Nanomaterials and the Environment Research Article • DOI: 10.2478/nanome-2013-0004 • nanome • 2013 • 48–57
Toxicity and stability of silver nanoparticles to the green alga Pseudokirchneriella subcapitata in boreal freshwater samples and growth media Abstract The toxicity of silver nanoparticles (AgNPs) to green alga Pseudokirchneriella subcapitata was evaluated in standard nutrient medium (ISO 8692), lake water samples from an oligotrophic and an eutrophic lake, and in lake waters supplemented with the standard nutrient medium. Prior to toxicity testing the agglomeration of polyvinylpyrrolidone (PVP) and starch-coated AgNPs was studied in each test medium. Agglomeration was studied by determining the hydrodynamic diameter (HDD). The HDDs for the PVP- and starch-capped AgNP dispersions in deionized water were 40 and 175 nm respectively, indicating the presence of agglomerates. The HDDs of AgNPs remained stable throughout the exposure time in all media used for the toxicity tests. The algae growth inhibition test was performed as a microplate modification of the ISO method using fluorescence detection. The effect of concentration at a 50% inhibition value for PVPcoated AgNPs in standard medium was 115 ± 3 µg/L, and for starchcoated AgNPs 51 ± 32 µg/L. The eutrophic freshwater conditions suppressed the toxicity of the PVP- coated AgNPs, but not the starchcoated NPs. This finding emphasizes the importance of using different AgNPs and natural waters in assessing the environmental risks of silver nanoparticles.
M. Tuominen*, E. Schultz,
M. Sillanpää
Finnish Environment Institute, Laboratory centre, P.O. Box 140, FI-00251 Helsinki, Finland
Keywords Nanosilver • Pseudokirchneriella subcapitata • Nanoparticle fate • Nanoecotoxicology • Biotests
Received 04 January 2013 Accepted 29 May 2013
© Versita Sp. z o.o.
1. Introduction Nanotechnology is rapidly developing, and nanomaterials are used in an ever-increasing number of products and applications. Of the commercial products employing nanomaterials, silver nanoparticles (AgNPs) are utilized in the highest number of applications1. The extreme toxicity of silver towards bacteria is well known, and these antimicrobial qualities are applied in numerous applications. Furthermore, silver is one of the most toxic metals for phytoplankton, invertebrates and fish [1]. Silver has been found to be more phytotoxic to freshwater aquatic plant Lemna minor than mercury or cadmium [2]. However, the mechanism of silver toxicity remains unclear [3]. The three most common mechanisms of silver toxicity proposed are the disruption of ATP production and DNA replication following the uptake of free silver ions; the production of reactive oxygen species; and the direct damage to cell membranes [4]. In addition, silver ions have been shown to inhibit the enzymes for the N, P and S cycles in nitrifying bacteria [5] causing DNA to lose 1 Project on Emerging Nanotechnologies. 2011. Consumer Products Inventory. http://www.nanotechproject.org/inventories/consumer/
its replication ability, and damaging the cytoplasm membrane in bacteria [6]. Silver-based nanomaterials have already been shown to be released from their composites, within commercial applications such as textiles [7,8] and paints [9], via rinsing waters into the environment. It has not yet been fully established whether the toxicity of silver nanoparticles is caused by the nanoparticles themselves, or the silver ions dissolved from the NPs or a combination of both. The amount of dissolved silver in the solution can be controlled by adding complexing agents into the solution [10]. Navarro et al. [10] showed that the inhibitory effects of dissolved silver on photosynthesis of the freshwater algae Chlamydomonas reinhardtii were eliminated with the presence of cysteine, a strong silver ligand. The capacity of silver ions to function as an antimicrobial agent is also limited in media where chloride (Cl-) is present as it is likely that AgCl will be formed. AgCl has low water solubility and can be rapidly precipitated out of solution [4]. It has been suggested that the surface oxidation of AgNPs leads to the release of dissolved silver ions [11]. The rate of ion release is generally proportional to the particle surface area, and nanomaterials can release ions more rapidly [4] and have enhanced surface reactivity compared to their bulk counterpart
