Cerium Biomagnification in a Terrestrial Food Chain: Influence of

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Cerium biomagnification in a terrestrial food chain: Influence of particle size and growth stage Sanghamitra Majumdar, Jesica Trujillo-Reyes, Jose A. Hernandez-Viezcas, Jason C. White, Jose R Peralta-Videa, and Jorge L Gardea-Torresdey Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04784 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 27, 2015

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Environmental Science & Technology

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Cerium biomagnification in a terrestrial food chain: Influence of particle size and growth

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stage

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Sanghamitra Majumdar†§, Jesica Trujillo-Reyes†, Jose A. Hernandez-Viezcas†, Jason C. Whiteɸ,

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Jose R. Peralta-Videa†§‡, Jorge L. Gardea-Torresdey*†§‡

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Department of Chemistry, The University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, USA

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‡

Environmental Science and Engineering PhD Program, The University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, USA

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§

University of California Center for Environmental Implications of Nanotechnology (UC CEIN), El Paso, Texas, USA

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ɸ

Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, Connecticut 06504, USA

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*Corresponding author: [email protected], Phone: 915-747-5359; Fax: 915-747-5748

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TOC Art

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Abstract

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Mass-flow modelling of engineered nanomaterials (ENMs) indicates that a major fraction of

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released particles partition into soils and sediments. This has aggravated the risk of

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contaminating agricultural fields, potentially threatening associated food webs. To assess

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possible ENM trophic transfer, cerium accumulation from cerium oxide nanoparticles (nano-

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CeO2) and their bulk equivalent (bulk-CeO2) was investigated in producers and consumers from

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a terrestrial food chain. Kidney bean plants (Phaseolus vulgaris var. red hawk) grown in soil

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contaminated with 1000 - 2000 mg/kg nano-CeO2 or 1000 mg/kg bulk-CeO2 were presented to

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Mexican bean beetles (Epilachna varivestis), which were then consumed by spined soldier bugs

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(Podisus maculiventris). Cerium accumulation in plant and insects was independent of particle

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size. After 36 days of exposure to 1000 mg/kg nano- and bulk-CeO2, roots accumulated 26 and

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19 µg/g Ce, respectively, and translocated 1.02 and 1.3 µg/g Ce, respectively to shoots. The

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beetle larvae feeding on nano-CeO2 exposed leaves accumulated low levels of Ce, since ~98% of

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Ce was excreted, in contrast to bulk-CeO2. However, in nano-CeO2 exposed adults, Ce in tissues

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was higher than Ce excreted. Additionally, Ce content in tissues was biomagnified by a factor of

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5.3 from the plants to adult beetles and further to bugs.

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INTRODUCTION The revolutionary development and implementation of nanotechnology in industrial

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sectors such as energy, electronics, environmental remediation, automotive, medicine, personal

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care, health and fitness, and agriculture has increased dramatically in the last two decades.1,2 The

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market value of engineered nanomaterials (ENMs) around the world is estimated to reach $3

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trillion by 2020.2 Exclusively, the production of metal oxide nanoparticles is estimated to exceed

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1.6 million tons by 2020, compared to 0.27 million tons in 2012.3 Owing to the remarkable array

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of diverse applications, the environmental build-up of ENMs or their transformed products is

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inevitable via their unregulated release from ENM producing or dependent sectors into the

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wastewaters and landfills,4,5 or due to their direct application for environmental remediation

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purposes.6 Also, ENMs are being designed for various applications in agriculture, including pest

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control, early disease detection, higher productivity and growth enhancement.7,8 However, this

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fast paced development has also led to concerns over the environmental risks and subsequent

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safety of ENMs to humans as a function of direct and/or indirect exposure.4,9 In order to gain

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conclusive evidence on the fate and long-term impacts of ENMs varying in physicochemical

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properties, standardized procedures and risk assessment models using a wide range of test

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species and media are currently being considered.10-12

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Although the number of exposure studies in a range of biota has increased rapidly as part

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of an effort to ensure sustainable development of ENMs, information on particle transfer along

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terrestrial trophic levels is very limited.13 There has been some progress in assessing ENM

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trophic transfer in aquatic systems,14-21 but comprehensive evaluation in terrestrial systems is

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limited to a small number of studies.13 Judy et al. provided evidence of gold nanoparticle (nano-

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Au) bioaccumulation in tobacco (Nicotiana tabacum)22 and tomato (Solanum lycopersicum)23, 4 ACS Paragon Plus Environment

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with subsequent transfer to tobacco hornworms (Manduca sexta).22 Although nano-Au was

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biomagnified in this soil-plant-insect system, a separate study showed that the nano-Au levels

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were reduced 100-folds upon transfer from earthworm (Eisenia fetida) to bull frogs (Rana

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catesbeina) in contaminated soil.24 The authors also reported that nano-Au were more

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bioavailable through dietary transfer than direct gavage.24 Hawthorne et al. showed that crickets

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feeding on zucchini (Cucurbita pepo) plants exposed to cerium oxide nanoparticles (nano-CeO2)

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accumulated twice the Ce than did individuals exposed to bulk-cerium oxide (bulk-CeO2).25

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Alternatively, De La Torre-Roche et al. recently reported that the accumulation and trophic

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transfer of La2O3 did not vary with particle size in a food chain consisting of lettuce (Lactuca

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sativa), crickets and mantids. 26 Notably, in both studies, the elemental concentration declined

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significantly at each subsequent trophic level, indicating that although trophic transfer was

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evident, biomagnification was not.25,26 Although these findings clearly suggest potential transport

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of ENMs within terrestrial food chains, the literature is quite thin and the effects on full life

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cycle, or even multigenerational exposures, are yet to be evaluated.

