Bioaccumulation of CeO2 Nanoparticles by Earthworms in Biochar

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Bioaccumulation of CeO2 nanoparticles by earthworms in biochar amended soil: A Synchrotron Microspectroscopy Study Alia D. Servin, Hiram A. Castillo-Michel, Jose A. Hernandez-Viezcas, Wout De Nolf, Roberto De La Torre Roche, Luca Pagano, Joseph J. Pignatello, Minori Uchimiya, Jorge L. Gardea-Torresdey, and Jason C. White J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04612 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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Journal of Agricultural and Food Chemistry

Bioaccumulation of CeO2 nanoparticles by earthworms in biochar amended soil: A Synchrotron Microspectroscopy Study Alia D. Servin a, Hiram Castillo-Michel b, Jose A. Hernandez-Viezcas c, Wout De Nolf b, Roberto De la Torre-Roche a Luca Pagano a,d,e, Joseph Pignatello f, Minori Uchimiya g, Jorge Gardea-Torresdey c, Jason C. White a* a

Department of Analytical Chemistry, Connecticut Agricultural Experiment Station, New Haven, Connecticut 06511, United States b European Synchrotron Radiation Facility, B.P.220 - 38043 Grenoble Cedex, France c Chemistry Department and Environmental Science and Engineering PhD Program, The University of Texas at El Paso, El Paso, Texas 79968, United States UC Center for Environmental Implications of Nanotechnology, El Paso, Texas d Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003, United States e Department of Life Sciences, University of Parma, 43124 Parma, Italy f Department of Environmental Sciences, Connecticut Agricultural Experiment Station, New Haven, Connecticut 06511, United States g USDA-ARS, New Orleans, Louisiana 70124, United States

*Corresponding author Dr. Jason C. White Department of Analytical Chemistry  The Connecticut Agricultural Experiment Station  123 Huntington Street P.O. Box 1106 New Haven, CT 06504-1106  Voice: (203) 974-8523 Fax: (203) 974-8502  E-mail: [email protected]

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Abstract:

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influence NP availability and toxicity to biota. In the present study, earthworms (Eisenia fetida)

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were exposed for 28 d to a residential or agricultural soil amended with 0-2000 mg CeO2 NP/kg

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and with biochar (produced by the pyrolysis of pecan shells at 350°C and 600 °C) at various

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application rates (0-5% [w/w]). After 28 d, earthworms were depurated and analyzed for Ce

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content, moisture content and lipid peroxidation. The results showed minimal toxicity to the

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worms; however, biochar (350°C or 600 °C) was the dominant factor, accounting for 94% and

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84% of the variance for moisture content and lipid peroxidation, respectively, in the exposed

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earthworms. For both soils with 1000 mg CeO2/kg, 600 °C, biochar significantly decreased the

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accumulation of Ce in the worm tissues. Amendment with 350 °C biochar had mixed responses

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on Ce uptake. Analysis by µ-XRF and µ-XANES was used to evaluate Ce localization,

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speciation and persistence in CeO2- and biochar (BC)-exposed earthworms after depuration for

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12, 48 and 72 h. Earthworms from the 500 mg CeO2/kg and 0% BC treatments eliminated most

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Ce after a 48 h depuration period. However, in the same treatment and with 5% BC-600

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(biochar pyrolysis temperature of 600°C), ingested biochar fragments (~ 50 µm) with Ce

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adsorbed to the surfaces were retained in the gut after 72 h. Additionally, Ce remained in

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earthworms from the 2000 mg CeO2/kg and 5% BC treatments after depuration for 48 h.

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Analysis by µ-XANES showed that within the earthworm tissues, Ce remained predominantly as

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Ce+4O2, with only few regions (2-3µm2) where it was found in the reduced form (Ce+3). The

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present findings highlight that soil and biochar properties have a significant influence in the

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internalization of CeO2 NPs in earthworms; such interactions need to be considered when

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estimating NP fate and effects in the environment.