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Toxicity and stability of silver nanoparticles to the green alga Pseudokirchneriella subcapitata in boreal freshwater samples and growth media
[12]. The toxicity of AgNPs is affected by the size [13] and shape [14] of the particles and also the modification of the surface. Capping agents are included in the synthesis of nanoparticles to increase the stability of the nanomaterials. Capping agents are chemicals that prevent agglomeration of the nanoparticle by either steric repulsion or electrostatic repulsion or both [15]. The capping agent has the potential to reduce or increase the toxicity of the nanoparticles [3]. PVP- coated AgNPs have been shown to enhance antibacterial activity compared to free silver ions [16]. In this research the high antibacterial activity was shown to be linked with the surface stabilization capacity of PVP [16]. The objective of this study was to determine the effects of both standardized growth media and natural waters from an oligotrophic and a eutrophic lake on AgNP dispersions and the toxicity of AgNPs on the green alga Pseudokirchneriella subcapitata. In both freshwater samples and growth media the agglomeration of AgNPs was monitored by measuring the hydrodynamic diameter (HDD). To prevent an ionic strengthdriven agglomeration, the growth media was diluted by a factor of 2 for the agglomeration studies. After the suitability of the growth media or growth media dilution and freshwater samples was assessed, the algal growth inhibition tests were performed and the toxicities were established. In this research natural water samples were used instead of synthetic fresh water and silver nitrate was used as a reference, as recommended [17] with regard to using nanomaterials in biotests. To our knowledge this is the first research where nanoparticulate silver toxicity has been tested using water samples from boreal lakes. These waters typically are slightly acidic (pH 6-7) have high humic content, and low biodiversity.
Silver nitrate (AgNO3) (Sigma Aldrich, MO, USA) was used as a reference material for the algal growth inhibition tests. The solution was made as a 100 mg/L stock solution with deionized water. All silver and AgNP solutions were stored in the dark at +4°C.
2.2 Analytical instruments The hydrodynamic diameter (HDD) and the zeta potential (ζ potential) were determined with a dynamic light scattering (DLS) device (Zetasizer Nano ZS, Malvern Instruments, United Kingdom). A 633 nm He-Ne laser source and a detection angle of 173° were used in the measurements. HDD and ζ potential were determined as the average of three and five consecutive measurements respectively. For the algal growth inhibition test, fluorescence measurements (excitation 450 nm, emission 680 nm) were performed with a multilabel counter (Victor3 model 1420, Perkin Elmer, Wallac, Finland). The silver ion concentration was measured with a silver selective electrode (PerfectION, Mettler Toledo, Switzerland).
2.3 Freshwater samples
2. Experimental Procedures
Freshwater samples were collected from two Finnish lakes: eutrophic Lake Enäjärvi (60° 50N, 24° 30E) and oligotrophic Lake Simijärvi (60° 10N, 23° 33E). Water samples were collected from a depth of 1m. Lake Enäjärvi and Lake Simijärvi belong to the Finnish monitoring programme guided by the European Union’s Water Framework Directive (Directive/2000/60/EC). The physical and chemical properties were characterized with standardized laboratory methods [18]. A summary of the techniques is provided (Table 1). The freshwater samples were filtered with a cellulose acetate filter (Puredisc30, pore size 0.450 µm, Whatman, Germany) prior to characterization.
2.1 Test chemicals
2.4 Algal growth inhibition test
Two types of commercial AgNPs with different capping agents were utilized. Polyvinylpyrrolidone coated silver nanopowder (lot number 7024-043010) was purchased from Nanostructured & Amorpheus Material Inc. (Houston, TX, USA). The silver nanopowder had 25% silver according to mass with silver purity of 99.99% and with a nominal size of
