"Nanotechnology" with new technical and utility advances, is a rapidly developing technology in the world. Nanotechnology allows the manufacture of improved products with better performance and better quality in cheap methods. Although this is considered as a new technology, various types of nano scale materials have produced for decades such as miniscule computer microchips (Nanosense project, 2004). Nanoproducts are generated by incorporating nanoscale particles, which are defined as compounds with at least one dimension less than 100nm. Nanomaterials exists in 1 dimensional (thin films or coatings), 2 dimensional (nanowires and nanotubes) or 3 dimensional (precipitates, colloids or quantum dots) aspects (Nanowerck, 2007). Properties of nanoparticles greatly vary with its bulk materials, due to high surface area to volume ratio, size, and quantum effects. These aspects enable changes of reactivity, strength, and electrical properties of the nanomaterials (Nowack and Bucheli, 2007).
Due to their tremendous economic and resource saving capabilities, Nanoproducts are used in pharmaceutical, biomedicine, remediation, cosmetics, electronic, engineering, and environmental sectors (Nanowerk , 2010 and Niemeyer, 2001). Engineered nanoparticles are utilized in a wide variety of consumer products. It has been estimated that over 15% of the products in the market will have nano based materials by year 2014 (Dawson 2008).
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Table 1 Examples for engineered nanoparticles, properties and incorporated consumer products.
Fridges, vacuum cleaners, textiles, paints, varnish
Schmid and Reidiker (2008), Choi et al., 2008
Cements, paints, sunscreens. Caralyst, batteries, solar cells
Schmid and Reidiker (2008)
Excellent electrical conductors, semiconductors
Electronics, rechargeable batteries
Large energy band gap energy, high dielectric constant, block broad UV rays
Optoelectronic and electronic devices, cosmetics, sunscreens
Singh et al., 2007. Huang et al., 2008
Large energy band gap energy, photocatalytic
Sunscreens, photo catalytic and photovoltaic devices
Reinjnders, 2008, Zhu et al., 2005
Catalytic and electrical properties
Oxygen sensors, fuel additive
Robinson et al., 2002, Zhang et al., 2001
High stability, inertness, tuneable magnetic and electrical properties
Electronics, medical applications
High flexural and tensile strength, mechanical, electrical and magnetic properties
Ke-long et al., 2006
Inventory of Woodrow Wilson International center for scholars for nanoproducts (2010) claimed 974 consumer based nanoproducts in the market. Table 1 tabulates some of the examples for engineered nanoparticles, consumer products, and their relative properties found in the U. S. Commerce. Due to the immense advantageous properties of nanoparticles, research and Development funding has raised and estimated US$10 billion worldwide from governmental and industrial sectors (Lux research, 2006). The rate of increase in quantity of engineered nanoparticles expected according to the Royal society and Royal Academy of Engineering report 2004, was 58,000 metric tones per year from 2011 to 2020 (Maynard et al, 2006). Therefore a potential increase in consumer based nanoproducts and research on development of synthesis and new technological uses are expected in future.
