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EPA Grants 1st Approval For Nanopesticide

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EPA Grants 1st Approval For Nanopesticide

More than a year after floating the idea, the U.S. Environmental Protection Agency has granted the first approval for a pesticide that’s based on a nanoscale material—a Swiss-made antimicrobial nanosilver product used in fabrics.

The EPA announced Thursday that it is moving forward with a four-year “conditional registration” for HeiQ Materials’ AGS-20 product, which is essentially a composite of nanosilver and nanoscale silica. According to HeiQ’s application—first filed in 2008—AGS-20 will be incorporated into textiles.

Nanosilver is being used more and more in workout clothing, touted as a way to fight that gym-rat smell.

The agency proposed the conditional registration—which comes with a laundry list of required tests—in August 2010, and opened a public comment period. Then there was radio silence, except for an EPA announcement last summer that it planned to seek more information from manufacturers using nanomaterials, potentially including nano-enabled products that got the green light before the agency sharpened its focus on ultra-tiny substances.

Nano-watchers have been waiting eagerly for the agency’s decision, wondering whether the move would signal a larger shift in how nanomaterials, including silver, are monitored and regulated.

Nanotechnology leverages the often-unique properties of super-small particles to create products with amazing qualities. These materials can make better batteries or lighter and stronger bike frames, as well as new medical instruments and medicines that can save lives. They’re increasingly common in consumer products, from “mineral-based” sunscreens to stain-repellent pants to boat paints that resist algae growth.

Nanomaterials are believed to hold great promise for a wide variety of applications. But shrinking these substances can change their properties, and scientists are struggling to figure out whether that shift can make them dangerous in the process, and how and why it happens.

HeiQ CEO Carlo Centonze said in a statement that the company is “pleased that that EPA has recognized the potential benefits provided by HeiQ AGS-20.” He said the EPA’s approval came “in part because it could lead to less silver released in the environment while providing longer protection against the growth of odor- and stain-causing bacteria.”

Lynn Bergeson, a lawyer at the Washington firm Bergeson & Campbell who works with nanotechnology companies on navigating the regulatory process, said she was “thrilled” by the EPA’s move, but wasn’t sure it portends additional action by the agency.

“It really is very difficult to speculate as to whether this means anything other than the decision was made early on to approve this and the agency has carefully considered the comments and is sticking by its decision,” said Bergeson, who doesn’t represent HeiQ.

The EPA considers nanosilver, and its larger-sized counterpart, a pesticide, and evaluates it under the Federal Insecticide, Fungicide and Rodenticide Act, or FIFRA. According to that law, pesticides must be registered before going on the market.

EPA officials weren’t available for comment Thursday, aside from a brief press release announcing the move. (Click here to read the rules it proposed in 2010; it’s unclear whether there have been significant changes.)

Nanosilver has drawn a lot of attention from scientists and regulators. It’s widely used as an antimicrobial, in toothbrushes and other products as well as sweat socks. Studies have shown nanosilver turns up in sewage sludge—and also sloughing off of larger silver objects, raising questions about how much silver has been all around us, and whether these new products might have an additional impact.

Environmental groups and other advocates have warned that nanosilver could be a problem for the environment, by building up in water, soil and marine life or by disrupting the natural bacteria that are all around us. Others have questioned whether the widening use of nanosilver might create problems with resistance, either in people or in bacteria.

The silver industry maintains that the metal, which has been used for centuries to fight germs, is safe in any size.

In his statement, Centonze said HeiQ will comply with all the EPA’s requirements: “It is our constant effort to produce and communicate scientific findings to EPA and other regulators in their efforts to adapt risk assessment to new policies and environmental findings.”

Jaydee Hanson, policy director at the International Center for Technology Assessment, said he was disappointed that the EPA decided to move ahead with HeiQ’s application before completing work on broader guidelines for nanoscale pesticides. The ICTA, in concert with other advocacy groups, has petitioned both the FDA and EPA to start regulating products that contain nanomaterials.

The company should get credit for going through the registration process, he said, noting that other manufacturers seem to be cloaking their nanopesticide ingredients in order to avoid EPA scrutiny.

But the agency should have waited, Hanson said, at least until it gets the results of the toxicology tests required under the conditional registration.

“It’s a little bit like, ‘OK, let your horses out, and maybe we can recall them afterwards,’‘’ Hanson said.

“There are serious potential environmental problems, if we get every fabric out there impregnated with nanosilver,” he said, adding that he expects to see more companies apply to register nanosilver pesticides in the wake of the EPA’s decision.

