Article pubs.acs.org/est
Silver NanoparticleAlgae Interactions: Oxidative Dissolution, Reactive Oxygen Species Generation and Synergistic Toxic Effects Di He, Juan José Dorantes-Aranda, and T. David Waite* School of Civil and Environmental Engineering, University of New South Wales, Sydney, NSW, Australia 2052 S Supporting Information *
ABSTRACT: The short-term toxicity of citrate-stabilized silver nanoparticles (AgNPs) and ionic silver Ag(I) to the ichthyotoxic marine raphidophyte Chattonella marina has been examined using the fluorometric indicator alamarBlue. Aggregation and dissolution of AgNPs occurred after addition to GSe medium while uptake of dissolved Ag(I) occurred in the presence of C. marina. Based on total silver mass, toxicity was much higher for Ag(I) than for AgNPs. Cysteine, a strong Ag(I) ligand, completely removed the inhibitory effects of Ag(I) and AgNPs on the metabolic activity of C. marina, suggesting that the toxicity of AgNPs was due to the release of Ag(I). Synergistic toxic effects of AgNPs/Ag(I) and C. marina to fish gill cells were observed with these effects possibly attributable to enhancement in the generation of reactive oxygen species by C. marina on exposure of the organism to silver.
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INTRODUCTION The production of nanomaterials is increasing dramatically with an expected resultant increase in the distribution of nanoparticles (NPs) in the environment. While many of these NPs aggregate and form larger assemblages,1 some particles are resistant to aggregation and retain high surface area and high mobility, particularly in aquatic environments.2 Our extensive understanding of the properties and reactivity of colloidal materials will, in most instances, provide a sufficient knowledge base from which to draw conclusions concerning the behavior, including toxicity of NPs. However, some properties, such as the ability of certain NPs to generate reactive oxygen species (ROS) as a result of rapid reaction with oxygen3,4 or lightmediated generation of electron−hole pairs, 5 and the dissolution (and, in some instances, reformation6,7) of NPs resulting from their high specific surface area3,4,8 are less well understood from existing knowledge and may require further investigation. An area of particular deficiency relates to the interaction of NPs and organisms with uncertainty remaining as to the precise cause of toxicity of NPs to organisms and limited insight exists with regard to the potential for enhancement or diminution in toxicity as a result of synergistic effects. Silver nanoparticles (AgNPs) are one of the most commonly used nanomaterials because of their strong broad-spectrum antimicrobial activity with applications in textiles, personal care products, food storage containers, laundry additives, home appliances, paints, and even food supplements.9 On the basis of these uses, it is likely that AgNPs will be released to the aquatic environment, be a source of AgNP aggregates and dissolved silver ions (Ag(I)), and possibly exert toxic effects on aquatic organisms.9 Although much evidence has shown that both AgNPs and Ag(I) are toxic to bacteria10,11 and some aquatic organisms12 and accumulate in phytoplankton and marine invertebrates,13 it is unclear whether toxicity is specifically © 2012 American Chemical Society
related to NP properties or is due to the effects of dissolved Ag(I). For example, AgNPs appear to cause cell death by pitting bacterial cell membranes, increasing permeability and disturbing respiration.14 In contrast, Ag(I) interacts with the thiol groups of proteins,15 resulting in inactivation of vital enzymes, disruption of bacterial membrane integrity and increase in permeability and likely affecting DNA replication.16 When compared on the basis of total mass added, the toxicity of AgNPs to organisms has generally been found to be lower than that of Ag(I).12,17 Species of the raphidophycean genus Chattonella, which consists of C. antiqua, C. marina, C. subsalsa, and C. ovata, are well-known for their contribution to toxic red tides in Australia, Canada, Japan, and New Zealand, and are recognized to be responsible for mass fish deaths.18 The precise mechanism of toxicity from these algae remains unclear, but the production of ROS is suggested to be one of the key factors contributing to fish mortality.19 Recent investigations in our own laboratory showed that ROS can mediate the transformation between AgNPs and Ag(I) via an electron-charging and discharging process.6,7 For example, superoxide can reduce Ag(I) to AgNPs,6 whereas H2O2 can oxidize AgNPs to Ag(I). In addition, some evidence exists showing that AgNPs and Ag(I) can enhance ROS generation which, in turn, affects bacterial activity.10,20 Therefore, the interplay of silver species with C. marina, the most prolific extracellular superoxide producer of these Chattonella species, may exert synergistic toxic effects on fish and other living organisms. Received: Revised: Accepted: Published: 8731
February 12, 2012 June 16, 2012 July 20, 2012 July 21, 2012 dx.doi.org/10.1021/es300588a | Environ. Sci. Technol. 2012, 46, 8731−8738
Environmental Science & Technology
Article
centrifugal ultrafiltration (Amicon Ultra-15 3K, Millipore, MA) and MQ addition in two cycles. The concentration of the stock solution was monitored by inductively coupled plasma (ICP) analysis (Varian AX, Varian, Australia). A 200 μM AgNP stock solution was stored at 4 °C until use. The hydrodynamic diameter (HDD) of AgNPs was determined by dynamic light scattering (DLS) using a nanoseries zetasizer (Malvern Instruments) with a 633 nm laser source and a detection angle of 173°.7 The same instrument was also used to measure electrophoretic mobility which was subsequently transformed to zeta potential using Smoluchowski’s approximation. Exposure of C. marina to AgNPs and Ag(I). Aliquots of algal cultures were transferred into triplicate wells of 48-well microplates (Greiner) and aliquots of the AgNPs and Ag(I) stock solution were added to achieve concentrations of 0.02, 0.2, 2, 10, and 20 μM (final concentrations) in a final volume of 1 mL, respectively. Since GSe medium is seawater-based with a salinity of about 35 (∼0.5 M), the dominant species of Ag(I) added or released from AgNPs are AgCl2− (aq) and AgCl32− (aq) (calculated using the equilibrium speciation program MINTEQ). The algae were exposed to AgNPs and Ag(I) for 1 h at 19 °C with light intensity at 100 μmol photons m−2 s−1. Dissolution of AgNPs in GSe and C. marina culture media was quantified by monitoring the appearance of dissolved Ag(I) by ICP analysis (Varian AX; Varian, Australia). Prior to ICP analysis, algae were centrifuged (800 × g for 2 min at room temperature) and removed, followed by removal of AgNPs in the supernatant using Amicon centrifugal ultrafilter devices containing porous cellulose membranes with a nominal pore size of 1−2 nm. The supernatant was centrifuged for 45 min at 1800g (Allegra X-15R, Beckman Coulter Inc.) The clear filtrates showed no detectable plasmon resonance optical absorption peak, showing that AgNPs were absent in the filtrates.23−26 In addition, AgNP stock solutions had very low concentrations of total silver (