Environmental and Health issues
Besides vast range of benefits, rapid growth of environmental and human health issues associated with nanomaterials has captured the attention of environmental scientists and researchers. Properties such as high surface area to weight/volume ratio and small size that creates nanoparticles more useful also linked with creation of environmental and health issues (Kirchner et al., 2005). Engineered nanomaterials are likely to enter to the environment during any stage from cradle to grave. Some of these engineered nanomaterial processes include: manufacturing, processing, packaging, transporting, consumer handling, washing and disposing (Oberdoester et al., 2005). Released compounds may introduce variety of hazards into the environment and human life. Most studies done on nanoparticles are related to toxic effects to mammalian cells (Nowack and Bucheli, 2007) and few studies have been reported on effects to other organisms. In fact since these studies have been carried out mainly as in vitro experiments, it cannot be directly related to the actual reactions occurring inside the body due to lack of various enzymes and hormones responsible for detoxification process (Nowack and Bucheli, 2007).The main mechanisms of the entrance of nanoparticles into human body are: inhalation, dermal contact, and minor mechanisms are ingestion and injection as reported by McAuliffe and Perry (2007). When exposed through inhalation route, nanoparticles easily penetrate cellular membranes and react with cellular tissues leading to detrimental damages (Duran et al., 2007). Few studies have suggested that Inhalation exposure translocates nanoparticles to vital organs such as, liver, heart, kidney, spleen, bone marrow, and brain (through blood brain barrier) of humans (Akerman et al., 2002, Pietropaoli et al., 2004). According to the epidemiological studies done so far, micron size particles are absorbed through pulmonary tract and have been dispersed to other organs of the body via the blood (Panacek et al., 2006, Shahverdi et al., 2007). Therefore, nano sized particles would more likely to cause significant damage as they are more easily penetrable than the micron size particles. Sayes et al (2006) has shown in vitro toxicity effects on human dermal fibroblasts and human lung epithelial cells on Titanium dioxide nanoparticle exposure. Findings on carbon nanotubes (Shvedova et al., 2003), fullerenes (Sayes et al., 2004), cadmium, lead quantum dots, (Chan et al., 2006) and silver nanoparticles (Kawata et al., 2009, AshaRani et al., 2009) have shown cellular mediated inflammation and toxicity responses in human cells. So far, little is known about the toxicity mechanisms of humans and have yet to be discovered in relation to the site of activity and the type of nanoparticle (AshaRani et al., 2009).
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Apparent cytotoxic and genotoxic effects of several nanomaterials to aquatic organisms have been reported from many scientific works. In aquatic environments nanomaterials rest as colloids (Klaine et al., 2008) and the colloidal behavior should be well-recognized of the specific nanocompound to explain the mechanisms of hazardous effects. Inhibitory effects of nanoparicles have well ascertained among zooplanktons (ex: Daphnia) (Oberdorster, 2004b), Fish, (Large mouth bass) (Oberdorster, 2004a) and algae Desmodesmos subspicatus (Hunde-Rinke and Simon, 2006). Clearly, more studies are in demand for the inhibitory effects of vertebrate and algal species to satiate the research gaps. No literature is available on the toxicity of terrestrial, marine, and fresh water invertebrates. Among these toxic nanomaterials Ag nano compounds are considered as in a high state of holding biocidal activity, where biocidal effects have been observed among numerous organisms. Accumulation of nanocompounds as nanowaste at end of service life options such as landfills, waste water treatment plants and sludge treatment plants, have also grasped the attention of public and scientific personals due to surmised toxic effects on bacterial populations. It is likely to hinder the decomposition capability of aerobic anaerobic soil microbes by engineered nanoparticles.
Importance of Ag nanoparticles and synthesis approaches
Silver nanoparticles are one of the nano particles that have been employed in widest range of applications (Rajeski and Lekas, 2008). Antibacterial, antifungal, photocatalytic, and electrocatalytic properties and application potentials in catalysis, biological and chemical sensing, nonlinear optics, electronics, and surface enhanced Raman spectroscopy have drawn an exceptional attention of scientists and industrial persons (Kelly et al., 2003) for the generation of products with novel, improved characteristics using nano Ag. Therefore nano Ag related products are one of the commonly found nanoparticles in nano commerce and have been reported 104 out of 502 nanoproducts associated with nano Ag, based on a survey carried out by Maynard and Michelson (2006). Woodrow Wilson International Center (2007) claimed that 20% of the nanoproducts are responsible for nano Ag related products by the year of 2007 (Breggin and Pandergrass, 2007). Moreover, Silver nanoparticles receive second highest funding for risk related research based on the inventory compiled by Project on Emerging Nanotechnologies (2006). (Maynard et al., 2006).