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Nano-CeO2 is one of the widely produced metal oxide nanoparticles,4,27 and has been

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reported to accumulate and translocate to aerial and edible tissues of agricultural crops, with

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minimal toxicity28-31 and little biotransformation.32-34 Nano-CeO2 is also used in fuel catalysis,

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energy, planarization, and biomedical applications because of its low-redox potential, radical

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scavenging activity, high ionic conductivity, and enhanced UV absorbing properties.35 Due to the

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increasing scope of applications and multiple routes of environmental release,35 in 2008 the

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Organization for Economic Cooperation and Development (OECD) listed nano-CeO2 as a

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priority ENMs in need of immediate testing and risk assessment.36 The primary objective of the

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current study was to investigate the possible transfer of nanoparticle and bulk forms of CeO2



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across three trophic levels in a terrestrial mesocosm. The food chain consisted of kidney bean

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plants as producers, Mexican bean beetles as herbivores, and predatory spined soldier bugs as

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carnivores.

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EXPERIMENTAL

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Particle characteristics and soil preparation. Commercially produced nano-CeO2 (Meliorum

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Technologies, Rochester, NY) were procured from The University of California Center for

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Environmental Implications of Nanotechnology (UC-CEIN). The nano-CeO2 were reported as

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rod-shaped 100% cubic ceria, 95.14% pure, measuring (67 ± 8) nm × (8 ± 1) nm, (≤10%

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polyhedra: 8 ± 1 nm), with a surface area of 93.8 m2g-1.37 The bulk equivalent cerium oxide

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particles (bulk-CeO2; 99.95% purity) were purchased from Sigma-Aldrich.

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The soil used in this study was prepared by amending a sandy loam soil from Texas

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A&M Agrilife Research Centre, El Paso agricultural field (64% sand, 31% silt, 5% clay; pH 7.3

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[water]; cation exchange capacity 33.1 meq/100 g) with Miracle Gro potting mix at a ratio of 2:1

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(10.1 % organic matter). Due to natural occurrence of cerium as a rare earth element,35 the

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untreated soil in this study contained 33.8 mg/kg Ce. For the exposure assays, two concentrations

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of nano-CeO2 (1000 and 2000 mg/kg) were selected. The concentrations were comparable with

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existing trophic transfer and plant studies.25,26,31,38 In addition, studies have reported background

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levels of 800-900 mg/kg Ce in soils around rare earth element-based industries.39 In toxicology

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studies with potential emerging contaminants, it is preferable to begin with higher exposure

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concentrations for efficient detection of elements of interest, extrapolating the observed effects to

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lower concentrations. To investigate particle-size dependent behavior on CeO2 transfer in the

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food chain, the lowest dose (1000 mg/kg) of nano-CeO2 was selected for comparison with bulk-

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CeO2. To achieve the 1000 and 2000 mg/kg of nano-CeO2 in soil, requisite amounts of nano6 ACS Paragon Plus Environment

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CeO2 were suspended in 100 ml millipore water (MPW) by bath sonication (Crest Ultrasonics,

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Trenton, NJ, USA) at 180 watts for 30 min at 25°C. For characterization purposes, the

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suspensions prepared for 1000 mg/kg nano-CeO2 were used to measure size and zeta potential

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using NanoSizer 90 (Malvern Instruments, Worcestershire, UK) in quadruplicates, each with

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three consecutive readings. The average hydrodynamic diameter and the zeta potential of the

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particles in the unfiltered nano-CeO2 suspension were reported to be 45.4 ± 2.5 nm

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(polydispersity index (PDI) ≤ 0.3) and 11.6 ± 2.5 mV, respectively. The bulk-CeO2 solution was

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prepared in MPW by stirring for 30 min to avoid nanoparticle formation in the solution to obtain

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a final concentration of 1000 mg/kg in soil. The suspensions were slowly added to 800 g of air-

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dried soil and mixed manually in order to achieve homogeneity and desired concentrations. The

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soil wetted with MPW was regarded as the untreated control. Since the CeO2 exposures in the

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field soil were significantly high compared to the untreated control, the residual Ce content in the

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soil was considered inconsequential as a factor.

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Selection of test species and respective exposure scenario

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Plant species. Kidney bean (Phaseolus vulgaris var. red hawk) (KBP) seeds were

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obtained from Michigan State University, and were selected as the producers in this study. They

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are widely consumed legumes with high protein, folate and iron contents.40 They are also fast

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growing plants and produce sufficient aerial biomass to feed the primary consumers. The seeds

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were rinsed thrice with NaOCl and MPW, and soaked in MPW overnight before planting in soil.

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After 24 h of amending the soils with nano- and bulk-CeO2 suspensions, 4 KBP seeds were

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equidistantly placed at a depth of 2.5 cm in each pot. All the treatments were conducted in

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quadruplicates. Four separate sets of plantations were prepared to sustain the food supply for the

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different stages of primary consumers. The pots were placed in a growth chamber



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(Environmental Growth Chamber, Chagrin Falls, OH) with 14 h photoperiod (340 µmole m-2s-1),

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25/20°C day/night temperature and 65-70% relative humidity. The plants were watered daily

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with 80-100 ml MPW. A separate experiment with the same exposure regime was established to

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measure Ce accumulation in root, stem and leaf biomass after 22, 29 and 36 days of growth.

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These days are relevant to the feeding intervals at different developmental stages of the primary

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consumers, as discussed below.

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Primary consumer species. Mexican bean beetles (Epilachna varivestis) (MBB) were

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selected as the primary consumers, because they are voracious herbivores that are a common pest

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for KBPs. They feed on KBP stems and leaves, at their larval and adult stages.41 Batches of

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MBB eggs and second instar larvae were obtained from the New Jersey Department of

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Agriculture. The MBB larvae and adult cultures were enclosed in rearing cages in the growth

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chamber, and fed continuously on live uncontaminated KBPs. MBB larvae from the second filial

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and subsequent generations were used for the trophic transfer study.