HeiQ’s application, and the EPA proposal for the conditional registration, drew a variety of comments from industry, environmental and consumer advocates and the public (click here to see all of the background information). Many were negative, and it’s unclear how—or if—they affected the EPA’s decision.

“I think it’s going to be when we see their new regulations before we know whether they really paid attention to the comments or not,” Hanson said.

Bergeson said she also anticipates more applications for nanopesticides. The conditional nature of the registration is not ideal for a commercial product, since there’s the potential that after the four-year trial period, the EPA could pull it from the market. But it’s better than the stop sign companies have been getting, she said.

The wait for this decision “was a signal to pesticide registrants that the agency had now to find its sea legs” on nano-enabled products, she said. “Now, with the issuance of the final registration, I think you can expect EPA to be more confident and for pesticide registrants to be more hopeful.”

Antimicrobial Properties of a Novel Silver-Silica Nanocomposite Material

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ABSTRACT

Nanotechnology enables development and production of novel silver-based composite materials. We used in vitro tests to demonstrate the antimicrobial activity of a silver-silica nanocomposite compared to the activities of conventional materials, such as silver nitrate and silver zeolite. A silver-silica-containing polystyrene material was manufactured and shown to possess strong antimicrobial properties.

Many applications, including medicine and food production and storage, would benefit greatly from incorporation of safe and inexpensive long-lasting biocides into polymers, paints, or textiles (). The antimicrobial effect of silver additives is broadly used in various injection-molded plastic products, in textiles (), and in coating-based applications, including air ducts, countertops, and food preparation areas (). Some important advantages of silver-based antimicrobials are their excellent thermal stability and their health and environmental safety (). However, like the use of all biocide products, the use of silver is strictly controlled by various national laws and control agencies. In the United States, the Environmental Protection Agency has regulated the use of silver as a biocide since 1954 () under the Federal Insecticide Fungicide and Rodenticide Act. In the European Union, a European biocide product directive (EU/BPD/98) imposes regulatory requirements on the use and claims associated with all biocide products ().

In the past few years, there has been a tremendous push for development of inorganic nanoparticles with structures that exhibit novel physical, chemical, and biological properties (). In particular, the potential benefits of nano-silver materials have been recognized by many industries due to the strong antimicrobial activity of silver against a broad spectrum of bacteria, viruses, and fungi and the low frequency of development of resistance ().

Generally, silver-based antimicrobial additives consist of silver ions integrated into inert matrices consisting of ceramic, glass, or zeolite. Other silver additives based on silver salts or metallic silver may be readily incorporated into thermoplastic polymers, such as polyethylene, polypropylene, polystyrene, or nylon (). The bactericidal efficacy of silver-containing polymers is based on the release of silver ions (Ag+) through interaction with a liquid watery phase (). Although the antimicrobial effects of silver ions and salts have been intensively studied, the mechanism of the inhibitory action of silver on microbes is still not fully understood. It has been proposed that silver ions interact with disulfide or sulfhydryl groups of enzymes, causing structural changes that lead to disruption of metabolic processes followed by cell death (). The inhibitory action of silver nanoparticles is also based on the release of Ag+ (). Exposure of microorganisms to silver nanoparticles was shown to result in strong antimicrobial activity (). In addition to the increased surface area and associated increased potential for the release of Ag+, when dispersed in liquid suspensions, silver nanoparticles may accumulate in the bacterial cytoplasmic membrane, causing a significant increase in permeability and cell death (), and penetrate bacterial cells (). Recently, it has been suggested that the antimicrobial mechanism of silver nanoparticles may also be related to membrane damage due to free radicals that are derived from the surface of the nanoparticles (). This bactericidal activity also appears to be dependent on the size and shape of the silver nanoparticles ().