Numerous researches has been reported based on Ag nanoparticles related to synthesis, modification, maintenance of the intact properties in order to obtain above stated properties for specific application potentials. Discoveries on nanoparticles in the recent past, demonstrate that these properties are greatly influenced by size and shape (Pal et al., 2007). Therefore, there is an upsurge of research interests on various synthesis methodologies to control the size and shape of nanoparticles to gain the desired characteristics (Jana et al., 2001, Zhou et al., 1999). Size and shape specific characteristics are also apparent with regard to nano Ag (Pal et al., 2007). Recent literature demonstrates various synthesis methodologies for nano Ag such as chemical, electrochemical, Î³ radiation, photochemical, and laser ablation (Guzman et al., 2008 ). Various modifications that are employed with regard to the specific primary synthesis method during and after the preparation of nanoparticle, could give rise to particles of variable sizes and shapes. As examples variations in temperature, pH, and agitation; may influence inherent properties of nanoparticle significantly in chemically synthesized method. Incorporation of "capping /coating/ stabilizing agents" to the synthesized particle contribute the stabilization of colloids by hindering the aggregation, and controlling the size and shape of the synthesized product (Olenin et al., 2008). Colloidal stabilization mechanism is gained through electrostatic or /and steric stabilization. Electrostatic stabilization is functionalized via formation of an electrical double layer using surface charge of the particles. Steric stabilization is functionalized via adsorption of the stabilizing agent onto the particle surface as a protective layer (Hassell et al., 2007. Sun and Luo 2008). Range of stabilizing agents are in use, including Polyvinyi pyrrolidone (PVP), poly vinyl alcohol (PVA), Sodium dodecyl sulphate (SDS) like surfactants and thiol, carboxyl, amino, and cyano like functional groups (Si and Mandal 2007)
Synthesis of nano Ag is primarily based on reduction of Ag ions utilizing a specific reductant in order to form 0 valent stage of Silver (The resultant is the nanoparticles since specific conditions are applied to gain nano size simultaneously). Majority of the reported studies on synthesis have used silver nitrate as the salt precursor (Tolaymat et al., 2009). A widely applied method for nano Ag generation is the chemical method due to simple nature and easy accessibility to the materials. However, care should be exercised for the specific conditions to be maintained in order to synthesize stable, reproducible colloids (Guzman et al., 2008). Chemical reducing agent may be a plant extract, chemical agent or a biological agent (Tolaymat et al., 2009). Widely applied chemical reducing agents are citrate (Turkevich et al., 1951), borohydride (Solomon et al., 2007, Lee and Meisel, 1982), hydrogen (Evanoff et al., 2004), Branched Polyethyleneimine stabilized method (Tan et al., 2007), ascorbic acid, hydrazine. Selection of the Ag precursor, reducing and the stabilizing agents depend on the characteristics of nano Ag expected.
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Toxicity of nano Ag
Silver and Silver incorporated compounds have been used for the treatment of wounds and burns for centuries due to antibacterial properties associated (Rai et al., 2009). Thus nano Ag is likely to be antimicrobial, use of nano Ag as an antimicrobial and biocidal substance is now in surge of use. Nano Ag has declared to be more toxic than that of other Ag species such as Ag ions and AgCl colloids (Choi et al., 2008, Pal et al., 2007). Discoveries related to toxicity studies have demonstrated noticeable inhibitory levels on bacterial species (Pal et al., 2007), algae (Miao et al., 2009), fish (Oberdorster 2004b), and mammalian species (Asha Rani et al., 2008, Kawata et al., 2009 ). According to the Silver Nanotechnology Commercial inventory compiled by Fauss (2008), 45% of the Ag nano products were reported in the size range of 0.3-250nm, which is the size likely to be inducing toxicity effects of bacteria. The average nanoparticle size of all products was estimated as 45nm. However the mechanisms that allow nanoparticles entering to the living cells are not well understood, which in turn facilitates bacterial toxicity (Navarro et al., 2008). Srivastav et al (2007) has studied for different gram negative and positive bacteria and has revealed gram negative bacteria are more toxic towards Ag nanoparticles. Kim et al., 2007 has reported more toxicity towards gram negative Escherichia coli than gram positive Staphylococcus aureus. The toxicity difference of gram negative and positive bacteria lied on the basis of molecular structure of the cell walls. Bacterial cell wall is consisted with a layer of paptidoglycan. Gram negative bacteria have a thin layer of peptidoglycan while gram positive bacteria have a thick layer making nanoparticle difficult to penetrate the cells. Studies have also shown the potential generation of Reactive Oxygen species, which damage the "cell membrane", resulting the inactive or dead bacterial cells (Limbach et al., 2007, Srivastav et al., 2007). Although there were numerous studies on potential hazardous effects of nano Ag towards bacteria, harmful effects to aquatic organisms remains unknown (Moore, 2006).
