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I. Linkov and J. Steevens (eds.), Nanomaterials: Risks and Benefits, 287 © Springer Science + Business Media B.V. 2009

SILVER NANOPARTICLES

Environmental and Human Health Impacts

R.R. KHAYDAROV Institute of Nuclear Physics Ulugbek, 100214 Tashkent, Uzbekistan [email protected]

R.A. KHAYDAROV Institute of Nuclear Physics Tashkent, Uzbekistan

Y. ESTRIN ARC Centre of Excellence for Design in Light Metals Department of Materials Engineering, Monash University CSIRO Division of Materials Science and Engineering Clayton, Victoria, Australia

S. EVGRAFOVA V.N. Sukachev Institute of Forest SB RAS Krasnoyarsk, Russia

T. SCHEPER, C. ENDRES Institute of Technical Chemistry Leibniz University Hannover, Germany

S.Y. CHO Yonsei University Seoul, South Korea

Abstract. The bactericidal effect of silver nanoparticles obtained by a novel electrochemical method on Escherichia coli, Staphylococcus aureus, Aspergillus niger and Penicillium phoeniceum cultures has been studied. The tests conducted have demonstrated that synthesized silver nanoparticles – when added to water paints or cotton fabrics – show a pronounced antibacterial/antifungal effect. It was shown that smaller silver nanoparticles have a greater antibacterial/antifungal efficacy. The paper also provides a review of scientific literature with regard to

Author's personal copyR.R. KHAYDAROV ET AL. 288

recent developments in the field of toxicity of silver nanoparticles and its effect on environment and human health.

1. Introduction

Medicinal and preservative properties of silver have been known for over 2,000 years. The ancient Greek and Roman civilizations used silver vessels to keep water potable. Since the nineteenth century, silver-based compounds have been

Over the last decades silver has been engineered into nanoparticles, structures from 1 to 100 nm in size. Owing to their small size, the total surface area of the nanoparticles is maximized, leading to the highest values of the activity to weight ratio. Due to this property being distinctly different from that of the bulk metal, silver nanoparticles have attracted much attention and have found applications in

antibacterial/antifungal agents in a diverse range of consumer products: air sanitizer sprays, socks, pillows, slippers, respirators, wet wipes, detergents, soaps, shampoos, toothpastes, air filters, coatings of refrigerators, vacuum cleaners, washing machines, food storage containers, cellular phones, etc. [6].

Numerous synthesis approaches were developed to obtain silver nanoparticles

electron irradiation [3], chemical reduction by inorganic and organic reducing

compared minimum inhibitory concentration (MIC) values for bacterial cultures, one can see that the antimicrobial activity of silver nanoparticles strongly depends on the method of their synthesis.

This paper deals with the authors’ research in the field of antimicrobial pro-perties of silver nanoparticles obtained by our recently suggested electrochemical

(MIC) values as well as a high efficacy of nanosilver as antimicrobial agent

also provides a review of the most recent scientific publications regarding the possible toxic effects of silver nanoparticles to the environment and human health.

2. Materials and Methods

using an inexpensive two-electrode setup in which the anode and the cathode made from the bulk Ag are placed vertically, face-to-face, 10 mm apart. The electrodes are immersed into an electrochemical cell filled with 500 ml of distilled water obtained with water distiller (DE-25, Russia). In the tests reported here, the

and optics [21]. Furthermore, currently silver nanoparticles are widely used as

agents [4], photochemical methods [19], microwave processing [31], and thermal decomposition of silver oxalate in water and in ethylene glycol [22]. Having

widely used in bactericidal applications, in burns and in wound therapy, etc. [11].

of various shapes and sizes, including laser ablation [13], gamma irradiation [18],

technique [10], which provides extremely low minimum inhibitory concentration

diverse areas, including medicine [26], catalysis [14], textile engineering [14], biotechnology and bioengineering [23], water treatment [30], electronics [12]

against a range of microbes on the surface of paints and fabrics [7]. This paper

The process of electrochemical synthesis of silver nanoparticles [10] is based on