The interactions of nanoparticles (NPs) with biochar (BC) and soil components may substantially


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Introduction

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last 10 years, with applications in agriculture, environmental remediation, medicine, catalysts,

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cosmetics, and electronics, among others.1,2 Although, there are many benefits to the use of NPs

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in commercial products, there is still concern over potential impacts to the environment. For

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example, recent studies have reported the cytotoxicity of CeO2 NPs to plants, mammals, and

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bacteria.3-5 There is still a pressing need to evaluate NPs behavior (fate, effects) under more

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environmentally relevant conditions, including chronic exposures assessed by robust and

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meaningful endpoints.6 Significant release of NPs into the environment will occur during

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manufacturing, use and disposal processes. It has been estimated that 63- 91% of over 260,000 -

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309,000 metric tons of global NPs production were disposed in landfills in 2010.7 Additionally,

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NPs may enter wastewater treatment plants and accumulate in biosolids.7 Approximately

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7,100,000 dry tons of biosolids are generated each year from municipal wastewater treatment

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facilities in the U.S., approximately 55% of which are land applied.8 Once NPs are released into

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the environment, their unique properties of high surface area and reactivity will dictate

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interactions with soil components (i.e. soil organic carbon, minerals, biochar, etc.); these

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processes will subsequently control particle aggregation, availability and toxicity.9 For example,

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studies in aqueous media have shown that natural organic matter significantly reduces CeO2 NPs

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aggregation10; in studies with soil, Ce accumulation in corn roots increased with the soil organic

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matter content.11 Furthermore, the addition of humic acid (HA) to exposure media significantly

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reduced toxicity and bioaccumulation of CeO2 NPs in C. elegans.12 Another soil component that

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will significantly impact NPs fate is black carbon. Black carbon (BC) includes biomass pyrolysis

The production and use of engineered nanoparticles (NPs) has increased dramatically in the



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byproducts that are present in soils by direct land application as an agricultural amendment

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(biochar) and/or due to natural or deliberate burning.13 Biochar application to agricultural soils

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has increased due to the positive impacts that have been observed on water holding capacity and

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nutrient retention.14 The physical and chemical properties of biochar will depend on the

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feedstock material and on the conditions of synthesis.15 However, given the intrinsic properties

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of biochar, especially its high porosity and surface area, significant impact on the fate, transport,

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and toxicity of NPs in soil could be possible. Previous studies on the interactions of biochar with

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NPs have shown a pH-dependent heteroaggregation of positively charged CeO2 NPs with

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negatively charged functional groups present on biochar surfaces.16 Furthermore, in a plant-

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based study from our group, micro X-ray fluorescence (µ-XRF) and micro X-ray absorption near

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edge structure (µ-XANES) showed the association of CeO2 NPs to biochar and soil surfaces after

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28 days of exposure.17 These results suggest that biochar soil amendments will influence the fate

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and behavior of NPs in the environment. However, the impact of biochar-soil-CeO2 interactions

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on exposure to terrestrial biota is unknown.

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Eisenia fetida is an epigeous earthworm species that is found mostly within litter of the

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soil surface, making it a suitable model organism for bioavailability studies given its likely direct

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contact with NPs and biochar components in soil. Moreover, earthworm species can significantly

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influence the physical, chemical and biological properties of soil. For example, earthworms are

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vital to the incorporation and fragmentation of organic material and to mineral nutrient

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recycling.18 Thus, negative impacts on earthworm populations may indirectly affect soil function

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and plant growth. Recent reports have indicated that some biochars may have negative effects on

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earthworms. Liesch et al.19 reported considerable earthworm (E. fetida) mortality and weight loss

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upon exposure to poultry litter biochar at 67.5 and 90 Mg ha-1; conversely, populations exposed


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to pine chip biochar did not differ significantly from the controls. Separately, some studies have

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focused on the direct effects of NPs on earthworms; for example, exposure to CeO2 NPs at 41-

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10,000 mg/kg had no effect on the survival or reproduction of Eisenia fetida species after 28 d of

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exposure, although histological analysis did show potential toxicity through cuticle loss from the

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body wall

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increase in the expression of the cyp35a2 gene (a xenobiotic metabolizing gene), as well as

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decreases in fertility and survival, in response to CeO2 NPs (15 and 45 nm) exposure on

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nematode growth medium plates at 1 mg/L21. However, the influence of NPs interactions with

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biochar in terrestrial environments on particle toxicity to soil invertebrates is currently unknown.

