Grantee Research Project Results
Final Report: Aquatic Toxicity of Waste Stream Nanoparticles
EPA Grant Number: R833317Title: Aquatic Toxicity of Waste Stream Nanoparticles
Investigators: Gordon, Terry , Chen, Lung Chi , Wirgin, Isaac
Institution: New York University School of Medicine
EPA Project Officer: Packard, Benjamin H
Project Period: October 1, 2006 through September 30, 2009
Project Amount: $399,827
RFA: Exploratory Research: Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured Nanomaterials: a Joint Research Solicitation-EPA, NSF, NIOSH, NIEHS (2006) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
The objective of this study was to determine the biological consequences of nanoparticle contamination of the aquatic environment. We hypothesized that there will be a particle type-dependent difference in the developmental toxicity of manufactured nanoparticles in aquatic species, and in testing this hypothesis, we: (1) measured the differential toxicity of several types of nanoparticles in an estuarine species of fish, Atlantic tomcod, and in mummichug (killifish); and (2) identified whether the embryo and larval stages of development of these two species are particularly susceptible to nanoparticle toxicity. Little research has been published on whether the physicochemical properties of nanoparticles influence their toxicity at developmental stages in an aquatic species. Thus, while an ever-growing database is being established to understand the influence of physico-chemical properties of nanoparticle toxicity in mammalian species, it is critical to understand the ability of various nanoparticles to produce toxicity once they have entered the waste stream and the aquatic environment. In our studies, a group of particle toxicologists collaborated with a fish toxicologist to explore the toxicity of a variety of manufactured nanoparticles in an established fish model of aquatic toxicity.
Summary/Accomplishments (Outputs/Outcomes):
The work on the project aims has proceeded smoothly and has been completed. As described in the Preliminary Results section (5) below, concentration-dependent changes in fish embryo mortality and hatching were observed. Importantly, the observed toxic effects were metal-specific and mediated by the nanoparticles and not released ions.
Embryos were exposed to metal nanoparticles (Ag, Cu, Fe, Ni, Zn) and carbon-containing nanoparticles (nC60, carbon black and two functionalized single walled nanotubes) as well as particles from the different manufacturing stages of fullerenes encasing erbium and yttrium atoms. Effects of nanoparticle exposure on embryo mortality, hatching success, time to hatch, and morphological abnormalities were determined. Increases in embryo mortality were observed in tomcod and mummichug exposed to Cu nanoparticles, with LC50s of 0.2321 and 1.594 µg/mL, respectively. Exposure to 10 µg/mL Fe and Zn nanoparticles resulted in 100 percent mortality for tomcod but not mummichug, while this same concentration of Ag did not affect mortality in either species. This suggests that tomcod embryo are more sensitive to the toxicity of metal nanoparticles. No increases in mortality were observed with any carbon-containing nanoparticles, although carbon black exposure did result in a decreased time to hatch. Erbium- and yttrium-containing manufacturing stage nanoparticles did cause increased mortality and alterations in hatching time.
Conclusions:
Results to date:
Tomcod embryos were exposed to metal and carbon-containing nanoparticles at 17 days post fertilization (dpf). The carbon nanoparticles included fullerene (nC60), carbon black, and two derivatized single wall nanotubes with either polyethylene glycol or poly-m aminobenzenesulfonic acid surface modifications. Doses of fullerenes ranged from 0.8 to 500 µg/L while doses of the other carbon-containing particles ranged from 0.08 to 50 µg/mL. Interestingly, the nanoparticles containing carbon did not result in changes in mortality, hatching success, or time to hatch in tomcod embryos.
Tomcod embryos were also exposed to metal nanoparticles, with copper nanoparticles proving to be the most toxic with 83 percent mortality at 0.4 µg/mL and 100 percent at higher doses. This finding was not unexpected considering copper is known to be toxic to marine organisms. Both iron and zinc were 100 percent lethal at 10 µg/mL, whereas silver and nickel mortalities did not differ from controls at all doses. In silver-exposed embryos that survived to hatch, a dose-dependent increase in the occurrence of pericardial edema was observed in hatched larvae. Similarly, there was an increase in pericardial and yolk-sac edemas in embryos exposed to zinc. Sublethal effects such as these are common and non-specific in embryos exposed to contaminants and indicate the growing larvae will probably not survive.
To expand our findings to another fish species, a select group of nanoparticles (fullerene, Ag, Cu, Fe and Ni) were chosen for exposures using mummichug embryos at the same doses used for tomcod. Mummichug have a shorter embryonic developmental period and so were exposed at 5 dpf, which corresponds developmentally to 17 dpf in tomcod. Mummichug embryos proved to be less sensitive than tomcod. The only nanoparticle with an observed effect on mortality was Cu, which resulted in a 70 percent increase in mortality at the 2 µg/mL dose and 100 percent at 10 µg/mL. These results indicate that nanoparticles can vary in their toxic effects in different species of embryonic fish. Naive early life-stages of tomcod have proven to be sensitive monitors of chemical contamination for a variety of toxicological endpoints while mummichug, a frequently utilized species in toxicology studies, was found not to be as susceptible.
