
Heart rate24 and 72No. of heart beats during 10 s of observations
Pericardial edema24 and 72Observation of abnormal fluid accumulation around the heart compared with controls
Chorion turbidity24Observation of abnormal opaqueness in chorion compared with controls
Otic vesicle-to-optic cup distance24Length (µm) between the otic vesicle and optic cup relative to controls
Motility72 and 120Observation of abnormal swimming, pectoral fin gyrations, or mouth movements compared with controls

Escherichia coli culturing and assay development

The amount of time necessary for E. coli (ATCC 25922) to reach mid-log growth and, specifically, a transmittance value corresponding to precisely 1.5 × 108 colony forming units (CFU)/ml was determined following standard methods 26. Briefly, tryptic soy broth was inoculated with E. coli and cultured at 35 ± 2°C in an incubator shaker at 200 rpm. Transmittance in the broth was determined at 625 nm at 30-min intervals during the incubation period. Numbers of CFU/ml in the culture were determined at the same time points as transmittance values by removing and diluting aliquots from the broth and spreading on 100-mm tryptic soy agar plates with a hockey stick spreader. The plates were then incubated for 24 h at 35 ± 2°C, and colonies were counted. It was determined that a transmittance value of 62% corresponded to approximately 1.5 × 108 CFU/ml, so at the initiation of each experiment a glass tube was inoculated and grown to this transmittance value and referred to as tube “A.”

Because the Ag NPs are stable in phosphate-buffered saline (PBS), the effects of Ag NP binding to tryptic soy broth components are not known, and E. coli numbers were determined to be static in PBS up to 60 min (data not shown), we chose to carry out all bacteria dosing studies in PBS (0.01 M phosphate buffer, 0.0027 M KCl, 0.137 M NaCl, pH 7.4; Sigma; no. P4417) at a 60-min dosing time. At the beginning of each experiment, bacteria from tube A were diluted to 4.5 × 106 CFU/ml in sterile PBS (tube “B”).

Bacterial dose–response assays

The bacterial dose–response assays were based on standard methods used to assess the minimum bactericidal concentration of antimicrobial agents to bacteria 26. Tubes A and B were prepared as outlined above. A series of dilutions was prepared in sterile PBS in capped glass culture tubes for each material being tested, such that the concentrations after the addition of bacteria would be (in mg/L): 20-nm particles for 5, 10, 25, and 50; 50-nm particles for 10, 25, 50, 75, and 100; 110-nm particles for 50, 75, 100, 220; and AgNO3 for 0.25, 0.50, 0.75, 1.0, 2.0. Just prior to adding the bacteria, each of the dilutions was probe sonicated as described above to ensure suspension of the particles. One hundred microliters of bacterial suspension (∼4.5 × 105 CFU) from tube B was then added to each of the culture tubes at time 0, resulting in a total volume of 1 ml. Tubes were placed in an incubator shaker at 35 ± 2°C. At the end of the 60-min dosing period, 100 µl was removed from each culture tube and plated individually onto 100-mm tryptic soy agar plates using a hockey stick spreader. The plates were incubated for 24 h at 35 ± 2°C, and colonies were counted. The dose–response data were plotted both as mass concentration (mg/L) of material versus CFU/100 µl and also as total surface area (nm2/L) of particles versus CFU/100 µl. Three replicates were run for each dose with each particle size and AgNO3. Both positive and negative controls were prepared identically to all treatments, except without silver (positive control) and without bacteria (negative control) and demonstrated appropriate bacterial growth.

Statistical analysis

Statistical analysis was performed in SigmaStat Version 3.1 (Systat Software). One-way ANOVA using a Tukey's post hoc test and normality testing, with a significance value set at 0.05, was used to compare the zebrafish endpoints of mortality, heart rate, and OV-OP with control values.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Ag NP characterization

Transmission electron microscopy, dynamic light scattering techniques, and ultraviolet-visible spectroscopy analysis resulted in nominal mean particle sizes for 20-, 50-, and 110-nm particles with differing amounts of variation (Table 2, Fig. 2). Transmission electron microscopy imagery showed all particles having an approximately spherical shape (Fig. 2). Average NP mass and surface area for each size class varied, with the smallest NPs (20 nm) having the largest surface area per volume. A lack of Ag NP aggregation was verified via TEM imaging over a 120-h period for PBS and aquarium water treatments employed in the present study. It was found that probe sonicating all treatments for 30 s prior to dosing was sufficient to maintain Ag NPs within each suspension.