In this study, we evaluated the properties of a novel silver-silica nanocomposite material (HeiQ AGS-20; HeiQ Materials, Bad Zurzach, Switzerland) used as an antimicrobial additive and compared its efficacy to the efficacies of the conventional silver additives silver nitrate (AgNO3; 63.5% Ag) and silver zeolite (38% Ag bound to type A zeolite; Sigma-Aldrich, Buchs, Switzerland). The novel silver-silica nanocomposite material was produced using an industrial flame spray pyrolysis process. This process involves combustion of a flammable solvent containing homogeneously dissolved compounds as the source of components for the synthesis of the material (). A representative transmission electron micrograph of the silver-silica material is shown in Fig. Fig.1.1. The nanocomposite consists of silver nanoparticles embedded in a matrix of amorphous silicon dioxide (SiO2). The SiO2 fine structure consists of aggregate matrix particles with an average diameter of approximately 1 μm (Fig. (Fig.1A).1A). Silver metal particles are located on the surface of the silica and are also embedded within the matrix (Fig. (Fig.1B).1B). High-magnification scanning transmission electron microscopy imaging of a localized region of a nanoparticle indicated that each silica particle contains many small silver metal particles with a typical diameter between 1 and 10 nm (Fig. (Fig.1C).1C). The specific surface area of the nanocomposite powder, as measured by nitrogen adsorption (), is typically about 250 m2/g, a value which is consistent with the open structure of the silica aggregate shown in Fig. Fig.1.1. It can be concluded that, upon contact with moisture, the pure silver particles act as a source that releases silver ions, which represent the active antimicrobial principle (). Some key advantages of the novel nanocomposite are the dispersion of the discrete silver particles throughout the silica (which prevents agglomeration of the silver particles), the small diameter of the silver particles (which results in a large surface area and release of a large amount of Ag+, which results in high antimicrobial efficiency), and the small size of the silver-silica composite (ca. 1 μm) (which allows the material to be uniformly dispersed and readily incorporated into a variety of substrates, including synthetic fibers, plastics, and other thin or delicate materials). The silica structure acts as a convenient carrier for incorporating the fine silver particles into plastics, textiles, and coatings. A further advantage is that the immobilization of silver nanoparticles within the silica structure limits the potential for release and disposal of the nanoparticles themselves. This property may be highly desirable because of the possible abilities of nanoparticles to cross biological membranes and other barriers ().

An external file that holds a picture, illustration, etc. Object name is zam0090998820001.jpg

(A) Transmission electron micrograph showing an amorphous silicon dioxide aggregate particle (gray structure) together with numerous supported silver metal particles (dark spots). (B) Scanning transmission electron micrograph of the structure shown in panel A, providing better contrast between the silica structure (gray) and the silver metal particles (bright spots). (C) Higher magnification of the region in panel B enclosed in a box. The silver metal particles are typically between 1 and 10 nm in diameter. Transmission electron microscope and scanning transmission electron microscope images were obtained using an FEI Tecnai F30 FEG microscope operated at 300 kV.

The microorganisms and growth conditions used for antimicrobial testing are shown in Table Table1.1. The MICs for all combinations of silver materials and microorganisms were determined by preparing twofold serial dilutions of the additives in an appropriate growth medium (Table (Table1).1). The tubes were then inoculated with 107 CFU/ml from overnight cultures of the bacteria or 106 CFU/ml for Candida albicans and incubated on a shaker (180 rpm) for 24 h. The MIC was defined as the lowest concentration of the silver additive at which no visual turbidity of the growth medium developed. The minimal bactericidal concentration (MBC) was determined by surface plating 0.2-ml aliquots from the nonturbid tubes, followed by incubation at 37°C for 24 h. The MBC was defined as the lowest concentration of silver additive resulting in less than 200 colonies per plate (corresponding to a killing rate of more than 4 logs). Aspergillus niger spores were harvested by floating the spores in densely grown lawns on malt extract agar plates in an extraction buffer (0.1% [vol/vol] Tween 20, 145 mM sodium chloride, 20 mM sodium phosphate; pH 7.4) and removing them. The MIC was determined by spreading approximately 200 spores on malt extract agar plates containing serial dilutions of the silver additives (Table (Table1)1) and was defined as the lowest concentration that prevented visible growth after 72 h of incubation at 30°C.

TABLE 1.

Comparison of the antimicrobial activities of silver nanocomposite powder, silver nitrate, and silver zeolite

MicroorganismSilver nanocomposite


Silver nitrate


Silver zeolite


MIC (μg/ml)aMBC or MFC (μg/ml)bMIC (μg/ml)aMBC or MFC (μg/ml)bMIC (μg/ml)aMBC or MFC (μg/ml)b
Escherichia coli ATCC 2732c62.51257.815.63.915.6
Klebsiella pneumoniae ATCC 4352c62.51253.97.87.831.2
Pseudomonas fluorescens LME 2333d62.52507.87.815.631.2
Salmonella enterica serovar Enteritidis D1c62.52503.97.815.662.5
Salmonella enterica serovar Typhimurium DB 7155c62.52503.915.615.631.2
Enterococcus faecalis ATCC 19433e62.52503.97.87.87.8
Bacillus cereus ATCC 14579e25050031.231.262.5250
Listeria monocytogenes Scott Af5001,00031.231.231.262.5
Staphylococcus aureus ATCC 29213f2501,00015.615.615.6125
Candida albicans ATCC 10259g1252,00031.225062.5250
Aspergillus niger ATCC 9642h2,000NDi15.6ND125ND
aThe MIC was determined at least in duplicate.
bThe MBC or minimum fungicidal concentration (MFC) was determined at least in duplicate.
cCultured in Luria-Bertani broth (10 g/liter peptone from casein, 5 g/liter yeast extract, 10 g/liter NaCl; Merck, Darmstadt, Germany) at 37°C.
dCultured in Biotone tryptose broth (Biolife, Milan, Italy) at 30°C.
eCultured in Biotone tryptose broth (Biolife, Milan, Italy) at 37°C.
fCultured in half-strength brain heart infusion broth (Biolife, Milan, Italy) at 37°C.
gCultured in malt extract broth (Merck, Darmstadt, Germany) at 37°C.
hCultured on malt extract agar (Merck, Darmstadt, Germany) at 30°C. The MIC for A. niger was defined as the lowest concentration not associated with visible growth on malt extract agar after 72 h of incubation at 30°C.
iND, not determined.