The raw combustion soot material and sludge byproduct obtained during the processing and purification of carbon fullerenes with erbium and yttrium catalysts were also tested and all resulted in dose-dependent toxicity in tomcod embryos. Among the erbium-containing particles, soot proved to be the most toxic with 95 percent mortality at 2 µg/mL, yet the yttrium mix proved to be the most toxic amongst the yttrium group with 92 percent mortality at 10 µg/mL. The sludge byproduct, which as a waste product might be expected to have the greatest risk for environmental exposure, was similar for both metals with over 50 percent mortality at 10 µg/mL and 100 percent and 95 percent for erbium and yttrium, respectively at 50 µg/mL.
Aquatic exposure to nanoparticles in natural aquatic ecosystems occurs under complex conditions and a variety of chemical and physical factors influence the solubility, chemical surface interactions (i.e., charge) and bioavailability of nanoparticles in aqueous environments. Thus, the test media used to assess the toxicity of nanoparticles can be extremely important. In order to determine if the effects of nanoparticle exposure to tomcod embryos differ under various water conditions, exposures were performed with a subset of nanoparticles previously tested: fullerene, silver, copper, and yttrium sludge. These four different particles were each suspended in four different types of water: artificial seawater at 5 parts per thousand (ppt) salinity prepared with laboratory distilled water, artificial seawater at 5 ppt with 5 mg/L Suwanee River Natural Organic Matter (SR-NOM), natural water collected at a brackish lake in Westchester, NY and diluted to 10 ppt with laboratory distilled water, and natural water collected from the ocean at Sandy Hook, NJ diluted to 15 ppt. No differences in mortality or hatching success were observed with tomcod embryo exposure to fullerenes dispersed in any water type. Tomcod embryos exposed to silver nanoparticles dispersed in both natural seawater and brackish lake water, however, exhibited 60 percent and 40 percent increases in mortality, respectively, with 50 µg/mL Ag compared to only 10 percent mortality observed in artificial sea water, with or without SR-NOM. Copper nanoparticle exposure of tomcod embryos resulted in increases in mortality, compared to artificial seawater, when Cu was suspended in natural seawater, natural brackish water and SR-NOM amended waters with the largest increase in mortality occurring in natural seawater. Yttrium-containing sludge particles were most toxic to embryos when suspended in natural seawater with 50 percent mortality occurring at 2 µg/mL, and significant differences in mortality were also observed at 10 µg/mL in all natural waters compared to artificial 5 ppt water. These results indicate that the extent of nanoparticle toxicity at early life stages of fish is highly dependent upon the characteristics of the waters in which the nanoparticles are suspended for the bioassay.
It has been suggested that the toxicity observed with metal nanoparticle exposures might be the result of metal ions that dissolve from the nanoparticles and that under different conditions of salinity and natural organic matter, solubility differences might be responsible for the different observed toxicities. To test this hypothesis, Ag and Cu nanoparticles were suspended in 5 ppt, 15 ppt or natural seawater and allowed to dissolve for 24 hours. Ions were then separated from nanoparticles by high speed ultracentrifugation, followed by filtration through a 0.02-µm filter. The filtrate was then used as a stock solution. Tomcod embryos were exposed to this filtrate or nanoparticle suspensions prepared as previously described. Copper NP exposure resulted in dose dependent increases in mortality with 100% mortality occurring at 2 µg/mL with all three water types. However, mortality and hatching success in embryos exposed to soluble Cu ions were not different than controls, indicating that the toxicity of Cu NP is not due to the dissolution of Cu ions. Silver NP suspensions prepared in 15 ppt and natural SW resulted in significant increases in mortality (75% and 72%) at the highest dose of 50 µg/ml compared to those prepared in 5 ppt water (15%). However, there were no increases in mortality with any water type when embryos were exposed to soluble Ag ions only. It was later determined with graphite-furnace atomic absorption spectroscopy that the concentration of dissolved Ag was similar in all three waters: 79.4, 85.2 and 79.6 µg/mL in 5, 15 and natural SW which was less than 0.05 percent of the total mass of Ag nanoparticles added. Dissolved Cu concentrations were 502, 429 and 178 µg/mL in 5 ppt, 15 ppt and natural SW, accounting for less than 0.25 percent of total Cu mass in all three water types. Dissolution of metal ions from nanoparticles did not significantly differ between water types and, overall, constituted a minor portion of the mass of Ag and Cu added which seems to indicate that the toxic effects observed are due to the metal nanoparticles and not dissolved ions.
Journal Articles:
No journal articles submitted with this report: View all 6 publications for this projectSupplemental Keywords:
Nanoparticle toxicity, killifish, early life stage toxicity, Health, Scientific Discipline, Health Risk Assessment, Risk Assessments, Biochemistry, bioavailability, nanomaterials, carcinogenic, genetic analysis, human exposure, biological pathways, nanoparticle toxicity, nanotechnology, human health risk, toxicologic assessmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.