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The objective of the present study was to evaluate the effects of co-exposure to CeO2 NPs (0-

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2000 mg/kg) and biochar (pyrolysis temperatures of 350 °C and 600 °C) at several application

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rates (0-5%) on earthworms (E. fetida), including Ce accumulation, moisture content and lipid

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peroxidation. Additionally, µ-XRF and µ-XANES techniques were used to evaluate Ce

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internalization and speciation in worm tissues after exposure and to evaluate the persistence of

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NPs CeO2 in worms after several depuration times (12, 48 and 72h).

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. Additionally, studies with the nematode Caenorhabditis elegans showed an

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Materials and Methods

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Cerium oxide NPs (99.99%, < 25 nm) were purchased from Sigma-Aldrich (Newburyport MA).

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Previous characterization by bright-field TEM images showed cubic, pyramidal, or bipyramidal

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CeO2 NPs in sizes varying between 20 and 200 nm.16 Suspensions of CeO2 NPs were prepared

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by dilution with deionized water (DI) to levels that yielded 0, 500, 1000 and 2000 mg/kg in soil.

CeO2 NP Suspensions



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The suspensions were ultrasonicated with a probe sonicator (Fisher Scientific, FB- 505) at 30%

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amplitude for 1 min.

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Biochar preparation

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Biochar was produced by the pyrolysis of pecan shells at 350 (BC-350) and 600 °C (BC-600) for

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4 h under a stream of nitrogen (1600 mL/min) in a 22 L box furnace (Lindberg, Type 51662-HR)

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as previously described.16 The granules were exposed to air for 2 weeks to complete oxygen

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chemisorption. The properties of the biochars (BC-350 and BC-600) were evaluated by zeta

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potential characterization of nano-biochar and CeO2 NPs as a function of pH and were presented

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earlier

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increased from 3 to 1116. Biochar elemental composition (CHNSO), volatile matter (VM),

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moisture and ash content of biochars (BC-350 and BC-600), have been previously described

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Biochar was added to CeO2 NP-amended agricultural and residential soil at 0, 0.5 and 5% w/w

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(dry weight) levels as described below.

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. Briefly, the ζ- potential gradually became negative, reaching -51 mV as the pH

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.

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Soil preparation

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Residential soil (sandy loam; 69.4% sand, 22.0% silt, 8.6% clay; 4.3% organic matter; pH 5.9;

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cation exchange capacity 18.6 cmol/kg; Ce= 40.7 + 2.6 mg/kg) was collected from the top 50 cm

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soil layer of the Connecticut Agricultural Experiment Station (CAES) in New Haven, CT. An

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agricultural soil (fine sandy loam soil; 56% sand, 36% silt, 8% clay; 1.4% organic matter; pH

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6.7; cation exchange capacity 18.6 cmol/kg, Ce= 21.2 + 0.63 mg/kg) was collected from the top

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50 cm of the Connecticut Agricultural Experiment Station Lockwood farm in Hamden, CT. The

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two soils were sieved to 2 mm, and individual portions of 100 g were manually mixed with


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biochar. Nanoparticle CeO2 suspensions were added and mixed thoroughly to ensure

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homogeneity. Over the duration of the experiment (28 d), soils were watered as needed to

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maintain the appropriate soil moisture level (approximately 80% of field capacity).

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Earthworms

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E. fetida were purchased from Carolina Biological Supply Company (Burlington, NC). Ten

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worms per replicate (5 replicates per biochar level and NP concentration) were added to CeO2

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NP treatments (0, 500, 1000 and 2000 mg/kg) with different biochar levels (0%, 0.5% and 5%)

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(total of 600 worms per experiment). The worms were added to plastic containers with a

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homogenous mixture of 100 g of residential or agricultural soil amended with biochar (0%, 0.5%

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and 5% [w/w]) and CeO2 NPs (0-2000 mg/kg). At harvest, earthworms were removed from each

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replicate container and rinsed with DI water on a sieve to remove surface-adsorbed soil and

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CeO2 particles. The earthworms were depurated for 48h in petri dishes containing moistened

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filter paper as previously described.23 After 48 h, live worms were rinsed with DI water, weighed

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and prepared for analysis as described below. Percent moisture content was determined

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gravimetrically by heating samples at 100 °C for 48 h.