Author's personal copySILVER NANOPARTICLES 289

electrolysis was performed during 1 h at the temperature range of 325–340 K with a constant voltage of 20 V. Periodical changing the polarity of the direct current between the electrodes with a period of 4 min and vigorous stirring during the process of electrolysis were applied in order to reduce the agglomeration of particles. Synthesized silver nanoparticle solutions were stored under ambient conditions in glass containers. The morphology of the silver nanoparticles/ powders obtained was studied using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS) measurements. The concen-tration of silver nanoparticles in solutions was determined by neutron activation

To evaluate the antibacterial and fungicidal properties of Ag nanoparticles Escherichia coli was used as a representative Gram-negative bacterium; Staphylococcus aureus was used as a Gram-positive bacterium; Aspergillus niger and Penicillium phoeniceum were used to represent cosmopolitan saprotrophic fungi. To assay the antimicrobial activity of silver nanoparticles in aqueous solution against E. coli on solid media, the agar disk diffusion method was used. Bacteria inocula were prepared from a log-phase culture of E. coli K12 grown in LB-media on a rotary shaker (120 rpm) at 37°C. The inocula were diluted with 0.9% NaCl to the 0.5 McFarland standard and 100 μl were applied onto 9 cm Mueller-Hinton agar plates with a depth of approximately 5 mm. Disks of absorbent paper (5 mm in diameter) were impregnated with 10 μl of silver nanoparticle solutions (47.5, 42.5, 22.6 and 11.3 ppm). For comparison, disks of the same diameter with 10 μl Tetracycline, Penicillin G and Ampicillin (1 g/l each) were used, leading to a concentration of the respective substance of 10 μg/disk. The freshly prepared disks were placed on the surface of the inoculated agar plates. After incubation at 37°C for 18 h the zones of bacterial inhibition were measured optically. In order to impregnate a cotton fabric with silver

commercially available water paint was mixed with silver nanoparticles solutions in a ratio of 7:1 in order to impart antimicrobial properties to the paint. To evaluate the antibacterial and fungicidal properties of Ag nanoparticles added to a cotton fabric and a water paint, samples (1.5 × 1.5 cm) treated by different compositions of Ag nanoparticles as well as control samples were immersed in a thin layer of beef-extract agar. A 1 ml of suspension of approximately 105 CFU/ml density of the microorganisms to be tested were distributed uniformly on agar surface and incubated at 28°C (CFU = colony forming units). Antimicrobial activity was evaluated according to the presence or absence of microbial growth just above the sample after a 24-h incubation for bacteria and a 72-h incubation for fungi. All microbiological tests were performed in triplicate.

MICs of silver nanoparticle solutions for various microbes were determined using the macrodilution broth susceptibility test. Nutrient broth used in the macrodilution method contained peptic digest of animal tissue 50.00 g/l; beef extract 1.5 g/l; sodium chloride 5.00 g/l; glucose 5 g/l; pH 7.4 ± 0.2. A standardized suspension of approximately 106 CFU/ml density was obtained by inoculating the culture in nutrient broth (Hi-Media) and incubating the tubes at 37°C for 3 h. A serial dilution of our silver nanoparticles solution was prepared

analysis [29].

nanoparticles, the simple padding procedure [12] was used. In a separate exercise,


within a desired range. Ten milliliter of the standardized culture suspension was then inoculated and tubes were incubated at 37°C for 24 h. MIC was defined as the lowest concentration of the inhibiting agent that completely inhibited bacterial growth, the unit for MIC was chosen as mg(Ag)/l. MIC was examined visually, by checking the turbidity of the tubes.