Daphnia magna and toxicity testing
Due to many reasons, Use of aquatic organisms is a common aspect in toxicity studies. Aquatic environments are the ultimate destination for many types of ecologically significant chemicals (Van der Oost et al., 2003). It provides precise detectability of toxicity effects of biological organisms, than chemical methods available. Also current risk assessment methods are in need for the use of species for hazard detection (USEPA 2002). Daphnia magna a cladoceran which the study has been focused, is a well-known zooplanktonic freshwater dweller who's considered as an aquatic model species and also used as a bioindicator by organizations including U. S. Environmental Protection Agency (Lovern and Klaper, 2005). Daphnia interacts with large portions of an aquatic environment, filtering approximately 16.6 ml/h. Hence greater potential exists being affected by ingestion of particulates in water than in other aquatic organisms (McMahon and Rigler, 1965). As well as D. magna, this organism is a main component in an aquatic food chain and consumed by many higher animals including fish, snails etc.
D. magna is able to uptake aquatic nano compounds during feeding and likely to undergo physiological, biological, and behavioral alterations. Literature reports on nano related toxicity effects of TiO2 (Rosenkranz 2010, Lovern et al., 2007, Weinch et al., 2009), Fullerene (Rosenkranz 2010, Lovern et al., 2007), ZnO (Weinch et al., 2009), Cerium dioxide (Rosenkranz 2010). However studies on toxicity effects of Ag nanoparticles are scarce. Rosenkranz (2010) has carried out acute and chronic toxicity tests incorporating nano Ag, to D. magna, which has produced promising results, inferring high inhibitory levels even at low concentrations. Moreover, Rosenkranz has dictated a size dependent inhibition of D. magna after exposure to nano and micro sized nanoparticles.
Extent of Toxic effects, are likely to be altered based on the various procedures of synthesis as well as capping agents employed. Present study will mainly discuss the inhibitory differences of various Ag nanoparticles on the basis of the method synthesis, capping agent, and the size using E.coli and D. magna as model organisms.
The study hypothesized that (i) Ag nanoparticles cause inhibitory effects on Daphnia magna and Metplate E.coli bacteria (ii) Ag nano particles exhibit inhibitory effects based on size, synthesis procedure and capping agent (iii) D. magna can be used as a model organism to evaluate the nano Ag contamination in an aquatic environment to assess the environmental quality.
2.0 Goals and Objectives
Study will be carried out broadly to synthesize and characterize Ag nanoparticles. This study will incorporate the usage of different methods available in literature and study the inhibitory effects of D. magna and E. coli (from Metplate kit) considering the size, method of synthesis, and coating materials.
Synthesis and Characterization of Ag nanoparticles.
Numerous techniques have been published in relation to the synthesis of Ag nanoparticles. Ag nanoparticles will be synthesized by using 4 different chemical reduction methods which are well- defined for the long term stability, even without applying external coating substance. Physical characteristics such as size distribution, mobility, and zeta potential will be dictated.
Approach: Synthesis and stabilization of Ag nanoparticles will be accomplished differentially using citrate, borohydride, hydrogen, and BPEI reduced methods. Nanoparticles will be characterized using UV-vis spectrometry (DR 5000, HACH company), particle size analyzer (NICOMP 980ZLS, Particle sizer Laboratory), TEM (Transmission Electron Microscopy), and SEM (Scanning Electron Microscopy) equipped with EDS (Energy Dispersive X-ray Spectroscopy).