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Matrix digestion

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After depuration, earthworm tissues were oven dried (100 °C; 48 h), weighed and transferred

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into 50 mL DigiPREP polypropylene digestion vessels (SCP Science, Champlain, NY)

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containing 2 mL of 65% HNO3. The tissue samples were pre-digested for 20 min and were then

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transferred to a hot block digester (SCP Science, Champlain NY) for 45 min at 115° C. One mL

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of H2O2 (Fisher Scientific, Pittsburgh PA) was added and the samples were heated an additional



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20 minutes. After digestion, 25 mL of DI water were added and the digest was analyzed by

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inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7500ce) for Ce (140 amu).

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Lipid peroxidation content

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Lipid peroxidation in earthworm tissues was determined by the thiobarbituric acid assay (TBA)

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as described by Li et al.,24 with minor modification. Briefly, samples were frozen in liquid

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nitrogen, and then homogenized in 0.1% (v/v) trichloroacetic acid (TCA). The samples were then

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centrifuged at 10,000 rpm for 10 min and the supernatant was amended with 1.5 mL of 20%

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TCA and 1.5 mL of 1% TBA in DI water. The solutions were incubated in a water bath at 95°C

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for 30 min. Malondialdehyde (MDA) was determined spectrophotometrically across all

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treatments as previously reported.24

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Micro-XRF and Micro-XANES Data Acquisition

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Micro X-ray fluorescence (µ-XRF) and micro X-ray absorption near edge structure (µ-XANES)

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were used to analyze Ce localization and speciation in earthworms that had been depurated for

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48 h after 28 days of exposure to soils amended with 5% biochar (600 °C) and 0, 500, 1000 and

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2000 mg/kg CeO2 NPs . Additionally, in order to evaluate persistence and elimination time of

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NPs CeO2 in earthworms, five worms from the lowest CeO2 NP treatment level (500 mg/kg)

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were depurated for 12, 48 and 72h. After depuration, all samples were cleaned with DI water ,

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anesthetized using chloroform (Fisher Scientific, Pittsburgh PA) embedded into Tissue Tek resin

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(Sakura Finetek USA, Torrance, CA), and flash-frozen with the use of liquid nitrogen chilled

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isopentane (-159° C) (Fisher Scientific, Pittsburgh PA). Samples were then axially sectioned at

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50 µm thickness with a Microtome plus cryostat (Triangle Biomedical Sciences, Durham, NC)


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and were lyophilized. Samples were after mounted in between 4 µm Ultralene (SPEX,

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Metuchen, NJ) window film for µ-XRF/µ-XANES analysis at beamline ID21 from the European

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Synchrotron Radiation Facility (ESRF, Grenoble, France).

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µ-XRF elemental maps were performed at 5.8 KeV with varying step sizes (35, 3, and

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1µm2) and 100-200 ms dwell time. Focus was achieved using fixed-curvature Kirkpatrick–Baez

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mirror optics. The photon flux was 5.7 × 1010 ph s−1 at 5.8 keV with a beam size of 1.0 × 0.5 µm2

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(H × V). The fluorescence signal was detected using an 80mm2 active area SGX Si drift detector

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with a Be window. Two photodiodes were used to measure the incident and transmitted beam

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intensities. The µXRF acquisition was done in hyperspectral mode for which the XRF spectrum

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for each pixel in the image was registered. All maps were fitted using PyMCA software to obtain

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the intensity distribution of the elements.25 For µ-XANES data acquisition, the energy was

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selected using a Si111 monochromator and scanned from 5700 to 5850 eV. The final Ce LIII

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edge spectra were the sum of 3-5 individual scans with 0.1 s integration time and 0.5 eV energy

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steps. The µ-XANES spectra of CeO2 NPs and Ce+32(CO3)3 (Sigma Aldrich, St. Louis, MO)

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were used as reference materials and were analyzed as powdered pellets in both transmission

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and fluorescence mode. In order to achieve an in-depth characterization of the Ce speciation in

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selected regions, µ-XANES spectra were acquired in fluorescence mapping mode as reported by

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Ferraro et al (2017). Briefly, images were recorded by scanning the beam with a 0.7 × 0.7 µm2

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step size, 80 ms dwell time per pixel, and 0.5 eV energy steps from 5700 to 5760. This resulted

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in 120 images recorded using a region of interest selective for Ce L3M4 and L3M5 emission

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lines, corrected for the detector dead time and corrected for changes in incident flux. The stack of

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images was aligned using elastix 26 and saved to an hdf5 file containing intensities and the energy

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values for each map to be processed using PyMCA for XANES spectra extraction. The Athena



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software was used for background removal, normalization of the spectra and linear combination

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fitting.