Table 2. Particle characterization of silver treatmentsa
Parameter20 nm50 nm110 nm
  • a

    Silver nanoparticles (Ag NPs) (20, 50, 110 nm) were suspended in deionized water at a concentration of 0.02 mg/L for characterization. All values are represented as mean ± standard deviation.

  • b

    Total surface area calculated for 1 mg/L.

Mean particle diameter (nm; transmission electron microscopy)20.3 ± 1.953.1 ± 4.1112.6 ± 7.8
Hydrodynamic diameter (nm; dynamic light scattering)27 ± 10.857.9 ± 23.5111.6 ± 29.9
Min/max diameter (nm; transmission electron microscopy)16/25.441/64.995.3/148.9
Nanoparticle mass (g)4.39E–176.87E–167.31E–15
Total surface area (nm2/L)b2.82E + 161.08E + 165.08E + 15
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Figure 2. Ag NP particle characterization using transmission electron microscopy and ultraviolet visible spectroscopy. (A) 20-nm Ag NP exhibited a mean diameter of 20.3 ± 1.9 nm and displayed maximal absorbance at 400 nm, (B) 50-nm Ag NP exhibited a mean diameter of 53.1 ± 4.1 nm and displayed maximal absorbance at 420 nm, and (C) 110-nm Ag NP exhibited a mean diameter of 112.6 ± 7.8 nm and displayed maximal absorbance at 520 nm. Silver nanoparticles were suspended in 0.02 mg/ml deionized water.

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Silver nanoparticle and AgNO3 toxicity in zebrafish

Silver nanoparticles (20, 50, 110 nm) induced a concentration-dependent response in survivorship (percentage alive at a given time) at 72-hpf (Fig. 3A). Differences in survivorship were observed among doses for each Ag NP (20 nm: f4,11 = 20.076, p < 0.001; 50 nm: f4,11 = 30.021, p < 0.001; 110 nm: f4,11= 4.049, p = 0.029). Significant differences from controls (p < 0.05) were detected by post hoc tests in 20-nm particles at 1 and 5 mg/L; 50 nm at 0.5, 1, and 5 mg/L; and 110 nm at 5 mg/L. Control embryos exhibited 95% survivorship at 72 hpf. Zebrafish treated with AgNO3 showed nearly 100% survivorship for mass concentrations of 0.001 to 0.05 mg/L, although a sharp decrease in survivorship was observed at 0.1 mg/L, with zebrafish displaying 100% mortality (Fig. 3C). It is noteworthy that the mass concentrations of AgNO3 necessary to cause 100% mortality were at least 10-fold lower than any Ag NP. For 20-nm Ag NPs, 100% mortality was reached at a mass concentration of 5 mg/L and a total surface area of 1.4E + 17 nm2/L, and 50-nm particles reached 100% mortality at 1 mg/L and 1.1E + 16 nm2/L, respectively (Fig. 3A and B). The only Ag NP not to induce 100% mortality at a mass concentration of 5 mg/L was 110 nm (Fig. 3A). Among the Ag NPs employed in the present study, 50 nm was the most toxic, inducing 100% mortality at the lowest concentration on both a mass concentration and a total surface area basis (Fig. 3A and B).

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Figure 3. Percentage of surviving zebrafish (Danio rerio) at 72 h postfertilization after exposure to Ag NPs using mass concentration (mg/L; A) and total surface area (nm2/L; B). (C) Percentage of surviving zebrafish at 72 h postfertilization exposed to AgNO3 using mass concentration. All values are represented as mean ± SE.

