Soybean susceptibility to manufacturednanomaterials with evidence for foodquality and soil fertility interruptionJohn H. Priestera,b,c, Yuan Gea,b,c, Randall E. Mielkea,b,c,d, Allison M. Horsta,b,c, Shelly Cole Moritzb,Katherine Espinosae, Jeff Gelbf, Sharon L. Walkerg, Roger M. Nisbetb,c,h, Youn-Joo Ani, Joshua P. Schimelb,c,h,Reid G. Palmere,j, Jose A. Hernandez-Viezcasc,k, Lijuan Zhaoc,k, Jorge L. Gardea-Torresdeyc,k, and Patricia A. Holdena,b,c,1aBren School of Environmental Science and Management, bEarth Research Institute, and cUniversity of California Center for the Environmental Implications ofNanotechnology, University of California, Santa Barbara, CA 93106; dDivision of Geological and Planetary Sciences, NASA/Jet Propulsion Laboratory, CaliforniaInstitute of Technology, Pasadena, CA 91101; eDepartment of Agronomy, Iowa State University, Ames, IA 50011; fXradia, Pleasanton, CA 94588; gDepartmentof Chemical and Environmental Engineering, University of California, Riverside, CA 92521; hDepartment of Ecology, Evolution and Marine Biology, Universityof California, Santa Barbara, CA 93106; iDepartment of Environmental Science, Konkuk University, Seoul 143–701, Korea; jCorn Insects and Crop GeneticsResearch Unit, Agricultural Research Service, US Department of Agriculture, Ames, IA 50011; and kDepartment of Chemistry, University of Texas at El Paso,El Paso, TX 79968

AUTHOR SUMMARY

Manufactured nanomaterials (MNMs),manmade materials with at least onedimension less than 100 nm, are in-creasingly used in consumer goods (e.g.,sunscreens, fuels, and paints), and thusare entering the atmosphere and soil.Some MNMs stress the cells of plants,causing growth inhibition, MNM uptake,DNA damage, and death. For instance,hydroponically grown plants (i.e., plantsgrown in mineral nutrient solutions) aredamaged when they accumulate someMNMs. Also, some MNM-exposed soilmicrobial communities become lessdiverse. Therefore, soil-grown food cropscould be impacted by MNMs, althoughthere is a lack of direct evidence. Westudied soybean growth from plantingthrough seed production in farm soil towhich MNMs were added. For two high-production MNMs, we observed the bio-accumulation of MNM metals and thecessation of symbiotic nitrogen fixation.Soybean is an important crop world-

wide, with nearly all tissues and extractedoil used for food, feed, and commerce.Soybean is a plant that is able to increaseavailable nitrogen by fixing nitrogen gasfrom the atmosphere via root nodulesymbioses (i.e., interactions) with nitro-gen-fixing bacteria. This characteristic ofleguminous crops is important to soilfertility because it reduces the use of en-ergy-intensive, and therefore polluting,synthetic fertilizers. Soybean plants werecultivated in greenhouses with three con-centrations of nano-ZnO (i.e., zinc oxide)or nano-CeO2 (i.e., cerium oxide) addedto organic farm soil. These metal oxideMNMs, each with a primary particle sizeof ∼10 nm, are in high productionworldwide, and thus are probably accumulating in soils via land-applied wastewater biosolids, which are the final residues fromwastewater treatment. Previous research has indicated that thesemetal oxides may affect hydroponic soybean (1), and that nano-ZnO alters soil microbial community composition and biomass(2). Several plants grown hydroponically are affected by myriad

MNMs, raising concerns regarding thelong-term effects of these materials onthe food supply (3). However, MNMsmay not be bioavailable (i.e., accessibleto organisms) in soil (4), and hydroponicstudies do not provide information onbioavailability constraints or the effectson symbiotic nitrogen fixation. There-fore, we evaluated plant growth respon-ses, the effects on symbioses related tosoil fertility, and the propensity for MNMbioaccumulation, i.e., the uptake ofMNMs from the surrounding soil intoplant tissues. We used EM and X-raymicroscopy to visualize MNM-associatedmetal accumulations in plant tissue,and energy dispersive spectroscopy toidentify elements in electron-densemicrograph features.Our results prove that these MNMs

are indeed bioavailable to plants, andhave the potential to dramatically affectfood crops. Although aboveground bio-mass yield was not affected by nano-ZnO, plants grown with high nano-ZnOproduced more belowground biomass(i.e., root and nodule). Most strikingly,Zn bioaccumulated throughout theplants, with the amount increasingaccording to nano-ZnO dose. Althoughthe amount of Zn in soybean pods was