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Statistical Analysis

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A two-way ANOVA followed by Tukey-HSD was used to compare more than two treatments.

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All tests were performed using the statistical package SigmaPlot 13. Statistical significance was

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based on probabilities of p ≤ 0.05. Principal component analysis (PCA) was performed using the

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R software (https://www.r-project.org/).

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Results and Discussion

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Earthworm mortality was unaffected by CeO2 NPs or biochar exposure. The moisture content of

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earthworms exposed to CeO2 NPs (0-2000 mg/kg) and BC-350 and BC-600 (0-5%) is shown in

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Figure S1. PCA analysis was used to determine variances in moisture content of worms as a

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function of treatment (SI, Figure S2). The PCA results showed that biochar type (350 °C or 600

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°C) and soil type (agricultural or residential) were the dominant variables, accounting for 94% of

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the variance among the exposed earthworms. The moisture content in worms from the

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agricultural soil (SI, Figure S1A-B) did not vary as a function of BC-350 or BC-600

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concentration in the absence of CeO2. However, at 500 mg/kg of CeO2 NPs (0% BC-600), the

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percent moisture content significantly decreased (3.8%) in worms as compared to controls

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(Figure S1-B). The moisture content in worms from the residential soil with BC-350 showed

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mixed responses; there was an increase (3.6%) in percent moisture in control worms with 5%

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BC-350 and no CeO2. Conversely, at 1000 mg/kg of CeO2 NPs, earthworms exhibited decreases

Physiological effects from CeO2 NPs and biochar exposure.


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in moisture content of 3.1% and 3.4% with 0.5% and 5% BC-350 levels, respectively (Figure S1-

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C). Worms from the residential soil amended with 500 mg/kg CeO2 and BC-600 showed

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increases of 10.7% and 34.7% in moisture content as the biochar level increased (SI, Figure S2-

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D). Minimization of water loss is a critical factor in earthworm survival since water constitutes

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about 75-90% of earthworms’ body weight and is important to respiration.27 Since earthworms

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lack mechanisms to maintain a constant internal moisture content, the water potential of soil is a

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critical factor for survival.27 The physical characteristics of biochar (i.e. pore size) that result

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from the synthesis conditions and feedstock material utilized during its production are known to

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directly influence the water potential and redox environment of soil organisms.28 Additionally,

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biochar macropores serve as gas exchange channels that control the redox environment for soil

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biota.28 In the current study, earthworms from the amended agricultural soil did not experience

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overtly negative effects with regard to moisture content. However, in some cases, earthworms

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from the residential soil amended with BC 350/600 and CeO2 NPs exhibited changes in moisture

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content as biochar levels increased, suggesting that soil composition was likely responsible for

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subsequent differences in earthworms. For example, the two soils differ in organic matter content

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but from these data it is not possible to determine whether organic matter played any role in the

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observed effects.

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Biochemical responses in biota such as the production of reactive oxygen species (ROS)

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could lead to oxidative stress, lipid peroxidation, and cell death.29, 30 Malondialdehyde (MDA) is

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the final product of lipid peroxidation and has been previously used as an indicator of oxidative

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damage in different biological systems upon exposure to NPs.31-33 Exposure to CeO2 NPs and

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biochar had limited effects on lipid peroxidation in earthworm tissues (SI Figure S3). The PCA

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results showed that biochar type (BC-350 or BC-600) was the dominant variable, accounting for



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84% of the variance among the exposed earthworms (SI, Figure S4). Results from BC-350

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treatments showed that, in some instances (500 and 2000 mg/kg), earthworms had higher MDA

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levels in treatments without biochar (0%) compared to worms exposed to different amendments

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of BC-350 (SI, Results and Discussion section). Results from the BC-600 treatments showed

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minimal effects on lipid peroxidation; the only significant differences across both soils were in

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earthworms exposed to BC-600 at 5% (SI Figure S3B, SI Results and Discussion section).