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Heart rate was affected in a dose-dependent manner in Ag NP exposures at 72 hpf. Differences in heart rate were observed among doses for each silver treatment (20 nm: f4,54 = 55.482, p < 0.001; 50 nm: f4,40 = 390.256, p < 0.001; 110 nm: f4,47 = 4.05, p < 0.001; AgNO3: f4,74 = 3.030, p = 0.023; Fig. 4). Significant differences from control (p < 0.05) were detected by post hoc tests in 20-nm particles at 0.1, 0.5, and 1 mg/L; 50 nm at 0.5 and 1 mg/L; 110 nm at 0.1 and 0.5 mg/L; and AgNO3 at 0.001 and 0.005 mg/L. The general trend showed the heart rates of treated zebrafish to decrease as Ag NP mass concentrations increased.

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Figure 4. Average number of zebrafish (Danio rerio) heartbeats/10 s relative to control at 72 h postfertilization after exposure to silver treatments. Relative heart rates were calculated by subtracting the average control heart rate from each individual zebrafish tested. Control values were set to zero; negative values indicate slower heart rates relative to controls, and positive values indicate faster heart rates. All values are mean ± standard error. Lack of data for 1 and 5 mg/L is due to a high rate of mortality.

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Otic vesicle-to-optic cup data, observed at 24 hpf, revealed that zebrafish developed more slowly at higher mass concentrations of 50-nm Ag NPs and developed more quickly at all mass concentrations of 110-nm Ag NPs and AgNO3, with the exception of 110 nm at 5 mg/L, which induced slower development (Table 3). Differences in OV-OP were observed among doses for silver treatments (20 nm: f4,53 = 4.381, p = 0.004; 110 nm: f4,72 = 4.82, p = 0.002; AgNO3: f4,57 = 7.525, p < 0.001). However, significant differences from control (p < 0.05) were detected by post hoc tests only for 110-nm particles at 0.5 mg/L and AgNO3 at 0.001 and 0.01 mg/L. A significant relationship for OV-OP data was not observed by dose with 50-nm Ag NP. On average, the smallest Ag NP (20 nm) elicited the slowest development in zebrafish, with high variability observed across treatments. A dose-dependent relationship was not observed for pericardial edema in zebrafish, although the greatest number of instances was observed in 20-nm Ag NP and AgNO3 (Table 3). Controls exhibited 6% pericardial edema. All three Ag NPs (20, 50, 110 nm) and AgNO3 caused chorion turbidity in treated zebrafish embryos (Table 3), with zero instances of chorion turbidity observed in controls. Abnormal motility (Table 3) was most pronounced in embryos dosed with 20-nm Ag NP at concentrations 0.5 to 1 mg/L.

Table 3. Analysis of Ag NP and AgNO3 toxicity in exposed zebrafish using multiple endpointsa
Ag treatment (nm)Concentration (mg/L)Chorion turbidity (24 hpf; %)Pericardial edema (%)Abnormal motility (72 hpf; %)OV-OPb (%)
24 hpf72 hpf
  • a

    Percentage of zebrafish survivors exhibiting chorion turbidity, pericardial edema, and abnormal motility compared with controls at a given time point.

  • b

    Otic vesicle-to-optic cup (OV-OP) represents the length (µm) between the otic vesicle and optic cup relative to controls at 24 hpf. Negative values for OV-OP indicate faster development; positive values indicate slower development.

    hpf = hours postfertilization; N/A = data not applicable because of the high rate of zebrafish mortality.

200.12250297−8
 0.5207710020
 121296710017
 51717N/AN/A11
500.12201302
 0.52100334
 1220N/AN/A22
 522N/AN/AN/AN/A
1100.1430620−2
 0.54121320−19
 14415022−2
 538014257
AgNO30.00127332013−17
 0.0052772713−8
 0.012933437−27
 0.052775327−4

Ag NP and AgNO3 toxicity in E. coli

Assays demonstrated a clear dose- and size-dependent response of E. coli to Ag NPs and AgNO3 on a mass concentration (mg/L) basis, with smaller particle sizes showing greater toxicity (Fig. 5A and C). A dose-dependent but not size-dependent response was maintained when the toxicity was investigated on a surface area of particle (nm2/L) basis (Fig. 5B).