Study the inhibitory effects of D. magna and Escherichia coli on exposure to Ag nanoparticles
The study will primarily evaluate the inhibitory effects of D. magna using static renewal, 48 hr acute toxicity tests observing mortality as the endpoint. Metplate test will be executed to determine inhibitory effects of Metplate bacteria. Thus, LC50 will be calculated as a measure of toxicity.
Approach: Inhibitory effects will be determined for 4 types of chemically synthesized Ag nanoparticles using D. magna and Metplate E. coli, for the following modified solutions of Ag np prepared from each synthesis method. LC50 values will be used to determine the toxicity levels associated.
Unpurified Ag np solution with both Ag ion and Ag np (Original solution resulted after synthesis)
Purified Ag np solution
PVP coated purified Ag np
Tween 20 coated purified Ag np
3.0 Benefits expected
Studies on nanoparticles (CeO2, TiO2, Fullerene and ZnO) demonstrate toxicity over the range of terrestrial and aquatic organisms including: fish, bacteria, protozoa and algae. However, toxicity studies on Ag nanoparticles are scarce for any type of organism except for bacterial species, where high levels of inhibition have been reported during exposure. Chiefly, this study will satiate the loopholes of nano Ag toxicity, by exploring the toxicity towards freshwater dweller, D. magna and Metplate bacterium, E. coli; where no studies have reported in terms of nano Ag toxicity. Further synthesis method and coating material dependent toxicity of nano Ag to D. magna and Metplate bacteria (E. coli), will be determined The study will satiate the loopholes of nano Ag studies as well as aquatic toxicology of nano Ag.
Results will be used to predict maximum levels of nano Ag that should exist in a fresh water environment in the aspect of environmental impact assessment and demonstrate the importance of data in terms of fresh water criteria development. The study will be evaluating the use of D. magna as a model organism to detect nature/condition of an aquatic environment with regard to nano Ag. The approach will provide reliable, economical, and easy way to perform aquatic nano Ag toxicology compared to existing chemical methods since Daphnia is an ubiquitous organism in any fresh water environment.
Synthesis and characterization of Ag nanoparticles
Borohydride reduced method (Solomon et al., 2007)
AgNO3 + NaBH4 Ag + 1/2 H2 + 1/2 B2H6 + NaNO3
10ml of 1.0mM Silver nitrate will be added to 30ml of 2.0mM Sodium borohydride solution, chilled in an ice bath while stirring vigorously on a magnetic stir plate. Bright yellow color solution will be the result, when all silver nitrate is added.
Citrate reduced method (Badawy et al., 2010)
Bluish green color colloidal (19 Â± 5nm) solution will be prepared and with a hydro dynamic diameter of 10nm. 1x 10-3 M AgNO3 solution will be mixed with 1x10-2 Na3C6H5O7.2H2O (99% , SAFC supply solution) in a volume ratio of 2:1 respectively. Mixture will be heated for 4 hrs at 700C in water bath.
Branched polyethyleneimine reduced method (Badawy et al., 2010)
Polyethyleneimine (BPEI) (99%) and AgNO3 will be dissolved separately in 1 x 10-4 M solution of N- (2-hydroxylethyl) piperazin-N-2 ethanesulfonic acid. The 2 solutions will be then mixed in a volume ratio of 1:1 to give a final molar ratio 0.5: 1: 0.1, AgNO3: BPEI: HEPES, respectively. Then the solutions will be exposed for 2 hrs to UV irradiation using a standard low pressure Hg are lamp. 10 Â± 4 nm Ag NP will be
Hydrogen reduced method (Badawy et al., 2010)
3g of Ag2O will be added to 3L of DI water in a 5L round bottomed flask. The solution will be heated on a hot plate and will be maintained at 700C. 48L of ultra pure hydrogen gas per L of solution will be bubbled through the solution at standard pressure.
Characterization of AgNPs.
Ag NPs synthesized from each above method, will be characterized via UV-vis spectrometry (DR 5000, HACH company), particle size analyzer (NICOMP 980ZLS, Particle sizer Laboratory), TEM (Transmission Electron Microscopy), and SEM (Scanning Electron Microscopy) equipped with EDS (Energy Dispersive X-ray Spectroscopy.