3. Results and Discussion

3.1. MORPHOLOGY OF THE SYNTHESIZED Ag NANOPARTICLES

It was shown by DLS measurements that a typical sample of silver nanoparticles solution obtained by the two-electrode setup described above contains not only nanoparticles, but also a small amount of large (>100 nm) colloidal silver particles. In order to remove these coarse particles and to provide reduction of silver ions present in the solution, we used filtration of the solution through a 3-μm pore size paper filter. The filter narrows the range of size distributions of synthesized silver nanoparticles while providing additional reduction of Ag ions according to the following reaction: Ag+1 + e → Ag0. As a result, the ratio of the concentrations of silver ions and silver nanoparticles suspended in the solution is reduced. A final stage of Ag nanoparticle synthesis involves additional treatment of the smallest-size fraction of silver nanoparticles remaining in solution after the filtering stage. It consists of adding hydrogen peroxide to a level of up to 0.005% concentration of H2O2 to the solution. Due to the reaction Ag2O + H2O2 → 2Ag + H2O + O2 silver oxide is reduced to Ag which is released in the solution. By this process the size of the silver nanoparticles is reduced, while new Ag nanoparticles may be forming as well. Examination of TEM images taken 2 weeks after the addition of H2O2 revealed that silver nanoparticles suspended in water solution were nearly spherical and that their size distribution fell in the range of 2–20 nm, the average size being about 7 nm, cf. Figure 1.

Figure 1. Typical TEM image and size distribution of silver nanoparticles obtained by electrochemical synthesis.


3.2. ANTIBACTERIAL ACTIVITY OF SILVER NANOPARTICLES

In Figure 2 the antibacterial effect of silver colloids with the concentrations of 47.5, 42.5, 22.6 and 11.3 ppm is presented vis-à-vis to that of known antibiotics. The concentrations of silver were selected in such a way as to correspond to maximum Ag concentrations used in consumer nanoproducts which are currently available on the market. Comparing zones of growth inhibition around the disks impregnated with various antibiotics and Ag nanoparticles, one can see that silver nanoparticle solution demonstrates a certain antimicrobial effect. The intensity of the effect is increased with the concentration of the solution.

Figure 3 demonstrates zones of growth inhibition around the disks impregnated with various antibiotics and the disk with the largest Ag nanoparticles concen-trations that we have used. Considering that the Ag concentration used in the experiment was approximately 20 times lower than that of the antibiotics, one can expect that silver nanoparticles would outperform Ampicillin, Penicillin and Tetracycline antibiotics of the same concentration.

Ampicillin Tetracycline Penicillin G Ag 47.5 ppm Ag 42.5 ppm Ag 22.6 ppm Ag 11.3 ppm0

2

4

6

8

10

12

14

16

18

20

0.113 μg/disk0.226 μg/disk0.425 μg/disk0.475 μg/disk10 μg/disk10 μg/disk10 μg/disk

zone

of i

nhib

ition

diam

eter

[mm

]

Figure 2. Antibacterial activity of silver nanoparticles in aqueous solution against E. coli K12 deter-mined by the agar disk diffusion method.

In order to reveal an effect of the size of silver nanoparticles on their bactericidal efficiency the minimum inhibitory concentration (MIC) assays were conducted against the gram-negative bacterium E. coli and the gram-positive bacteria S. aureus and B. subtilis. The results for MIC assays shown in Table 1 demonstrate that smaller silver nanoparticles had a greater antibacterial efficacy. The conducted MIC assays have also shown clearly that the proposed electrochemical technique


Figure 3. Zones of growth inhibition around disks impregnated with silver nanoparticles and various antibiotics.

provides very high antimicrobial activity of synthesized silver nanoparticles. For

nanoparticles that provided the same MIC values for E. coli and S. aureus varieties as in the present study. Sarkar and coauthors claimed that “such a low value of MIC showed by silver nano particles is unprecedented”. The results obtained for larger nanoparticles (with a mean size of 70 nm) are in good agreement with MIC assays for E. coli for colloidal silver (32.2 mg/l in case of

higher than those presented in Table 1, although they studied smaller silver nanoparticles (3.32 ± 1.129 nm). We suppose that it is mainly connected with the high purity of nanoparticles obtained by our electrochemical technique without surfactants. Unfortunately, existing studies on nanotoxicity were concentrated on empirical evaluation of the toxicity of various nanoparticles, with less regard

Table 1. Minimum inhibitory concentration (MIC) assay results for silver nanoparticles.