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The MBB larvae were starved for 24 h prior to the onset of the experiment both to ensure

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feeding and to void contents from the gut. After 22 days of KBP growth, the leaves and stems

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were manually infested with 30 second-instar MBB larvae. The MBB-infested plants were

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enclosed in rearing and observation insect cages measuring 14 x 14 x 24" (Bioquip Products,

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CA). The replicates in each cage were separated with cardboard sheets. The experimental design

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is shown in Figure 1. The soil was covered along the rim of the pot with a net to avoid

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contamination by direct contact of MBBs with nano-CeO2 or bulk-CeO2 amended soil. The

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second-instar larvae developed into pupae in 7-8 days, and the pupae metamorphosed into the

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adult form in about 5-7 days. This interval of approximately 7 days between the MBB growth

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stages justifies the harvesting of KBP tissues on the 22nd, 29th, and 36th day of plant exposure. 8 ACS Paragon Plus Environment

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These correspond to the days at which the MBB (larvae, pupae, adult) start feeding (day 0,

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larvae) or metamorphose (day 7, pupae) and continue feeding (day 14, adults) on the KBPs.

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Seven MBB larvae, 5 MBB pupae and 5 MBB adults were sampled per replicate at 7 day

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intervals on 29th, 36th, and 43rd day of KBP exposure, respectively. The insects were starved for

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48 h to void the gut contents and euthanized in liquid nitrogen. Feces were collected from the

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larvae and adults during the depuration period for further analysis. Feces from all the replicates

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were composited into one sample to have sufficient biomass for elemental analyses.

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Secondary consumer species. Spined soldier bugs (Podisus maculiventris) (SSB) were

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chosen as the secondary consumer in this food chain, because they prey on MBB larvae by using

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their proboscis to predigest the prey and consume their contents, leaving the carcass behind.

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Eggs of SSB were purchased from GreenMethods.com and were maintained in small cubical

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cages until adulthood. The SSBs cultures were supplied with untreated MBB larvae and KBPs as

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food after the appearance of the second instar nymphs. For the exposure assay, the adult SSBs

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were starved for 24 h to void gut contents and to ensure feeding on the control or treated MBBs.

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Three adult SSBs were kept in enclosure with ten MBB third-instar larvae, which had been

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feeding on plant shoots exposed to control, nano-CeO2, and bulk-CeO2 for preceding 10-15 days.

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Once the SSBs had completed feeding, the MBB remains and the SSBs were collected for

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elemental analysis.

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Analyte quantification in KBP, MBB and SSB tissues

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After 22, 29 and 36 days of nano-CeO2 or bulk-CeO2 exposure, the plants grown

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separately for determining Ce accumulation were divided by tissue (roots, stems, and leaves).

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The KBP tissues, MBB tissues and feces, SSB tissues and MBB remains (after being fed to the



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SSB) were oven-dried at 70 °C for 96 h. Percent moisture content in the different KBP tissues

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were calculated. The dried tissues were digested using plasma pure HNO3 and 30% (w/v) H2O2

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(1:4) mixture in a microwave accelerated reaction system (CEM Marsx, Mathews, NC).42 Cerium

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content in the KBP tissues was determined by inductively coupled plasma-optical emission

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spectroscopy (ICP-OES) (Optima 4300 DV, Perkin Elmer), and in the MBB and SSB samples by

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inductively coupled plasma-mass spectrometry (ICP-MS) (Perkin Elmer ELAN DRC II, Shelton,

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CT). Cerium recovery from a National Institute of Standards and Technology (NIST) certified

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standard reference material (peach leaves NIST-SRM 1547, Gaithersburg, MD) was 98%,

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validating the digestion process. Blanks were used to determine the detection limit of Ce in ICP-

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OES (1 µg/L) and ICP-MS (1e-4 µg/L). All samples were above the detection limits. For quality

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assurance/quality control, 0.5 mg/L and 10 µg/L Ce standards were analyzed every 20 samples

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by ICP-OES and ICP-MS, respectively to monitor matrix effects on the analytes. Ce contents in

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the KBP, MBB and SSB tissues were expressed as µg Ce/g tissue dry weight. Absolute values of

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Ce contents in different KBP tissues were also calculated to shed light on the distribution of Ce

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in the entire plant when exposed to a certain concentration.

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Statistical Analysis

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All the analyses were carried out in four replicates and were reported as mean ± standard

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error, except in feces where all the samples from individual replicates were combined to one

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composite set due to low biomass. A one-way ANOVA was performed, followed by Tukey’s

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multiple comparisons test (IBM SPSS Statistics 19, Chicago, USA) at p ≤ 0.05.

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RESULTS AND DISCUSSION

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Effect on plant growth


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Figure 2 shows the percent moisture content, dry biomass, and root and shoot lengths of KBPs

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exposed to nano-CeO2 and bulk-CeO2 for 22, 29 and 36 days. The soil grown KBPs in this study

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did not show any visible symptoms of toxicity in response to nano- or bulk- CeO2 exposure.