2. Toxicity tests
(1) 48 hour Daphnia magna toxicity test
Static renewal 48hr/96hr toxicity tests will be performed to total Ag concentrations of 0.001, 0.025, 0.05, 0.10, 0.3125, 1.25, 1.25, 2.5, 5.0, 10, 100mg/L concentrations to following modified AgNP solutions obtained from each method of synthesis. (i) Unpurified AgNP solution (Resulted solution immediately after synthesis) (ii) Purified Ag NP solution (iii) PVP coated solution (iv) Tween 20 coated solution.
Daphnia starter cultures will be obtained and will be scaled up and maintained (EPA, 2002) (Appendix B) accordingly to the requirement. Alga, Selenastrum capricornutum, YCT (Yeast, Cerophyll and Trout chow) will be provided as food following the standard procedures (Appendix C). Standard synthetic water (Dilution water) (Appendix A), will be used to culture and maintenance of all Daphnia and food cultures. Less than 24 hr old neonates from Daphnia cultures will be used as the test organisms. 15mL aliquots of test solution will with 10 replicates will be analyzed for each concentration. Standard synthetic water will be used as the medium for controls. 10neonates will be transferred to each replicate. Each following day a new set of precleaned or new test chambers will be prepared with the original set and 15mL solutions will be placed. Test organisms will be transferred to the new set of chambers carefully using a dropper hindering air trapping under the carapace. Mortality will be detected at the end of the incubation period and required physical measurements: pH, conductivity and temperature will be carried out. LC50 value will be calculated for each test as a measure of toxicity.
(2) MetPlate Toxicity test (Appendix D)
Traditionally, Metplate kit is used to test heavy metal toxicity which will be used to test Ag toxicity. Silver, Copper, and fullerene NPs have shown toxicity towards Metplate bacteria in study done by Gao et al., 2008. In this study Ag NP toxicity will be extensively studies on Metplate bacteria. The toxicity assay is based on Î²-Galactosidase enzyme inhibition in a bacterial strain by bioavailable Ag NPs. Test will be carried out in a 96 well microplate and intensity of the color will be detected in a microplate reader at 575nm wavelength, after 0.5-2hrs of incubation with the substrate. LC50 value will be calculated according to the standard methods.
5.0 Statistical Analysis
LC 50 values will be calculated using Probit analysis for each test. Comparisons will be done employing ANOVA/mANOVA based on the variables.
Today Ag nanoparticles are one of the widely used nanoparticles in U.S. commerce primarily due to its antibacterial, anti-fungal, and photocatalytic properties. These properties have also drawn the great attention of both scientific personals and public. Reports have been published on AgNP toxicity to numerous bacterial species, where inturn advantageous in medical and health fields. Since the applications of Ag Nps rise continuously, it is likely to enter to major compartments of the ecosystem, soil, air, and water. Studies on AgNP toxicity to biological organisms (except for common bacterial species) are sparse and the study attempts to satiate the loopholes of Ag NP ecotoxicity, especially considering common methods of synthesis and commonly applied coating materials. Toxicity studies of AgNPs to Metplate bacterial strain and Dapnia magna (48hr acute toxicity assay), a common aquatic dweller will be used to accomplish the objectives of the study. Toxicity will be measured in terms of LC50 for both tests. Metplate test is a measure of bacterial enzyme (Î²-Galactosidase) activity, where the absorbance is measured and LC50 is detected accordingly. In D. magna test LC50 will be calculated considering mortality as the endpoint. Tests will be carried out for a broad range of total Ag concentration, thus the LC50 values or toxicity will be projected to predict criteria for environmental risk assessment processes.
7.0 Time Line
Synthesis of Ag nanoparticles and Characterization.
Set up Anaerobic Digester
BMP assay using synthesized Ag nanopartcles
MEA assays for synthesized Ag nanoparticles
Laboratory fabrication of Ag nanowaste
BMP assay using laboratory fabricated Ag nanoawaste
MEA assays for synthesized Ag nanoparticles
Analysis using MYGRT model
Data analysis and Thesis Preparation
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