Uptake of Nanoparticles of Cerium Oxide and Yttrium Oxide by Acanthamoeba castellanii (Protozoa) and Daphnia magna (Crustacea)

Currently, nanoparticles are synthesized and used at an unprecedented rate for industrial, medical, and research applications. The use of cerium oxide nanoparticles (CeONP) and yttrium oxide nanoparticles (YtONP) results in their spread as contaminants into the environment. Once in the environment, CeONP and YtONP can be taken up by organisms in the food chain where they may pose a public health risk. In this study we determine whether Acanthamoeba castellanii and Daphnia magna uptake CeONP or YtONP from their environment and thereby play a role in the transmission of the nanoparticles. Using electron microscopy, organisms exposed to the nanoparticles were examined. Our results indicate that the nanoparticles are associated with cell and organelle membranes. These findings have implications for the health risks associated with environmental contamination by CeONP and YtONP. INTRODUCTION In this study we determine whether protists and crustaceans play a role in the transfer of cerium oxide nanoparticles (CeONP) and yttrium oxide nanoparticles (YtONP) from the environment to other organisms within the aquatic food chain. Acanthamoeba castellanii, a common protist, and Daphnia magna, a planktonic crustacean, are important components in many aquatic ecosystems. Because acanthamoebae, such as A. castellanni, are aggressive feeders they consume inorganic and organic compounds from their environment, thereby serving as a link by transferring normally unavailable inorganic components to the food chain (Weekers et al. 1993). D. magna feeds on acanthamoebae and other protists found in lower trophic levels. Because of the high reproductive potential of D. magna, these planktonic crustaceans can substantially alter the structure and functioning of microbial food webs in freshwater ecosystems , such as acidic swamps, freshwater lakes, ponds, rivers and streams (Guisande 1993). D. magna and other aquatic crustaceans have the ability to filter particles of a variety of sizes including nanoparticles (Rosenkranz et al. 2009, Kim et al. 2010, Zhu et al. 2010). The authors hypothesized that CeONP or YtONP in 1 Corresponding author: jpalmieri@vcom.vt.edu Virginia Journal of Science, Vol. 62, No. 1, 2011 https://digitalcommons.odu.edu/vjs/vol62/iss1 4 VIRGINIA JOURNAL OF SCIENCE the natural environment could be taken up by these organisms. Particles engineered at dimensions between 1-100 nm, are referred to as nanoparticles. Currently, nanoparticles are synthesized and used for industrial, medical, and research applications. CeONP are used as diesel fuel additives, in automotive catalytic converters, and are a by-product of many industrial processes, including the polishing of glass and semi-precious stones. CeONP also have potential uses for medical applications acting as an antioxidant (Elswaifi et al. 2009). Yttrium oxide and cerium oxide belong to the rare earth elements. Yttrium oxide nanoparticles (YtONP) are used in the manufacturing of cathode ray tubes for computer monitors and televisions and have potential medical applications due to their ability to act as antioxidants (Schubert et al. 2006, Cotton 2006, Okuyama et al. 2007, and Gilmore et al. 2008). The use of CeONP and YtONP results in their release into the environment (Biswas and Wu 2005, Chow et al. 2005) where they may exist in concentrations and forms that are toxic. This release may result from the process of their synthesis, as a by-product of their use, or from their indiscriminate disposal after use. Presently, CeONP and YtONP are released into the environment from diesel engine emissions, from improper disposal of automotive catalytic converters, and from improper disposal of old TV and computer monitors. These nanoparticles may then make their way into air, soil, or ground water (Biswas and Wu 2005, Chow et al. 2005). As with many of the engineered nanoparticles, CeNOP and YtONP may also have toxic effects on humans and animals (Gatti and Montanari 2008). The toxicity of CeONP and YtONP has been recently investigated in vivo and in vitro (Gojova et al. 2007, Gatti and Montanari 2008, Andelman et al. 2009, Hardas et al. 2010). Toxic effects include reduced cell viability, increased cellular oxidative damage, and apoptosis. Effects also include vascular inflammation that may lead to pulmonary thromboemblism resulting in stroke or myocardial infarction. Chronic inflammation may also lead to rare earth pneumoconiosis or lung cancer (Gojova et al. 2007, Gatti and Montanari 2008, Andelman et al. 2009, Hardas et al. 2010, Lin et al. 2006). However, the routes of exposure of humans to these nanoparticles are poorly understood. Examples of routes of exposure of CeONP and YtONP may be through the contamination of organisms in the food-chain or through contamination of drinking water (Holbrook et al. 2008). In a typical food chain, there is usually a maximum of four or five trophic levels, although