Author contributions: J.H.P., Y.G., S.C.M., S.L.W., R.M.N.,Y.-J.A., J.P.S., J.L.G.-T., and P.A.H. designed research; J.H.P.,Y.G., R.E.M., A.M.H., J.G., J.A.H.-V., L.Z., J.L.G.-T., andP.A.H. performed research; R.E.M., J.G., J.L.G.-T., andP.A.H. contributed new reagents/analytic tools; J.H.P.,A.M.H., J.A.H.-V., L.Z., J.L.G.-T., and P.A.H. analyzeddata; and J.H.P., Y.G., R.E.M., A.M.H., S.C.M., K.E., J.G.,S.L.W., R.M.N., Y.-J.A., J.P.S., R.G.P., J.A.H.-V., L.Z., J.L.G.-T.,and P.A.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: holden@bren.ucsb.edu.

See full research article on page E2451 of www.pnas.org.

Cite this Author Summary as: PNAS 10.1073/pnas.1205431109.

Fig. P1. Outcomes of growing soybean in farmsoil amended with nano-ZnO (Right) or nano-CeO2

(Left). With nano-ZnO, plants bioaccumulated Znin the belowground and aboveground parts ofthe plant, with the highest concentrations in theleaves (indicated by the darkest shading). Withnano-CeO2, MNMs did not translocate aboveground, but rather bioaccumulated in the rootsand root nodules, where, in the latter, symbioticnitrogen fixation was decimated (not depicted).

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high, the highest concentrations were in the leaves. In com-parison, plants grown with low nano-CeO2 had reduced leafcover, were shorter, and grew more slowly, whereas plantsgrown with high nano-CeO2 yielded less soybean biomass. Al-though Ce was not translocated into aboveground plant tissue,it did bioaccumulate in the roots and root nodules. Then, al-though potential N2 fixation rates were not affected by nano-ZnO, the potential rates were near zero in medium and highnano-CeO2 treatments. The root nodules of plants grown withhigh nano-CeO2 appeared to be devoid of nitrogen-fixing bac-teria. However, these plants grew faster and larger than plantswith low nano-CeO2, implying that, with the loss of atmosphericnitrogen fixation capacity, plants were able to successfullycompensate by using the soil as an alternative nitrogen source.One implication of this shift is that, under nitrogen-limited fieldconditions, the application rate of synthetic fertilizer wouldneed to be increased. The broad implication is that food quality,as related to metal content, is significantly altered by nano-ZnOin soils. Furthermore, nitrogen fixation is impaired in plantsgrown with nano-CeO2. In this study, we demonstrated thebioaccumulation of MNMs in a soil-grown food crop andhighlight the potential risks to soil fertility and the harvestablefood supply.

This study did not evaluate plant responses to the metals inplant tissues, the speciation of MNM metals in plant tissues,or the definitive source of nitrogen in plant tissue. Nevertheless,the implications of this study are profound: we provide evi-dence that two commonplace metal oxide MNMs can changesoybean growth, thereby suggesting the eventual necessity forshifts in current agricultural practices. Our results reinforcethe importance of the idea of making MNMs sparingly bio-available by design and thus more environmentally compatible(i.e., likely to cause minimal impact on the environment, andhence agriculture) (5), and of managing waste streams toprevent the crop-damaging soil buildup of toxic MNMs.

1. López-Moreno ML, et al. (2010) Evidence of the differential biotransformation andgenotoxicity of ZnO and CeO2 nanoparticles on soybean (Glycine max) plants. EnvironSci Technol 44:7315–7320.

2. Ge Y, Schimel JP, Holden PA (2011) Evidence for negative effects of TiO2 and ZnOnanoparticles on soil bacterial communities. Environ Sci Technol 45:1659–1664.

3. Rico CM, Majumdar S, Duarte-Gardea M, Peralta-Videa JR, Gardea-Torresdey JL (2011)Interaction of nanoparticles with edible plants and their possible implications in thefood chain. J Agric Food Chem 59:3485–3498.

4. Tong Z, Bischoff M, Nies L, Applegate B, Turco RF (2007) Impact of fullerene (C60) ona soil microbial community. Environ Sci Technol 41:2985–2991.

5. Xia TA, et al. (2011) Decreased dissolution of ZnO by iron doping yields nanoparticleswith reduced toxicity in the rodent lung and zebrafish embryos. ACS Nano 5:1223–1235.

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