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However, on the 29th day of exposure to 2000 mg/kg nano-CeO2, the percent moisture content

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decreased significantly in the roots (p = 0.041), stems (p = 0.007) and leaves (p = 0.05),

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compared to control (Figure 2A). At 1000 mg/kg, nano-CeO2 did not significantly affect the

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KBP tissues throughout the 36 day exposure duration, but bulk-CeO2 exposure significantly

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reduced the percent moisture content in stems and leaves at p = 0.029 and p = 0.002,

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respectively, compared with control. The moisture content in the tissues was restored to normal

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levels with longer exposure to CeO2. Interestingly the dry biomass of the tissues remained

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unaffected upon nano- and bulk-CeO2 exposure for 22, 29 or 36 days (Figure 2B). The plant

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growth with respect to root and shoot length was not significantly affected by exposure to nano-

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or bulk- CeO2 (Figure 2C). In recent years, nano-CeO2 has invited significant attention in

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agriculture and biomedicine, due to their evident positive and protective responses in plants and

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in vitro animal tissues with respect to growth, physiology or fighting oxidative stress.43 Earlier

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findings suggest that plants exposed to a wide range of nano-CeO2 concentrations (10-2000

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mg/L) in cultured medium have varied and conflicting responses depending on the test species,

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exposure duration, dose and age of the plant.25, 32, 43 Evidences of negative impact at biochemical

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and molecular level in plants have been reported on short-term exposure to nano-CeO2,

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especially at high concentrations (125-4000 mg/L) in cultured media, due to direct interactions at

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the nano-bio interface.32, 43, 44

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Although the nanotoxicity studies in artificial media aid in providing mechanistic

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information, from a regulatory and holistic perspective, more information is needed on exposure



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and fate assessments in natural conditions. As observed in the current study, reductions in root

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moisture content, compared to untreated control, with no associated effects on the dried tissue

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biomass were also noted in farm soil grown soybeans exposed to 100-1000 mg/kg nano-CeO2

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(48 days).31 In the current study, the decrease in the percent moisture content in the tissues

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became statistically significant only at a higher dose of nano-CeO2 (2000 mg/kg), but bulk-CeO2

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exposure resulted in a reduction even at 1000 mg/kg in the aerial tissues (Figure 2). Hawthorne

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et al. also demonstrated significant reduction in zucchini root fresh mass on exposure to 1000

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mg/kg CeO2 in soil, irrespective of particle size.25 However, the zucchini stems and leaves

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showed a reversed trend upon exposure to 1000 mg/kg nano- and bulk-CeO2.25 Soil grown

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lettuce showed a hormetic trend when exposed to a 0-1000 mg/kg nano-CeO2, with enhanced

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dried root mass at 100 mg/kg, but again, reduction was noted at 1000 mg/kg.38 Conversely, nano-

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CeO2 at 500 mg/kg enhanced growth in barley (Hordeum vulgare) plants in terms of length and

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biomass, but eventually, deterring grain production.45 In agreement to minimal effects of nano-

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and bulk-CeO2 on KBPs, cucumber,46 and cilantro (Coriandrum sativum)47 exposed to nano-

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CeO2 at 1000 mg/kg showed no significant effects, either detrimental or enhancement, on plant

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growth. Radish (Raphanus sativus) plants also experienced no effects on nano-CeO2 exposure,

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but increased biomass when exposed to bulk-CeO2 in soil.48 Thus previous, as well as current

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study, suggests that although nano-CeO2 in soil environment may cause alterations in some

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physiological parameters in different plant species, it does not cause overt toxicity even on

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prolonged exposure. This could be due to the chemical stability of nano-CeO2 in biological

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environments, owing to the transition between Ce oxidation states, resulting in buffering of

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redox processes in situ.49 However, studies have provided evidences of negative impacts on

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several plant species at biochemical and cellular level.31,38,50 Also, nano-CeO2 exposure in the


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presence of soil organic matter alleviates the toxicity as a function of particle interactions with

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soil constituents.51

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Cerium accumulation in the producers

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Accumulation of Ce in KBP tissues exposed to nano- and bulk-CeO2 for 22, 29 and 36

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days, expressed as µg/g tissue dry wt, is shown in Figure 3. Although exposure period dependent

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studies are extremely valuable to understand the root accumulation and translocation of a

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toxicant over time, it is often difficult to explain decreases in the toxicant concentration in a

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growing tissue, when expressed as gram per dry weight. Thus, to effectively show the average

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distribution of Ce in the roots, stems and leaves, the absolute values of Ce content in different

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tissue of KBPs are presented in SI Table S1. Due to varying levels of Ce in individual aerial

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tissue compartments (stems, cotyledoneous leaves and true leaves), as a function of time (SI

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Table S2), the Ce accumulation was summed as the total shoot concentration (Figure 3).

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Kidney bean roots from all the CeO2 treatments accumulated significant levels (p ≤ 0.01)

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of Ce with respect to control, indicating uptake. After 22 days of exposure to 1000 and 2000

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mg/kg nano-CeO2, the roots accumulated 23 ± 2.2 and 26.7 ± 3.0 µg Ce /g tissue dry wt (Figure

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3). On longer exposures (29 and 36 days), the KBP roots exposed to 2000 mg/kg nano-CeO2

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accumulated significantly higher (p ≤ 0.01) amount of Ce (34.2 ± 1.5 and 40.7 ± 1.6 µg/g tissue

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dry wt), compared to 1000 mg/kg nano-CeO2 (12.9 ± 0.3 and 26.4 ± 1.8 µg/g tissue dry wt).

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Additionally, at all exposure periods, roots exposed to 1000 mg/kg bulk-CeO2 accumulated

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similar levels of Ce as in 1000 mg/kg nano-CeO2 (Figure 3, SI Table S1). At 1000 mg/kg nano-

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and bulk-CeO2 exposure, Ce accumulation in KBP roots did not increase with duration. This

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could be due to the aggregation of the CeO2 particles in the soil due to the presence of natural

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organic matter, thereby minimizing uptake and internalization of Ce in tissues.32 However, at



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higher nano-CeO2 concentration (2000 mg/kg), there may be more Ce available for interactions

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at the root surface,52 coupled with no biomass dilution (Figure 2), leading to significantly high

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Ce content in the roots after 36 days compared to 22 days (p ≤ 0.01) (Figure 3, SI Table S1).

