Environ Sci Pollut Res (2013) 20:8636–8648 DOI 10.1007/s11356-013-1798-3
RESEARCH ARTICLE
Environmental effects of nanosilver: impact on castor seed germination, seedling growth, and plant physiology Jyothsna Yasur & Pathipati Usha Rani
Received: 28 February 2013 / Accepted: 30 April 2013 / Published online: 24 May 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Increasing use of nanoparticles in daily products is of great concern today, especially when their positive and negative impact on environment is not known. Hence, in current research, we have studied the impact of silver nanoparticle (AgNPs) and silver nitrate (AgNO3) application on seed germination, root, and shoot length of castor bean, Ricinus communis L. plant. Silver nanoparticles had no significant effects on seedling growth even at higher concentration of 4,000 mg L−1, while the silver in bulk form as AgNO3 applied on the castor bean seeds inhibited the seed germination. Silver uptake in seedlings of the castor seeds on treatment with both the forms of silver was confirmed through atomic absorption spectroscopy studies. The silver nanoparticle and silver nitrate application to castor seeds also caused an enhanced enzymatic activity of ROS enzymes and phenolic content in castor seedlings. Highperformance liquid chromatography analysis of individual phenols indicated enhanced content of parahydroxy benzoic acid. These kinds of studies are of great interest in order to unveil the movement and accumulation of nanoparticles in plant tissues for assessing future applications in the field or laboratory. Keywords Silver nanoparticles . Silver nitrate . Castor plant . Seed germination . Phenols . Oxidative enzymes
Responsible editor: Elena Maestri J. Yasur : P. U. Rani (*) Biology and Biotechnology Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007( Andhra Pradesh, India e-mail:
[email protected] P. U. Rani e-mail:
[email protected] P. U. Rani e-mail:
[email protected]
Abbreviations AAS Atomic absorption spectroscopy AgNPs Silver nanoparticles AgNO3 Silver nitrate CAT Catalase DLS Dynamic light scattering HPLC High-performance liquid chromatography NP Nanoparticles POD Peroxidase ROS Reactive oxygen species SOD Superoxide dismutase TEM Transmission electron microscope XRD X-ray diffraction
Introduction The increasing level of production, use, and disposal of nanomaterials will unavoidably lead in to the environment or air, water, and soil. The progressive usage of nanoparticles (NPs) in several products including agriculture is gaining importance in recent years. The antimicrobial properties of silver have made it as major ingredient in several agricultural products from companies such as Monsanto, Bayer, Syngenta, etc., and also in detergents, plastics, and textiles (Benn and Westerhoff 2008; Blaser et al. 2008). However, use of these products has resulted in the release of NP into the environment. Though silver is known to be one of the most toxic trace metals (Ratte 1999), the mechanism involved in AgNP toxicity have not been fully studied. Although great effort has been made, it is still challenging to elucidate whether the toxicity is related to nanoparticles or because of dissolved forms of Ag (Ag+ ions) and their co-occurrence with AgNPs. There are only a few studies in which trials have been made to determine the effects of two species in environment. The present study was undertaken to find whether the toxicity of Ag is caused by
Environ Sci Pollut Res (2013) 20:8636–8648
AgNPs or Ag+ ions. Hence, the silver nanoparticles and their corresponding bulk form silver nitrate which is the major substrate other than silver chloride for the synthesis of silver nanoparticles were used to study their impact on castor seeds and castor plants. As plants contribute a significant area in the environment, there is a chance that they encounter the released nanomaterials. It is unclear what impact these nanomaterials will have on plant’s life, and it is essential to understand the effects of NP on plant growth. Such studies are important not only from the point of view of the application of NPs in plants, but also for understanding presumed effects on plants and causes for bioaccumulation. As of yet, the risks of NPs in environment have not yet been fully characterized. Though there is available literature on the effects of nanoparticles on seed germination on certain plant species. It differs with nanoparticles used, size, concentration, and plant species (Rico et al. 2011). However, there are no data available on the effects of silver nanoparticles and their bulk form on the castor plants. Especially, the literature regarding the change of phytotoxicity and oxidative stress by means of nanoparticles is very limited (Navarro et al. 2008a). Ecotoxicity of silver nanoparticles (AgNPs) are reported in a few studies earlier. They are known to reduce cell growth, photosynthesis, and chlorophyll production of a marine diatom (Thalassiosira weissflogii) and in freshwater alga (Chlamydomonas reinhardtii), and these toxic effects are implied to be due to the release of dissolved silver (Miao et al. 2009; Navarro et al. 2008a, 2008b). Previously, AgNPs less than 5 nm are reported to be more toxic to the nitrifying bacteria than larger AgNPs or dissolved Ag, signifying the toxicity of the dissolved silver (Choi and Hu 2008). Our recent studies on the biosynthesized AgNPs on the ecotoxicological effect on aquatic organisms Daphnia magna have shown it to be safer (Usha Rani and Rajasekharreddy 2011). However, there have been very few reports on the impact of AgNPs on certain higher plants (Kumari et al. 2009; Monica and Cremonini 2009; Stampoulis et al. 2009). Toxicity studies of several nanomaterials such as TiO2, ZnO, Mg, Al, Pd, Cu, Si, C60 fullerenes, and multiwall carbon nanotubes showed both negative and positive effects on plant growth on certain higher plants (Bernhardt et al. 2010; Monica and Cremonini 2009). However, nanoparticles show different effects on seed germination of different plants too (Rico et al. 2011), but their exact effect on trophic levels and the trophic transfer into plant is not known yet. Hence, the study on effects of silver nanoparticles and its salt form (AgNO3) application on plant seeds and further its impact on plant/seedling growth as well as on the plant reactive oxygen species (ROS) undertaken as an attempt to understand the NP penetration in to the plant and plant’s reaction to this.
