The Ecological Effects of Nanomaterials: Are New Stressors Associated with New Technologies?

L.Ziccardi, M. McArdle, Y. Lowney, J. Tsuji

The U.S. Environmental Protection Agency (EPA) defines nanotechnology as “research and technology development at the atomic, molecular, or macromolecular levels using a length scale of approximately one to one hundred nanometers in any dimension.” Nanomaterials include naturally-occurring particles, those that are produced from combustion byproducts, and engineered or manufactured nanomaterials. Nanoparticles can be released to the environment from deliberate application (e.g., remedial applications), and from unintentional or incidental releases, where they could come into contact with fish, wildlife, and plants. These organisms, termed “ecological receptors,” can potentially be exposed to nanoparticles through inhalation, ingestion, movement across gills, passive transport, and cellular absorption.

The unique physicochemical properties of nanomaterials that make them beneficial in commercial applications might also result in unexpected biological interactions. For example, their large surface area relative to mass may translate to enhanced chemical binding capacity and reactivity. Another consideration in aquatic environments is that smaller particles will remain in suspension longer, which may affect their environmental transport, bioavailability, and toxicity. On the other hand, nanoparticles’ high surface area and associated intermolecular forces may increase agglomeration and adherence to suspended matter or sediments, potentially reducing bioaccessibility.

nanotechBecause of the unique properties of nanomaterials, concerns have been expressed that their effects on aquatic and terrestrial organisms and ecosystems may be different from normal or fine-scale materials, although actual effects are likely complex and difficult to predict. Aquatic organisms and terrestrial plants have been the focus of concern for environmental effects.

nanotech2Studies specific to the aquatic and terrestrial effects of nanomaterials in environmentally relevant species have been few in comparison to mammalian studies targeted primarily at understanding potential effects to humans. The ecotoxicological studies that are available focus on metal oxide particles, carbon nanotubes, and fullerenes, primarily in aquatic and plant toxicity tests. These initial studies have also generally used high concentrations to maximize exposure and ensure that effects are observed. Aquatic tests have examined the uptake of nanoparticles by fish, filter feeders (invertebrates), and algae, and have provided evidence of toxicity or behavioral changes associated with exposure. Studies on terrestrial species are limited to experiments with plants (e.g., root elongation, germination), primarily to investigate effects on crop species and the use of nanomaterials in fertilizers. Some of these studies conclude that nanoparticles can be taken up by or produce effects in biota, and that dose-response relationships and patterns of relative toxicity among types of particles are emerging.

Toxicity testing of nanomaterials is not yet standardized and certain challenges need to be addressed. For example, preparation methods of nanoparticle test suspensions may influence particle behavior in solution and toxicity, thereby confounding conclusions with regard to the toxicity of the nanoparticles themselves. Nearly all studies of nanoparticles attempt to counteract the natural tendency of the particles to stick to the sides of the test vessel or agglomerate and form larger particles in solution by using techniques such as sonication, agitation, filtration, or addition of agents such as the solvent tetrahydrofuran (THF). For example, some research indicates that toxicity observed in aquatic exposure tests with C60 may in part be attributed to THF itself or its more toxic breakdown product (g-butyrolactone) rather than purely to the effects of C60. Studies that removed much of the THF by extraction before exposures and have controls to check on the influence of adding THF would more accurately indicate the toxicity of the test particles.

Toxicity studies may not be representative of the real world. Studies that artificially produce nanoparticles in solution, or in vitro studies involving tissue cultures and isolated cells, must also be interpreted with caution. Such tests may indicate the potential of nanoparticles to cause toxicity, but in the actual environment or within organisms, agglomeration would occur, thereby potentially reducing their transport, migration, and toxicity. Studies indicate that higher concentrations of nanoparticles in solution are associated with greater agglomeration. Toxicity and mobility of nanoparticles in the environment may thus, in part, be self-limiting.

For regulation of products containing substances such as nanoscale metallic compounds, the central question for ecotoxicity is whether the substance is more toxic in nanoscale form than in the dissolved form used in standardized tests and for which toxicity-based limits are available. Studies for certain metal oxides, zinc oxide for example, have indicated that the nanoscale form is no more toxic to aquatic organisms or in vitro than the same concentration of soluble zinc, although one study suggested that nanoscale iron oxide particles were more toxic in vitro than soluble iron.

Emerging findings in aquatic toxicity studies using invertebrates, fish, and algae indicate greater toxicity associated with more reactive nanoscale substances such as engineered fullerenes, relative to more inert substances such as titanium dioxide particles. Greater reactivity in nanoscale form has also been investigated for anti-bacterial applications of metal oxides such as silver, zinc oxide, and photo-catalytic forms of titanium dioxide. Such antibacterial effects, however, may have ecological implications. Therefore, the evaluation of regulatory limits for such materials will need to consider whether effects from exposure to the nanoscale forms are any greater than effects from these metals in ionic form or solution.