MICs and MBCs for the silver additives tested are shown in Table Table1.1. All experiments were performed at least in duplicate. For bacteria, the MICs of the nanocomposite material ranged from 62.5 to 500 μg/ml, corresponding to 12.5 to 100 μg pure Ag/ml. The MICs of silver nitrate varied from 3.9 to 31.2 μg/ml (corresponding to 2.4 to 19.8 μg Ag/ml), and the MICs of silver zeolite ranged from 3.9 to 31.2 μg/ml (corresponding to 2 to 12 μg Ag/ml). The MBCs determined were in the ranges from 125 to 1,000 μg/ml for the nanocomposite powder, from 7.8 to 31.2 μg/ml for silver nitrate, and from 7.8 to 125 μg/ml for silver zeolite. Growth of C. albicans was inhibited by 125 μg/ml silver nanocomposite, and the minimal fungicidal concentration was 2 mg/ml. Development of visible colonies of A. niger on agar plates was also completely inhibited by 2 mg silver nanocomposite per ml agar.

In general, gram-positive bacteria appeared to be more tolerant to silver than gram-negative cells (Table (Table1),1), except for Enterococcus faecalis, for which the MICs and MBCs were similar to those for gram-negative bacteria. It has previously been reported that gram-positive bacteria are less susceptible to the antimicrobial activity of silver (). It was speculated that this may be due to differences in the cell wall structure (). The cell wall of gram-positive bacteria contains multiple layers of peptidoglycan compared to the cell wall of gram-negative bacteria. Peptidoglycan is a complex structure and often contains teichoic acids or lipoteichoic acids which have a strong negative charge, which may contribute to sequestration of free Ag+ions. Thus, gram-positive bacteria may allow less Ag+ to reach the cytoplasmic membrane than gram-negative bacteria allow () and may therefore be less susceptible.

Susceptibility tests using different silver compounds in previous studies revealed that the MICs of silver particles for Escherichia coli ranged from 2 to 75 μg/ml (). However, because corresponding silver concentrations were not specified, it is not possible to compare these values to our results. For silver zeolite containing 1.9% (wt/wt) Ag, the previously reported MICs determined by using a similar protocol ranged from 256 to 2,048 μg/ml, corresponding to 4.8 to 38.4 μg/ml of Ag (). Here we used silver zeolite containing 38% (wt/wt) Ag to determine the MIC for E. coli. The MIC determined (1.9 to 3.9 μg/ml) was much lower than the previously reported MICs of the silver zeolite containing 1.9% Ag. The nanocomposite material had an MIC of 62.5 μg/ml (12.5 μg Ag/ml) for E. coli. Not considering the relative Ag content, silver nitrate and silver zeolite (38% Ag) resulted in inhibition that was approximately 10 times more effective than the inhibition observed with the nanocomposite. This can be explained by the fact that in aqueous systems silver nitrate dissolves completely and the silver is completely available in its biologically active ionic form. The silver ions held in the zeolite structure are also relatively rapidly released into solution. In contrast, the silver nanoparticles embedded within the silica matrix release Ag+ in a more gradual, controlled manner and at a much lower rate (). Thus, although silver nitrate and silver zeolite are more effective in applications where high Ag+ concentrations are required immediately, the effect is only short lived. In contrast, the nanocomposite powder allows slow and controlled release of Ag+, resulting in long-term antimicrobial activity. This should be a clear advantage in any long-term antimicrobial applications (e.g., contact surfaces, fibers, plastics, medical devices, food-manufacturing equipment, cutting boards, etc.).