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Figure 5. Escherichia coli bacteria mortality (colony-forming units [CFU]/100 µl) after exposure to Ag NPs using mass concentration (mg/L; A) and total surface area (nm2/L; B). (C) Escherichia coli bacteria mortality (CFU/100 µl) after exposure to AgNO3 using mass concentration. Data shown above the dashed line represent E. coli concentrations that are too numerous to count (TNC). All values are mean ± standard error.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

Our results suggest that Ag NPs are toxic to developing zebrafish in a mass-concentration-dependent manner within each size class tested. Toxicological endpoints, such as increased mortality, decreased heart rate, and increased observations of pericardial edema, were observed across all Ag NP treatments employed in the study (Figs. 3 and 4 and Table 3). Previous research has shown Ag NPs to penetrate tissues of the circulatory system and the heart in zebrafish 27, 28, leading to cardiac tissue malformations and edema. These findings correlate with our data, showing Ag NPs to hinder the heart rates of zebrafish and elicit an increase in observations of pericardial edema.

An Ag NP size-dependent response was not observed in zebrafish when viewing mortality across all Ag NP treatments based on individual particle size (Fig. 3). However, 20-nm Ag NP (the smallest NP used in the present study) did elicit the most drastic response in zebrafish motility and slower development (OV-OP) compared with the other Ag NPs in the present study (Table 3). For both mass concentration and total surface area, 50-nm Ag NP induced the highest rate of mortality. No clear trend was observed for zebrafish mortality when comparing mass concentration with total surface area (Fig. 3A and B).

Otic vesicle-to-optic cup data (Table 3) suggest that Ag NPs alter developmental timing of zebrafish embryos. As zebrafish develop, the distance between the otic vesicle and the optic cup becomes shorter. Kimmel et al. 29 showed this distance to be quantifiable through the first 24 h of development. Slowest development was observed in zebrafish treated with the higher concentrations of 20-nm Ag NP (0.1–5 mg/L) and 50 nm at 1 mg/L. Interestingly, AgNO3 treatments (0.001–0.05 mg/L) and the lower concentrations of Ag NPs elicited faster development in zebrafish embryos. A response, such as faster development, to low concentrations of Ag NPs may hint at a hormetic effect. Hormesis has been observed in green algae dosed with titanium-NPs 30, daphnia treated with carbon-NPs 31, and human cell lines dosed with Ag NPs 32 but has not been observed in zebrafish. Results from the present study are far from conclusive, and future research should consider the hormetic effect in Ag NP toxicity in order to elucidate these findings.

Our data show ionic silver (AgNO3) to be more toxic than the 20-, 50-, and 110-nm Ag NPs in both model systems used in the present study. Previous research has also shown that it may take from hours to several months for complete dissolution of Ag+ to occur from the surface of Ag NPs 8, 21. Given that our zebrafish study was terminated after 5 d, 20-, 50-, and 110-nm Ag NPs were likely not in a state of complete Ag+ dissolution compared with AgNO3. It therefore follows that the toxic effects observed in the present study with Ag NPs might not be restricted to Ag+ dissolution only, but might possibly occur with the Ag NPs themselves. However, if the study had been extended several weeks or months, allowing for complete Ag+ dissolution, zebrafish toxicity from Ag NPs might resemble that of AgNO3. Previous studies have shown Ag NPs to translocate into the chorion of zebrafish and also to enter various tissues, including the epidermis, gills, brain, liver, and heart 19, 22, 27, 33. In these tissues, toxic effects have been shown to be a consequence of Ag NP exposure, although dissolution of Ag+ might have been a contributing factor.

Chorion turbidity (Table 3) was observed in zebrafish exposed to all silver treatments. No study has definitively shown how Ag NPs cross cell membranes, but researchers have observed Ag NPs crossing the chorion and embedding within tissues 19, 27, 28. A possible explanation for chorion turbidity may be Ag NPs gaining access to blastomeres in early embryogenesis 28, as has been suggested previously. Another possible explanation is that Ag+ might be causing the chorion turbidity observed in treated zebrafish, as evidenced by our data showing all silver treatments (including AgNO3) to elicit instances of chorion turbidity.