Nanoparticle ecotoxicity can vary with particle type, size, and attached functional groups. Nanoscale application appears to increase the efficacy of some chemical formulations, such as micronutrients or fertilizers, and may therefore increase potential reactivity and toxicity. The relationship between toxicity and particle size, however, is complicated. Greater reactivity may also be beneficial; this is the case for the effect of nanoscale titanium dioxide on spinach seed germination and growth by affecting enzymes involved in nitrogen metabolism. In another example, one study on daphnids, a freshwater filter feeder, reported a hormetic effect (i.e., beneficial effect at low exposures and an adverse effect at higher exposures) with exposure to single-walled carbon nanotubes; and similar patterns have been observed in plants. In addition, while several studies indicate that particle size can influence biological effects, others suggest that toxicity is more related to changes in nanoparticle surface characteristics, and that smaller size does not always result in greater toxicity. Size is likely only one of many characteristics that influences the environmental fate and toxicity of nanoparticles. In addition to surface characteristics, other factors include chemistry and crystal type, shape, and electromagnetic properties.

daphniaSome of the properties of nanomaterials that make them useful in biotechnology and remedial applications may also result in negative biological effects at high doses. For example, fullerenes bind to lipids, which makes them useful as drug carriers and for other therapeutic applications when functionalized; however, this same binding characteristic and reactivity may have been the cause of adverse effects such as the lipid peroxidation observed in the brains of fish exposed to high concentrations. Therefore, as the use of nanotechnology increases, it is important to improve our understanding of the potential effects of these materials on biota, as well as increase our knowledge to be able to better design future tests on these materials.

While more research is clearly needed to understand the potential for impacts on ecological receptors and systems, further guidance would be useful regarding appropriate study design to ensure that meaningful conclusions can be drawn from the investment in future research. Several important considerations have emerged in our review of the available literature on ecological effects. Each unique nanomaterial (e.g., fullerenes, metal oxides, etc.) and derivatives of these materials may cause unique effects because of differences in particle size, shape, surface area, charge, solubility, and reactivity. Thus, the toxicity of each nanomaterial needs to be considered independently. Studies should also be designed to allow for an understanding of whether the nano-characteristics of the material are controlling toxicity, or whether toxicity is associated with the chemistry of the material being evaluated and is unrelated to particle size. Nanomaterials toxicity assessment should consider potential effects resulting from the test-material preparation method (e.g., solvent vehicles or sonication used to maintain aquatic dispersion), the presence of trace contaminants in commercial nano-products, and the potential influence of nanoparticle agglomeration. The ability of nanoparticles to increase the transport of other chemicals via adsorption, and potential influence on environmental fate, absorption, bioaccumulation, and biological effects, should also be considered.

The existing body of research focuses on evaluation of relatively few types of nanoparticles, although the particles are generally those with the most current interest and application in consumer products (metals and carbonaceous materials; www.nanotechproject.org/inv entories/ consumer/analysis/). Future research could benefit from early identification of nanoformulations, so that research can target materials of industrial, and potential environmental, importance. Several sources of funding to expand these investigations are beginning to emerge. For example, EPA is funding current research under their grant program, Science to Achieve Results (STAR). STAR grants have been awarded to study the environmental and human health effects of engineered nanomaterials, and nanomaterials for use in environmental remediation. For more information on this program and this research please go to es.epa.gov/ncer/nano/research/starawards.html.

Given the lack of standard methodology for quantifying nanoparticle exposure and limitations in knowledge on toxicity, a key focus for manufacturers should be on engineering processes and consumer products that encapsulate or limit liberation of free nanomaterials. Such materials must also be durable and maintain their encapsulation even during wear or weathering of the product. Design of such products or evaluations of their wear behavior is within current knowledge through the wellestablished discipline of materials science. In this way, Exponent toxicologists have teamed with our material scientists to evaluate the potential risks of products containing nanomaterials.

Exponent scientists have presented the results of their review of the ecological effects of nanomaterials at the International Symposium on Nanotechnology in Environmental Protection and Pollution (ISNEPP 2007), and will also be presenting an update of their review at the NanoEco conference in Monte Verità, Switzerland, March 2–7, 2008. For more information on these conferences please visit www.isnepp.org and www.empa.ch/nanoECO. For more information on the subject of nanomaterials, please contact Joyce Tsuji (tsujij@exponent.com), or Linda Ziccardi (lziccardi@exponent.com).