To examine the antimicrobial properties of a typical application product, silver-containing polystyrene plates were manufactured from commercially available polystyrene polymer (clear, unfilled) using a thermoplastic injection-molding process (). Test coupons that were 50 by 50 by 1.5 mm and contained the nanocomposite material (0.25% [wt/wt], corresponding to approximately 500 ppm Ag) were produced by dry blending the polystyrene polymer with the required amount of polymer concentrate containing the silver nanocomposite additive, which was followed by injection molding. The antimicrobial activity was determined by using the Japanese industrial standard test (JIS Z 2801:2000) (). In brief, the test samples were placed in petri dishes and inoculated with 0.4 ml of a bacterial culture containing 105 to 106 CFU/ml. The inoculum was covered with a polyester film (X-131 transparent copier film; Folex Imaging), and the petri dishes were incubated at 37°C for 24 h in a humid chamber to prevent desiccation. After the incubation period 20 ml of extraction solution (0.1% [vol/vol] Tween 20, 145 mM sodium chloride, 20.5 mM sodium phosphate; pH 7.4) was added to the petri dishes and shaken for 2 min. Subsequently, serial dilutions of the extraction solution were spread on agar plates in triplicate and incubated at 37°C overnight. Colonies were counted visually, and the numbers of CFU per sample were determined. The activity value was calculated from the mean value for the individual samples by subtraction of the log value determined for the test sample from the log value determined for the control. The results for viable counts determined for the control and the silver nanocomposite-containing samples are shown in Table Table2.2. The activity values determined by the JIS Z 2801:2000 method () were 4.4 for E. coli and 2.1 for Staphylococcus aureus (P < 0.05, Student’s t test [n = 3]). The results demonstrate that the silver-silica nancomposite-containing polystyrene material has significant antibacterial activity against both E. coli and S. aureus.

TABLE 2.

Antimicrobial activity of silver nanocomposite-containing polystyrene platesa

OrganismNo. of cells (CFU/sample)b


Activity
Polystyrene control after inoculationPolystyrene control after 24 hPolystyrene with silver after 24 h
Escherichia coli1.7 × 105 ± 4.9 × 1042.6 × 106 ± 6.5 × 104<1004.4
Staphylococcus aureus1.7 × 105 ± 7.9 × 1041.8 × 105 ± 6.9 × 1041.4 × 103 ± 9.0 × 1022.1
aActivity was tested by using Japanese industrial standard JIS Z 2801:2000 ().
bThe values are means ± standard deviations for measurements obtained for three independent sample pieces. The values for the polystyrene control after 24 h and polystyrene with silver after 24 h were significantly different according to the Student t test (P < 0.05; n = 3).

In this study, a silver-silica nanocomposite material with a novel structure and composition was investigated to determine its antimicrobial properties. The material exhibited very good antimicrobial activity against a wide range of microorganisms. The inhibition of microbial growth due to surface contact with the silver-silica nanocomposite-containing polystyrene demonstrated that materials functionalized with the silver nanocomposite have excellent antimicrobial properties. Further studies of the mode of action of the silver-silica nanocomposite material with gram-positive and gram-negative bacteria and also with yeasts and molds are required to fully evaluate its potential for use as an antimicrobial additive in various materials.

ACKNOWLEDGMENTS

We are grateful to Joos Kiener for excellent technical assistance and to Elisabeth Müller Gubler from the Electron Microscopy Center of ETH Zürich for the electron microscopy images. M.J.H. thanks S. E. Pratsinis for his suggestions and ideas concerning synthesis of the material and the Particle Technology Laboratory, Department of Mechanical and Process Engineering at the Swiss Federal Institute of Technology (ETH Zurich), for supporting initial development of the material.

FOOTNOTES

Published ahead of print on 6 March 2009.

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Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

Are Nanoparticles Safe?

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The rapid development of nanotechnology has increased fears about the health risks of nano-objects. Are these fears justified? Do we need a new discipline, nanotoxicology, to evaluate the risks? Harald F. Krug and Peter Wick of the Swiss Federal Laboratories for Materials Science and Technology discuss these questions in the journal Angewandte Chemie.

“Research into the safety of nanotechnology combines biology, chemistry, and physics with workplace hygiene, materials science, and engineering to create a truly interdisciplinary research field,” explain Krug and Wick. “There are several factors to take into account in the interaction of nano-objects with organisms,” they add.

The term nanotoxicology is fully justified. “Nanoscale particles can enter into cells by other means of transport than larger particles.” Another critical feature is the large surface area of nano-objects relative to their volume. If a similar amount of substance is absorbed, an organism comes into contact with a significantly larger number of molecules with nanoparticles than with larger particles.