8637
One of the main mechanisms through which NPs mediate their effects involves oxidative stress (Li et al. 2003; Nel et al. 2006; Xia et al. 2006). ROS can be formed via radicals, transition metals, or other chemicals on the particle surface, or as a consequence of the interaction between particles and cellular components (Nel et al. 2006). To overcome oxidative stress, plants can induce a series of detoxification reaction catalyzed by antioxidative enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) (Vitoria et al. 2001; Zhang et al. 2005). SOD enzymes are involved in antioxidation during stress which dismutates superoxide into hydrogen peroxide and molecular oxygen. Hydrogen peroxide, produced by superoxide dismutase, is scavenged by catalase and a variety of peroxidases with concomitant oxidation of co-substrates such as phenolic compounds and/or antioxidant molecules. Also, the flavonoids and phenols are reported to be induced by various types of stresses (Liu et al. 1993; Dixon and Paiva 1995). Our previous studies with plant defense mechanisms indicated that application of any stress in the form of chemicals or insect feeding can affect plant’s ROS enzymes and also causing changes in biochemical profile (Usha Rani 2008; Jyothsna et al. 2009). Owing to the important role of all these biochemicals and enzymatic changes on external modifications, an attempt was made to study the application of NP on castor plant seeds and the differences in ROS production, i.e., antioxidant enzymes: POD, CAT, and SOD in castor seedlings. In addition, the Ag content in plant tissues was determined by AAS. Phenolic metabolites are involved in antioxidant activity that depends on the availability and number of hydroxyl groups in the molecule. Furthermore, phenolic structures can act as metal chelators (Michalak 2006) and participate in ROS scavenging through peroxidases (Sgherri et al. 2003). Quantitative data available on phenolic acids accumulation in nanoparticles or metal-treated plants are scarce. Other than antioxidation, simple phenolics are involved in polymerization of p-coumaric, ferulic, and sinapic acids further leading to the formation of lignin. This is a normal process occurring in plants, while enhanced lignification in stress conditions serve as a barrier limiting metal/pathogen entry into tissue (de Ascensao and Dubery Ian 2003; Ederli et al. 2004). Castor Ricinus communis L. (Euphorbiaceae) is major crop in dry land areas of Andhra Pradesh, India. Castor seeds are used in many pharmaceutical preparations, having tremendous export value. We have used this plant as a model for the experiments. Studies on seed germination and root elongation effects are the widely used phytotoxicity tests that provide comprehensive impact of tested nanoparticles. This test has several advantages such as simplicity and suitability to various species. Hence, the current study aims at comparing the effects of Ag NPs treatment to
8638
their corresponding bulk material counterparts on castor seed germination. To date, there are many techniques in use for measuring the size of nanoparticles, which are based on fundamentally different scientific principles. In earlier literature, it is reported that no single technique is sufficient to accurately estimate the size of nanoparticles in different media types (Domingos et al. 2009; Macken et al. 2012). Therefore, in this study, a series of different techniques were employed to investigate the size and behavior of the AgNP suspensions prepared with Millipore water.
Materials and methods
Environ Sci Pollut Res (2013) 20:8636–8648
Dynamic light scattering (DLS) Dynamic light scattering (DLS) was used to measure the hydrodynamic diameter (Z average), the intensity-based size distribution, and the zeta potential of AgNP in Millipore water. The particle size distributions (PSD) and Zeta potential measurements of the AgNP were analyzed using a Malvern Instruments Zetasizer Nano Series (Malvern Instruments, UK). Typically, 1 mL of solution was placed into a disposable sizing cuvette, and folded capillary cells were used for PSD and Zeta potential studies, respectively. Measurements were conducted at 25 °C, using a concentration of 100 mg L−1 silver NP, and six measurements on triplicate samples were made.
Preparation of silver suspensions Germination experiments Silver nanoparticles (powder form) stabilized by polyvinyl pyrrolidone (PVP) were purchased from Sigma Aldrich Inc., USA, and were