Bacterium MIC (mg(Ag)/l) (average particle size of

7 nm)

MIC (mg(Ag)/l) (average particle size of 70 nm)

E. coli S. aureus B. subtilis

3 2

19

34 25

no data

example, Sarkar et al. [27] have recently proposed a method of synthesis of Ag

average Ag-particle size of 63 nm) stabilized by sodium oleate (cf. [32]). On the other hand, MIC values for the same bacteria obtained by Rupareli et al. [25] are


is an obvious need for further studies on the development of a database of bactericidal efficacy of silver nanoparticles as a function of their size and composition.

3.3. ANTIMICROBIAL EFFECT OF COTTON AND PAINT SAMPLES MODIFIED WITH SILVER NANOPARTICLES

Bactericidal activity has become a significant property of textiles and paints used in applications such as medicine, clothing, and household products. We have impregnated cotton fabrics and water paints with our nanosized silver colloids. As one can see from Figure 4, most of initial silver nanoparticles had agglomerated into clusters because of attractive interaction forces between them (6-month old samples).

Figure 4. Samples of water paint (left) and cotton fabric (right) with immobilized silver nanoparticles.

The bactericidal action of the cotton fabric with immobilized silver nanoparticles on S. aureus was also studied. Experiments with agar plates demonstrated that the modified fabric (1 µg/cm2) can inhibit the growth of S. aureus on beef extract agar (Figure 5).

Similar tests were conducted on S. aureus using pasteboard covered with water paint modified with silver nanoparticles. These tests demonstrated that the modified paint with the area concentration of Ag of 0.001 mg/cm2 could inhibit the growth of S. aureus on beef extract agar.

paint modified with silver nanoparticles on Aspergillus niger and Penicillium phoeniceum cultures. It was shown in particular that a 20 ppm concentration of Ag nanoparticles (mean size of 50 nm) and a 3 ppm concentration (mean size of 15 nm) have similar antifungal effects, i.e. smaller silver nanoparticles had a greater antifungal efficacy. Tests on nanosilver-modified cotton fabrics, in which a 20 ppm solution of Ag nanoparticles with the mean size of 50 nm was used, also confirmed their antibacterial/antifungal effect: growth of these species of fungi in the vicinity of samples treated with a colloidal solution of Ag nanoparticles was suppressed.

given to the relationship between nanoparticle properties and toxicity [6]. Thus, there

Our recent microbiological tests [7] confirmed antifungal effect of the water


Figure 5. Growth of S. aureus culture on a cotton fabric sample modified by silver nanoparticles on the first and the next day. (Note the white spots corresponding to S. aureus colonies.) The sample #1 is a control sample, i.e. non-modified; other samples are modified with silver nanoparticles having various average sizes.

4. Environmental and Human Health Impacts

Silver-based materials have been widely used over the last decades in medical organizations, photographic laboratories etc. Not long ago, the annual silver release into the environment from industrial wastes and emissions was estimated at approximately 2,500 t, of which 150 t ended up in the sludge of wastewater

The maximum concentrations of silver released into the environment are regulated at various levels in different countries by their appropriate environmental protection agencies. It was well documented in studies conducted in the twentieth century that the toxicity of silver in the environment occurred mainly in the aqueous phase

+

on human health, the scientific literature of the last century cited mainly cases of permanent bluish-gray discoloration of the skin (argyria) or eyes (argyrosis) occurring when the accepted threshold values for silver and its compounds were exceeded [1].