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Due to biomass dilution in the aerial tissues with time (22 to 36 days), the Ce

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concentration calculated as µg/g tissue dry wt, remained constant or declined in the shoots

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(Figure 3). This interpretation shows the normalized cerium concentration with an assumption

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that Ce was distributed evenly throughout the shoot system, suggesting decreased translocation.

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However, continuous translocation and further redistribution of Ce from older to younger tissues

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lead to decrease in µg Ce per g dry wt of the entire tissue as a whole. As shown in SI Table S2,

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the absolute mass of Ce in the stems significantly (p ≤ 0.01) increased at 2000 mg/kg nano-CeO2

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with exposure duration from 0.54 ± 0.02 µg (29 days) to 1.02 ± 0.09 µg (36 days) (SI Table S2).

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By the 36th day of exposure, the cotyledoneous leaves shed naturally and were not included in

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the Ce accumulation measurements, and were unavailable for feeding next trophic level.

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Although the Ce concentration in the stems decreased over time, other aerial tissues started to

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accumulate more. At 22nd day, KBP shoots from 1000 mg/kg nano-CeO2, 2000 mg/kg nano-

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CeO2, and 1000 mg/kg bulk-CeO2 exposures contained 1.31 ± 0.23, 1.45 ± 0.28, and 1.32 ± 0.23

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µg Ce/ g tissue dry wt, respectively, and at 29th day, the levels were 0.87 ± 0.08, 1.27 ± 0.16, and

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2.06 ± 0.69 µg Ce/ g tissue dry wt, respectively. Last, at 36 days the plants had accumulated

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1.03 ± 0.06, 1.38 ± 0.11, and 1.34 ± 0.25 µg Ce/ g tissue dry wt, respectively. Over the 36 days

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of exposure, no significant variation was noted in Ce accumulation in the shoots, similar to the

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findings in the roots. This suggests that after certain period of exposure, the relative rate of Ce

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uptake into KBP tissues became constant.


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Cerium accumulation in KBP roots was notably lower when compared to accumulation

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reported in soybean,31 zucchini,25 or lettuce38 (210, 567, and 449 µg/g tissue dry wt, respectively)

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grown in soil amended with 1000 mg/kg nano-CeO2. In contrast to our observations, Hawthorne

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et al. reported that Ce transport from soil to plant tissues was greater from nanoparticle form,

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compared to bulk particles.25 However, comparing across studies is confounded by the

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differences in experimental design, including plant species, exposure duration, and differences in

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soil type. Although nano-CeO2 has been reported to undergo minimal chemical transformation

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outside or inside plant tissues,32-34 particles may aggregate into clusters in the presence of

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organic matter,53 leading to similar Ce accumulation in roots as bulk-CeO2. However, species

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specific root exudation and plant-associated microbial activity may both serve to promote

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particle dispersion or dissolution into soil pore-water.52

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Interestingly, in spite of much higher Ce content in the roots of zucchini and soybean (10-

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30 times higher than KBP),25,31 the Ce content in stems of these plants (0.1 and 0.5 µg/g,

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respectively) was considerably lower than that present in the KBP stems (1.35 ± 0.15 to 2.13 ±

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0.30 µg/g tissue dry wt) at 1000 mg/kg nano-CeO2 (SI Table S2). In addition, KBP leaves

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accumulated higher amounts of Ce (0.28 to 0.51 µg/g tissue dry wt) than did zucchini (0.15 µg/g

322

tissue dry wt)25 and soybean (3x10-4 µg/g tissue dry wt) leaves.31 However, lettuce leaves

323

exposed to 1000 mg/kg nano-CeO2 accumulated 206 times more Ce than KBP leaves.38 Tissue

324

translocation factors (TF), calculated as TFsr = [Ce]stem/ [Ce]root and TFlr = [Ce]leaf/ [Ce]root,

325

were 0.071 ± 0.009 and 0.020 ± 0.005, respectively, in the 36 day-old tissues from plants

326

exposed to 1000 mg/kg nano-CeO2. However, in zucchini and soybean, the values were 78 and

327

23 times lower than TFsr in KBPs, respectively; and 20 and 104 times lower than TFlr in KBPs,

328

respectively.25,31



329

Cerium transfer from producers to higher trophic levels

330

The nano- and bulk-CeO2 exposed plants were infested with MBB second-instar, which

331

were subsequently fed to SSBs. Cerium accumulation in the MBB and SBB tissues at different

332

developmental stages is shown in Figure 4. The MBBs at the larval, pupal, and adult stages were

333

collected after 7, 14 and 21 respective days of feeding on untreated, nano- and bulk-CeO2 treated

334

KBPs. ICP-MS analysis of the digested MBB tissues suggests that the Ce was transferred from

335

the KBP to MBB larvae (Figure 4); but a significant fraction (97-98 %) of the ingested Ce was

336

excreted during the 48 h depuration period. The MBB larvae excreted 4.45, 15.5, 21.4, and 6.5

337

µg Ce/ g feces dry wt after feeding on leaves from KBPs exposed to 0, 1000, 2000 mg/kg nano-

338

CeO2 and 1000 mg/kg bulk-CeO2 exposures, respectively. The Ce content in MBBs larvae feces

339

from nano-CeO2 treatment was notably higher (2.4 times) than in bulk-CeO2, even at similar

340

exposure concentration (1000 mg/kg), although a statistical analysis is not possible because only

341

single replicates were available. The decreased Ce excretion resulted in 1.9 and 2.4 times higher

342

Ce in the MBB larval tissues from 1000 mg/kg bulk-CeO2 treatment (901.5 ± 60.1 ng/g tissue

343

dry wt) than corresponding nano-CeO2 treatment (483.9 ± 60.2 ng/g tissue dry wt) and control

344

(382.3 ± 138.8 ng/g tissue dry wt), respectively. Although the Ce content increases in the bulk-

345

CeO2 larvae tissues were statistically significant compared to control accumulation (p = 0.043),

346

but not to nano-CeO2 tissues (p = 0.108).