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Previous studies have reported a higher oxidative DNA damage (measured by 8-

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hydroxydoxyguanosine (8-OHdG) and catalase (CAT) activities) in earthworms exposed to soil

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amended with biochar produced at 550 °C in comparison with biochar produced at 350 °C, with

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the authors suggesting that the toxicity from the higher temperature char was due to increased pH

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and metalloid content.34 Li et al.24 reported a lack of lipid peroxidation and no increase in

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superoxide dismutase activity in E. fetida in the presence of biochar produced at ~ 400 °C from

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apple wood chips.24 In the current study, MDA content did not show excessive or consistent

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changes with treatment; however, results did show that in some cases earthworms from BC-600

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treatments had increased lipid peroxidation with high exposure (5%), while the opposite was

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observed in BC-350 treatments where the observed peroxidation effects were largely in

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treatments with no biochar exposure (0%). Further study is needed to evaluate the mechanisms

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responsible for the biochemical responses of earthworms upon co-exposure to biochar and NPs.

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The Ce content in 48 h-depurated worms exposed to various CeO2 NPs concentrations (0-

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2000 mg/kg) and BC-350 or BC-600 (0-5%) is shown in Figure 1A-D. The PCA results showed

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that CeO2 NP concentration (1000 and 2000 mg/kg) was the dominant variable, accounting for

Ce content in earthworm tissues.


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82% of the variance among the exposed earthworms (Figure S5). The results showed that at 0-

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500 mg/kg CeO2 NPs, Ce accumulation by earthworms was not significantly affected by biochar

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or NP concentration in either soil. However, significant differences in the Ce accumulation were

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observed at 1000 (Figure 1A-1D) and 2000 mg/kg (Figure 1C) of CeO2 NPs treatments with the

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various biochar levels. Earthworms in the agricultural soil amended with BC-350 accumulated

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from 1.36 to 36.02 mg/kg of Ce, unaffected by biochar concentration (Figure 1A). Conversely,

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earthworms exposed to the same soil with BC-600 accumulated from 4.87 to 100.28 mg/kg of

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CeO2, and Ce content was significantly decreased by 95.4% with 5% biochar exposure at 1000

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mg/kg of CeO2 NPs treatment (Figure 1B). The Ce content in earthworms from the residential

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soil with BC-350 ranged from 1.56 to 104.66 mg/kg; the results showed mixed responses in Ce

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content as a function of biochar. For example, at 1000 mg/kg of CeO2 and 5% biochar, there was

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a significant increase (235%) in Ce content (Figure 1C). Conversely, the opposite was observed

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in the 2000 mg/kg treatment; Ce content decreased as the biochar level increased. Ce content in

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worm tissues exposed to 0.5% biochar decreased by 51.2%, while worm tissues exposed to 5%

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BC-350 contained 2.5 times less Ce than unexposed earthworms (Figure 1C). The Ce content in

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worms from the residential soil amended with BC-600 was 0.38 to 9.51 mg/kg. At 1000 mg/kg,

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significant decreases of 1.77 and 2.32 times in Ce content were observed in worm tissues with

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0.5% and 5% BC-600, respectively (Figure 1D). Previous studies have reported Ce accumulation

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of 5.3 and 49 µg/g in earthworm (Lumbricus rubellus) tissues and feces, respectively, after 7 d

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exposure at 5000 mg/kg of CeO2 in soil.35 Additionally, other studies have shown the dose-

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dependent increase of Ce content in earthworms (Eisenia fetida) exposed for 28 d in soil with

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CeO2 NPs at different sizes (5 to 300 nm) and concentrations (41 to 10,000 mg/kg).20 Overall,

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the results from the present study also showed an increase of Ce content in worms as CeO2 NP



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exposure increased (0% biochar treatments). Furthermore, our results showed that worms

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exposed to 1000 mg/kg of CeO2 NPs were found to have altered Ce accumulation when exposed

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to 0.5% and 5% biochar (350 °C and/or 600 °C). The only exception was in worms exposed to

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agricultural soil and BC-350, where Ce accumulation was unaffected by NP and biochar

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exposure (Figure 1A-D).

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It has been reported that earthworms can ingest and redistribute biochar particles (

Bioaccumulation of CeO2 Nanoparticles by Earthworms in Biochar

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