In the twenty-first century the significant growth of applications of nanosilver in various branches of industry as well as its use in consumer products has caused new concerns that silver nanoparticles may have a toxic effect on the environment and human health. There is a public perception that silver nanoparticles do not discriminate between different strains of bacteria and are likely to destroy microbes beneficial to other organisms and ecological processes [2]. Unfortunately, only a few scientific investigations on cytotoxicity of nanosilver have been conducted to date. For example, in vitro toxicity assays of silver nanoparticles in rat liver cells

stress, cellular shrinkage and impaired mitochondrial function. Silver nanoparticles also turned out [5] to be highly toxic to in vitro mouse germline stem cells, as they drastically reduce mitochondrial function and cause increased leakage of ions

treatment plants with 80 t being released into surface waters [28, 24].

and depended on the concentration of active, free Ag ions. [24]. As for the impact

through cell membranes. According to studies conducted by Soto [31], nanoparticulate

by Hussain et al. [9] have shown that low level exposure resulted in oxidative


silver aggregates were more cytotoxic than asbestos. There is also an impact of nanosilver exposure on development of the lymphatic system of embryos of chickens, although the entire embryo development was not influenced by silver

Considering the studies on cytotoxicity of nanoparticles, it is important to keep in mind that in vitro results can differ from what is found in vivo and are not

cytotoxicity studies were performed using unrealistically high concentrations of nanosilver.

It would be fair to say that the mechanism of the bactericidal effect of silver

that “Nanosilver represents a special physicochemical system which confers their antimicrobial activities via Ag+”. If this conclusion is verified then most bioaccu-mulation and toxicity issues relating to silver nanoparticles can be considered from the point of view of the toxic potential of ionic silver, which is documented sufficiently well. As under natural environmental conditions the ionic silver is

ronmental risks of nanosilver toxicity is not as severe as the popular perception may suggest.

nanoparticles on micro-organisms is connected not merely with the release of silver ions in solution. Following their report, silver nanoparticles can also be attached to the surface of the cell membrane and disturb its proper function drastically. They are also able to penetrate inside the bacteria and cause further damage by possibly interacting with sulfur- and phosphorus-containing compounds such as DNA. It is interesting to note that silver nanoparticles have also demon-

Thus, there is an urgent need for further studies on the bactericidal mechanism of silver nanoparticles, which will be a step forward to better understanding of their environmental and human health impacts. As silver-based materials have a great commercialization potential, we anticipate a large amount of reports from various scientific groups in the field of nanosilver toxicity in near future. To quote a recent review: “A full understanding of the hazards of nanoparticles will make a major contribution to the risk assessment that is so urgently needed to ensure that products that utilize nanoparticles are made safely, are exploited to their full

5. Conclusion

An electrochemical technique for synthesis of silver nanoparticles with high anti-microbial activity has been developed. Our studies have revealed that silver nanoparticles suspended in water solution are nearly spherical, their average diameter being 7 ± 3 nm. Due to their high purity, very low inhibitory concentration (MIC) values for Escherichia coli (3 mg/l), Staphylococcus aureus (2 mg/l) and Bacillus subtilis (19 mg/l) cultures have been obtained. The tests

nanoparticles is not well understood as yet. Lok et al. [17] have recently reported

readily transformed to nonreactive compounds [24], this would mean that the envi-

By contrast, according to Morones et al. [20] the bactericidal effect of silver

strated synergistic effects with known antibiotics, such as amoxicillin [16].

nanoparticles [8].

necessarily clinically relevant [15]. It should also be noted that some reported silver

potential and then disposed of safely” [15].


conducted have demonstrated that synthesized silver nanoparticles added to water paints or cotton fabrics show a pronounced antibacterial/antifungal effect, despite the fact that they tend to be agglomerated into clusters. It has been shown that smaller silver nanoparticles have a greater antibacterial/antifungal efficacy. A brief review of the scientific literature on recent studies into the impact of silver nanoparticles on environment and human health has been provided.

Acknowledgments

R. R. Khaydarov acknowledges partial support of this work through the INTAS Fellowship Grant No. 5973 for Young Scientists under the “Uzbekistan – INTAS 2006” program.

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