347

Increased Ce accumulation in bulk-CeO2 larvae tissue combined with lower

348

concentrations in the feces suggests more rapid metabolic rate (including elimination) of the

349

element, although we do not have enough information to suggest the chemical form of the

350

particle in situ. However, 1000 mg/kg bulk-CeO2 MBB larvae feces weighed twice as more as

351

1000 mg/kg nano-CeO2 (SI Table S3), but less than 2000 mg/kg nano-CeO2. This could be 16 ACS Paragon Plus Environment

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352

argued as leading to lower values of Ce in feces from bulk-CeO2 MBB larvae compared to nano-

353

CeO2 MBB larvae. But simultaneous higher accumulation in the bulk-CeO2 MBB larvae tissues

354

compared to all other treatments, independent of feces volume, supports our claim that this

355

particle-size dependent trend was due to metabolism, and not because they may have consumed

356

more KBP biomass. The actual dietary accumulation of Ce in the MBB larval tissues from 1000

357

and 2000 mg/kg nano-CeO2 treatments were not significantly higher than the untreated controls.

358

Low accumulation in the larval tissues may result from the short feeding period. Also, the

359

metabolism in the larval stage is rapid, likely resulting in low residence time of the Ce in the

360

exposed MBB larvae.

361

In the MBB pupae tissues, 361 ± 79.8 , 461.2 ± 35.8, 1181.3 ± 69.9 and 1013.5 ± 175.5

362

ng Ce/g tissue dry wt were accumulated from 0, 1000, 2000 mg/kg nano-CeO2, and 1000 mg/kg

363

bulk-CeO2, respectively. When the MBB larva transform into an adult, it passes through the

364

sedentary and transitional pupa stage. At the pupa stage, feeding ceases and metabolic activities

365

are lowered.41 Accordingly, Ce accumulation in the pupae from 1000 mg/kg nano- and bulk-

366

CeO2 treatments was proportionally similar to the larval stage. However, at 2000 mg/kg nano-

367

CeO2, the pupae stored 2.4 times more Ce compared to larvae, but the increase was not

368

statistically significant. The pupae from 1000 mg/kg bulk-CeO2 and 2000 mg/kg nano-CeO2 thus

369

accumulated significantly higher levels of Ce, compared to untreated controls (p = 0.002 and

370

0.005, respectively) and 1000 mg/kg nano-CeO2 (p = 0.009 and 0.023, respectively).

371

As the MBB further develops into the adult form, the rate of feeding and metabolic

372

processes increase. Therefore, Ce accumulation in all the tissues feeding on nano-CeO2 exposed

373

plants showed significantly higher levels (p= 0.003, 0.001 in 1000 and 2000 mg/kg, respectively)

374

than the background controls (347.1 ± 64.8 ng/g tissue dry wt), but was not significantly affected 17 ACS Paragon Plus Environment


375

in response to particle size or nano-CeO2 exposure concentration tested in this study. As seen in

376

Figure 4, Ce accumulations in the MBB adults from 1000, 2000 mg/kg nano-CeO2 and 1000

377

mg/kg bulk-CeO2 were 5358.6 ± 451.2, 6276.2 ± 819.5, and 3039.5 ± 879.5 ng/g tissue dry wt,

378

respectively. The Ce contents in the MBB adult tissues were 12, 5, and 3 times higher when

379

compared to pupae from 1000, 2000 mg/kg nano-CeO2, and 1000 mg/kg bulk-CeO2. This could

380

be due to time dependent accumulation as well as increased feeding habits in the adult stage for

381

expending energy towards movement and growth. Notably, the fold-increase in Ce accumulation

382

in MBB adults were significant in nano-CeO2 treatments, but not statistically significant in bulk-

383

CeO2 treatment. On comparing fold increases along the growth stages at same exposure

384

concentration, it is interesting to note that Ce from bulk and nano-CeO2 forms accumulate

385

differently over time in insects. The finding that draws even further attention was that upon

386

depuration, the MBB adults from 1000, 2000 mg/kg nano-CeO2 and 1000 mg/kg bulk-CeO2

387

treatments excreted 34, 32 and 36% of Ce from their bodies respectively, which is considerably

388

lower than that excreted by the MBB larvae (97, 98, 88%, respectively). The adult feces from 0,

389

1000 and 2000 mg/kg nano-CeO2 and 1000 mg/kg bulk-CeO2 contained 119.1, 2707.6, 2950.2,

390

and 1673.7 ng Ce/g feces dry wt. A lower fraction of Ce in the feces correlated with higher Ce

391

accumulation within the adult MBB tissues when compared to the MBB larvae exposed plants. A

392

lower accumulation and increased excretion in the larvae (Figure 4) suggests that a major

393

fraction of the Ce was retained in the gut of the early stage of MBB growth. With

394

prolonged/chronic feeding on CeO2 in both forms, the absorption of Ce from the gut to the MBB

395

tissues increased significantly, resulting in “bioconcentration” (uptake higher than excretion).