food chains in aquatic ecosystems frequently contain more levels than those in terrestrial ecosystems (Pimm and Lawton 1977). As organisms in lower trophic levels are consumed by those in a higher level, nanoparticles may become concentrated in top level consumers, level consumers, namely fish and animals that consume fish, including humans. Many protists, including acanthamoebae, are voracious feeders of organic and sometimes inorganic materials as they occupy the bottom of aquatic and some terrestrial ecosystems (Khan 2009). Acanthamoeba spp. are free-living amoebae that are ubiquitous in aquatic and terrestrial ecosystems. Acanthamoebae exist as trophozoites, the feeding stage, and as cysts. Presently, 23 species of Acanthamoeba are reported and their biology and pathogenicity reviewed by Marciano-Cabral and Cabral (2003) and Khan (2009). At least three species of Acanthamoeba have been reported as parasites of animals, and humans (Marciano-Cabral and Cabral 2003 and Khan 2009). It is unknown whether CeONP or YtONP particles are taken up and concentrated by protists or by crustaceans in contaminated aquatic ecosystems. In this report we investigate whether CeONP and Virginia Journal of Science, Vol. 62, No. 1, 2011 https://digitalcommons.odu.edu/vjs/vol62/iss1 UPTAKE OF NANOPARTICLES 5 YtONP may be incorporated into the protest Acanthamoeba castellanii (Fig. 1) and into the arthropod Daphnia magna, two integral components representing organisms at two different trophic levels of the aquatic food chain. MATERIALS AND METHODS Exposure of Organisms to Nanoparticles: CeONP and YtONP stock solutions were prepared by suspension of nanoparticles in distilled water. Samples of the nanoparticles suspended in distilled water were placed in a vortex apparatus for 5 minutes before and after preparation of the solution to minimize formation of nanoparticle aggregates. D. magna cultures were exposed to nanoparticles by the addition of the respective solution to make a final concentration of 10 μM of CeONP or YtONP. D. magna viability was determined by observing motility and gill movement. Distilled water without nanoparticles was used in control samples. CeONP or YtONP were added to cultures of A. castellanii containing >95% trophozoites, making a final nanoparticle concentration of 100 nM. Equal amounts of distilled water were used for exposure of control samples. Cultures were incubated for 24 hours at 25C, washed two times in Page’s saline solution (Petry et al. 2006), then processed for electron microscopy using standard techniques. Control groups consisted of organisms treated with only distilled water. To define the appearance CeONP and YtONP alone, suspensions were used to prepare samples for observation by transmission and scanning electron microscopy. FIGURE 1. Scanning electron micrograph showing two trophozoites feeding stages of Acanthamoeba castellanii. Virginia Journal of Science, Vol. 62, No. 1, 2011 https://digitalcommons.odu.edu/vjs/vol62/iss1 6 VIRGINIA JOURNAL OF SCIENCE The appearance of CeONP and YtONP in cells was compared to those reported in other studies. Each experiment was performed twice including control groups. There were sixteen experimental group cultures; eight cultures of A. castellanii and eight cultures of D. magna. There were four control groups run for each species; two cultures each, exposed to CeONP for D. magna and A. castellanii and 2 cultures each, exposed to YtONP for D. magna and A. castellanii . Electron Microscopy: After exposure of A. castellanii and D. magna to CeONP or YtONP, specimens were prepared for electron microscopic examination. A. castellanii was washed two times using Page’s saline solution, centrifuged and fixed in 0.5% buffered glutaraldehyde at 4C for 72 hours. D. magna was pre-fixed in FAA fixative solution for 5 minutes at 4C and post-fixed in 0.5% buffered glutaraldehyde at 4C for FIGURE 2. Outer membrane of Acanthamoeba castellanii demonstrating accumulations of yttrium oxide nanoparticles attached to outer surface. Arrows indicating yttrium oxide nanoparticles (Bar = 500nm ). Virginia Journal of Science, Vol. 62, No. 1, 2011 https://digitalcommons.odu.edu/vjs/vol62/iss1 UPTAKE OF NANOPARTICLES 7 72 hours. A. castellanii and D. magna were then processed for ultra-thin sectioning and examination using transmission electron microscopy according to standard methods used by the Virginia-Maryland Regional College of Veterinary Medicine Morphology Services Laboratory. Electron micrographs of thin sections of A. castellanii and D. magna were examined for densely stained nanoparticles of CeONP and YtONP to determine if nanoparticle uptake had taken place. For scanning electron microscopy, samples of A. castellanii were isolated and processed according to standard methods used by the Virginia-Maryland Regional College of Veterinary Medicine Morphology Services Laboratory. RESULTS Our results demonstrate that CeONP and YtONP can be readily taken up by A. castellanii and D. magna. We have also demonstrated that after uptake, Ce