Though abnormal motility (Table 3) was not seen in a dose-dependent manner, 20-nm Ag NPs had the most drastic effect on motility in concentrations ranging from 0.5 to 1 mg/L. A recent study has shown that silver, especially Ag+, can result in neurobehavioral and neurotoxic affects in zebrafish, causing persistent swimming problems in larvae 34.

We chose E. coli as a standard gram-negative bacterium that has been used often for NP studies 24, 35. The bacteria dose–response data from the present study support the hypothesis that toxicity may be due to dissolution of silver ions from the surface of the particles 21, 23. Although mortality curves based on both a mass concentration basis (mg/L) and on a total surface area of particles basis (nm2/L) clearly demonstrate a dose response, the two ways of looking at the data show a striking difference. In fact, the data show that looking solely at toxicity on a mass concentration (mg/L) basis can be quite misleading. The dose response on a mass concentration basis indicates that toxicity to bacteria is particle size dependent (Fig. 5A), with toxicity increasing as particle size decreases, as has been shown in other studies 36, 37. However, the dose response on a total surface area (nm2/L) basis tells a different story (Fig. 5B), showing little or no difference in toxicity among particles of different sizes when total surface area is the same. This indicates that, in bacteria exposed to Ag NPs in PBS, approximately 100% mortality occurs once a total surface area of approximately 1E + 18 nm2/L is reached, regardless of particle size.

From the surface area-based toxicity data collected on E.coli in the present study, it appears that the amount of exposed total surface area of the particles is what is driving the toxicity in bacteria. This implies that it may simply be dissolution of Ag+ from the surface of the particles causing toxicity. This possible explanation is supported by Radniecki et al. 21, who concluded that silver ions contributed significantly to the toxicity of Nitrosomonas europaea and observed a higher rate of Ag+ dissolution from 20-nm Ag NPs compared with 80-nm Ag NPs. Of course, toxicity of Ag+ to bacteria would not be surprising; Ag+ has been used as an antimicrobial agent for centuries 38.

It appears that a more efficient way of gauging Ag NP toxicity in bacteria might be to use total surface area (nm2/L). It must be recognized, however, that differences in bacterial strains 39, media 21, and NP shapes 35 might not make this approach universal. In zebrafish dosed with Ag NPs, the data for total surface area were similar to mass concentration data (Fig. 3A and B), unlike the results seen with E. coli. These results may imply that prokaryotes and eukaryotes inherently respond differently to Ag NP exposures, and the mode-of-action for the two systems might also be different.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

We conclude that first Ag NPs are toxic in a concentration-dependent manner to zebrafish and bacteria based on mass concentration. Second, although zebrafish did not respond to Ag NP treatments in a size-dependent fashion when viewing mortality, 20-nm (the smallest) Ag NP elicited the highest incidence of abnormal motility and induced slower development; bacteria did respond to Ag NPs in a size-dependent manner, with 20 nm causing the highest rate of lethal toxicity at the lowest concentration. Third, AgNO3 (ionic silver) was more toxic than 20-, 50-, and 110-nm Ag NPs for both zebrafish and bacteria. Forth, zebrafish development is hindered by high concentrations of 20-nm and 50-nm Ag NPs, and faster development occurs when zebrafish are exposed to lower concentrations of 110-nm Ag NP and all concentrations of AgNO3.Lastly, when total surface area of Ag NP toxicity is used to gauge bacterial mortality, a dose-dependent—but not size-dependent—response was observed for all three Ag NPs (20, 50, 110 nm) used in the present study, with nearly 100% mortality observed once a total surface area of approximately 1E + 18 nm2/L was reached, although this trend was not apparent when measuring total surface area for zebrafish mortality.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES

For technical assistance and support, we thank V. Kalaimani, K. Hulsey, N. Boupharath, A. Schaffer, C. Graham, S. Staley, A. Mohan, K. Miller, A. Clowers, and K. Averett.

REFERENCES

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  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. REFERENCES
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