396

Judy et al. reported that tobacco hornworm bioaccumulated nano-Au when exposed to surface

397

contaminated tomato plants, and the nanoparticles localized primarily in the medial and posterior


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398

midgut regions, as opposed to the anterior part.22 Similar to plant tissues and early growth stages

399

of MBB, i.e larvae and pupae, Ce accumulation in MBB adults were also not significantly

400

affected by particle size. In contrary, higher Ce accumulation in cricket tissues from nano-CeO2

401

exposed zucchini leaves compared to bulk-CeO2 was reported in a similar study with a different

402

food chain.25 The Ce concentration in the MBB adult tissues was 157 folds higher compared to

403

that transferred to crickets (33.6 ng/g tissue dry wt) from zucchini plants in a separate study,

404

considering that Ce accumulation in KBP stems and leaves were also higher than in zucchini

405

leaves by 4.6 and 3.4 folds.25 However, the higher variation in Ce accumulation in consumers

406

could be due to varying feeding habits and metabolism between species, as well as longer

407

feeding period of the MBB (21 days) compared to the crickets (14 days).

408

Further along the food chain, the third instar-MBB larvae feeding on 0, 1000, 2000

409

mg/kg nano-CeO2 and 1000 mg/kg bulk-CeO2 for 10-15 days were presented to three SSB adults

410

for 5 h. This was sufficient time for the SSBs to paralyze the MBBs with rostrum and consume

411

their contents. At 0, 1000, 2000 mg/kg nano-CeO2 and 1000 mg/kg bulk-CeO2, the SSBs

412

accumulated 1113.7 ± 78.4, 2567.1 ± 213.2, 3245.1 ± 432.1 and 1465.4 ± 145.2 ng/g Ce in its

413

tissues. With an exception of the bulk-CeO2 treatment, the SSBs from the nano-CeO2 treatments

414

accumulated significantly higher Ce concentration (p ≤ 0.05), on comparison to untreated

415

controls. In this study, the Ce transfer to the third trophic level was 60 times higher than what

416

was reported in Ce transfer from crickets to spiders.25 Spiders contained only 5.4 ng/g Ce in their

417

tissues after feeding on crickets that had accumulated 33.6 ng/g Ce from zucchini plants exposed

418

to 1000 mg/kg nano-CeO2.25 This variation could be due to differences in experimental design,

419

such as feeding duration, feeding frequency, number of insects consumed, or species-specific

420

physiological features of spiders and SSBs.



421

In spite of rapidly growing literature on effects of ENMs on individual biota and related

422

mechanistic studies, the information on the bioaccumulation and biomagnification of ENMs and

423

related ions in a food chain is very limited. In an aquatic trophic system, mussels (Mytilus

424

galloprovincialis) bioaccumulated lesser Au after feeding on nano-Au exposed microalgae

425

(Dunaliella salina), than feeding directly on the nano-Au suspension.20 Currently, no information

426

is available on the toxicity of nano-CeO2 to terrestrial insect species; however, studies with

427

Daphnia species in freshwater ecosystems have provided evidence of sublethal toxicity

428

associated with physiological functions.19 A recent aquatic mesocosm study with snails

429

(Planorbarius corneus) demonstrated that bare nano-CeO2 induced oxidative stress, but the

430

coated particles had no such effect.21 In the current study, no observable mortality,

431

morphological alterations or physiological deficits were noted in the MBBs or SSBs as a

432

function of CeO2 treatments. Our findings suggest that Ce was biomagnified from the CeO2

433

exposed KBPs to MBB adults, and also further magnified to the SSBs feeding on MBB larvae

434

(Table 1). Specifically, on CeO2 exposure, irrespective of particle size, trophic transfer of Ce was

435

observed from the KBPs to MBB larvae, but biomagnification was not noted until reaching the

436

adult stage, which fed on the treated tissues for two more weeks. The biomagnification factors in

437

adult MBB from nano-CeO2 exposures (5.32 ± 0.29 and 4.33 ± 0.49 at 1000 and 2000 mg/kg,

438

respectively) were similar to higher than from 1000 mg/kg bulk-CeO2 exposure (4.51 ± 1.62).

439

Also, biomagnification was noted from nano-CeO2 exposed MBB larvae to SSBs by mean

440

factors of 5.32 ± 0.41 at 1000 mg/kg and 6.7 ± 0.46 at 2000 mg/kg. Whereas the

441

biomagnification factor from MBB larvae to SSBs in bulk-CeO2 exposure was lower (1.62 ±

442

0.12) compared to nano-CeO2 treatments. Biomagnification of Ce observed in this study is in

443

accordance with another trophic transfer study where 5, 10 and 15 nm sized Au nanoparticles


Page 20 of 36

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444

suspended in distilled water (100 mg/L) were observed to biomagnify by factors of 6.2, 11.6 and

445

9.6, respectively, from tobacco plants to tobacco hornworms. Biomagnification of Cd was also

446

observed in a microbial food chain consisting of bacteria (Pseudomonas aeruginosa) and

447

protozoa (Tetrahymena thermophile) exposed to CdSe quantum dots by a factor of 5.4.17 On

448

contrary, Unrine et al. reported “trophic dilution” or a decrease in the concentration of nano-Au

449

in juvenile bullfrogs from earthworms upon exposure to artificial soil media.24 Also, Hawthorne

450

et al. and De la Torre-Roche et al. reported no biomagnification of Ce and La in a terrestrial food

451

chain.25,26

452

In summary, this study provides direct evidence of higher translocation of Ce compared

453

to previous studies and their trophic transfer in a terrestrial food chain, with further particle size-

454

dependent biomagnification at the consumer level. The findings suggest that the duration of

455

ENM exposure and growth stage of the consumer are critical factors in particle bioaccumulation

456

and biomagnification in a terrestrial food chain. During the developmental stages with active

457

metabolism and shorter exposure, the vast majority of the ingested Ce was excreted from the

458

body. However, mature adults systemically absorbed greater levels of Ce into their tissues. For

459

the exposed receptor, the excretion of Ce is beneficial as it suggests that although nano-CeO2

460

was transferred along the food chain, a large fraction may be discarded as waste by the metabolic

461

processes of the organism. However, the ecological significance of the excreted Ce, including the

462

chemical form and bioavailability to decomposers remains completely unknown. Importantly,

463

the uptake and translocation of Ce by plants and accumulation in insect tissues was observed to

464

be independent of particle size, but biomagnification of Ce in the adult stages of primary and

465

secondary consumers was observed to be higher in the nanoparticle form. This study clearly

466

demonstrates that intentional or accidental release of nano-CeO2 in sewage sludge, waste-water



467

or agricultural fields may result in the contamination of agricultural crops, leading to potential

468

dietary uptake and transfer of Ce within food chains. In the future, additional studies are needed

469

to localize and speciate Ce in biota tissues and feces to gain better insight into mechanism of

470

contaminant transfer through the food chain, and to better inform the regulatory framework for

471

ENMs risk assessment.

472

Supporting Information. Absolute values of Ce (µg) in different tissue compartment of kidney

473

bean plants exposed to 0-2000 mg/kg nano-CeO2 and 1000 mg/kg bulk-CeO2 (Table S1). Cerium

474

accumulation in above-ground biomass of kidney bean plants exposed to 0-2000 mg/kg nano-

475

CeO2 and 1000 mg/kg bulk-CeO2 (Table S2). Dry weights (g) of feces from Mexican bean beetle

476

larvae and adults (Table S3). This material is available free of charge via the Internet at

477

http://pubs.acs.org.

478

ACKNOWLEDGEMENT

479

This material is based upon work supported by National Science Foundation (NSF) and

480

Environmental Protection Agency (USEPA) under Cooperative Agreement Number DBI-

481

0830117. Any opinions, findings, and conclusions expressed in this material are those of the

482

author(s) and do not necessarily reflect the views of NSF or USEPA. This work has not been

483

subjected to USEPA review and no official endorsement should be inferred. The authors

484

acknowledge the USDA 2011-38422-30835 and NSF grants CHE-0840525 and DBI-1429708. J.

485

L.G-T acknowledges the Dudley family for Endowed Research Professorship, Academy of

486

Applied Science/US Army Research Office, Research and Engineering Apprenticeship program

487

(REAP) at UTEP, grant # W11NF-10-2-0076, sub-grant 13-7, and STARs programs of the UT

488

System. This work was also supported by Grant 2G12MD007592 from the National Institutes on

489

Minority Health and Health Disparities (NIMHD), a component of the National Institutes of 22 ACS Paragon Plus Environment

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Health. J.C.W. acknowledges USDA-AFRI #2011-67006-30181. We also acknowledge Dr.

491

James D. Kelly, Michigan State University for generously providing kidney bean seeds for this

492

research. We are grateful to Thomas W. Dorsey, NJ Department of Agriculture, for the generous

493

supply of MBBs.

494 495

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Figure legends

670

Figure 1. Schematic diagram of the trophic experiment. (A) Exposure of KBP to control and

671

CeO2 treatments; (B) Infestation of kidney bean plants with MBB larvae; (C, D) MBB larvae

672

feeding on kidney bean leaves (22nd day of plant exposure); (E) MBB collected for next trophic

673

level; (F) MBB being attacked and fed by SSBs); (G). MBB larvae metamorphose into dormant

674

stage, called pupae (25th day of plant exposure); (H) MBB adults feeding on kidney bean leaves

675

(36th day of plant exposure).

676

Figure 2. (A) Percent moisture content, (B) dry biomass, (c) length of roots and shoots of kidney

677

bean plants exposed to 0, 1000 and 2000 mg/kg nano-CeO2 and 1000 mg/kg bulk-CeO2 (1000

678

mg/kg) for 22, 29 and 36 days. Values are expressed as mean ± SE (n = 4). Bars with different

679

letters represent significant difference at p ≤ 0.05.

680

Figure 3. Cerium accumulation in roots and shoots (including stem and leaves) of kidney bean

681

plants exposed to 0, 1000 and 2000 mg/kg nano-CeO2 and 1000 mg/kg bulk-CeO2 for 22, 29 and

682

36 days. Values are expressed as mean ± SE (n = 4). Bars with different letters within days of

683

exposure represent significant difference at p ≤ 0.05.

684

Figure 4. Accumulation of cerium in Mexican bean beetle (MBB) tissues at different stages of

685

growth, MBB feces and spined soldier bug (SSB) tissues. All values are expressed as mean ± SE

686

(n = 4), except in feces. Bars with different letters within days of exposure represent significant

687

difference at p ≤ 0.05.

688



Figure 1.


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Figure 2.



Figure 3.


Page 34 of 36

Page 35 of 36


Figure 4.



Page 36 of 36

Table 1. Root to shoot translocation factors (TF) and mean biomagnification factors (BMF) from producers to primary consumers and from primary consumer to secondary consumer. Values are expressed as mean ± SE. Treatments

TF (root to shoot)

22 d

29 d

36 d

BMF (KBP to MBB larvae)

BMF (KBP to MBB adults)

BMF (MBB larvae to SSB)

nano-CeO2 1000

0.06 ± 0.01

0.07 ± 0.01

0.04 ± 0.00

0.42 ± 0.12

5.32 ± 0.29

5.32 ± 0.41

nano-CeO2 2000

0.05 ± 0.01

0.04 ± 0.01

0.03 ± 0.00

0.44 ± 0.16

4.33 ± 0.49

6.7 ± 0.46

bulk-CeO2 1000

0.07 ± 0.01

0.14 ± 0.04

0.07 ± 0.01

0.9 ± 0.18

4.51 ± 1.62

1.62 ± 0.12

*KBP= Red kidney bean, MBB= Mexican bean beetle, SSB= Spined soldier bug


Cerium Biomagnification in a Terrestrial Food Chain: Influence of

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