Carbonaceous Nanomaterials Have Higher Effects on Soybean

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Ecotoxicology and Human Environmental Health

Carbonaceous Nanomaterials Have Higher Effects on Soybean Rhizosphere Prokaryotic Communities During the Reproductive Growth Phase than During Vegetative Growth Yuan Ge, Congcong Shen, Ying Wang, Yao-Qin Sun, Joshua P Schimel, Jorge L. Gardea-Torresdey, and Patricia A. Holden Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00937 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Carbonaceous Nanomaterials Have Higher Effects

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on Soybean Rhizosphere Prokaryotic Communities

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During the Reproductive Growth Phase than During

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Vegetative Growth

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Yuan Ge1,2,3,4, Congcong Shen1, Ying Wang2,3,4, Yao-Qin Sun1, Joshua P. Schimel3,4,5, Jorge L.

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Gardea-Torresdey4,6, Patricia A. Holden2,3,4,*

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1

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Sciences, Chinese Academy of Sciences, Beijing 100085, China

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State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental

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Bren School of Environmental Science and Management, University of California, Santa

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Barbara, California 93106, United States

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3

12

States

13 14 15 16

Earth Research Institute, University of California, Santa Barbara, California 93106, United

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University of California Center for the Environmental Implications of Nanotechnology (UC

CEIN), University of California, Santa Barbara, California 93106, United States 5

Department of Ecology, Evolution and Marine Biology, University of California, Santa

Barbara, California 93106, United States

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Department of Chemistry, University of Texas at El Paso, El Paso, Texas 79968, United States.

*

Corresponding Author: [email protected]; Tel: 805-893-3195; Fax: 805-893-7612

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Abstract: Carbonaceous nanomaterials (CNMs) can affect agricultural soil prokaryotic

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communities, but how the effects vary with crop growth stage is unknown. To investigate this,

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soybean plants were cultivated in soils amended with 0, 0.1, 100, or 1000 mg kg-1 of carbon

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black, multi-walled carbon nanotubes (MWCNTs), or graphene. Soil prokaryotic communities

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were analyzed by Illumina sequencing at day 0 and at the soybean vegetative and reproductive

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stages. The sequencing data were functionally annotated using the Functional Annotation of

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Prokaryotic Taxa (FAPROTAX) database. The prokaryotic communities were unaffected at day

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0, and were altered at the plant vegetative stage only by 0.1 mg kg-1 MWCNTs. However, at the

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reproductive stage—when pods were filling—most treatments (except 1000 mg kg-1 MWCNTs)

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altered prokaryotic community composition, including functional groups associated with C, N,

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and S cycling. The lower doses of CNMs—which were previously shown to be less

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agglomerated and thus more bioavailable in soil relative to the higher doses—were more

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effective towards both overall communities and individual functional groups. Taken together,

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prokaryotic communities in the soybean rhizosphere can be significantly phylogenetically and

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functionally altered in response to bioavailable CNMs, especially when soybean plants are

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actively directing resources to seed production.

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TOC/Abstract Art

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Introduction

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Carbonaceous nanomaterials (CNMs), e.g., carbon black, carbon nanotubes, and graphene, are

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used in diverse commercial applications such as pigments, automotive tires, composite bicycle

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frames, antifouling coatings, solar cells, capacitors, and water filters.1-4 CNMs may be released

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into soils with field application of CNM-containing biosolids,5 or through the intentional use of

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CNM-containing agrochemicals (e.g., fertilizer products).6-8 The accumulation of CNMs in soils

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raises concerns about CNM effects on soil microorganisms—the main catalysts of soil nutrient

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cycling in terrestrial ecosystems.

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Most studies addressing CNM effects on soil microorganisms have been conducted in

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unplanted soil microcosms. Such studies have examined the effects of various CNMs including

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fullerenes,9-12 single-walled carbon nanotubes (SWCNTs),11, 13-17 multi-walled carbon nanotubes

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(MWCNTs),11, 17-21 graphene,21, 22 graphene oxide,23, 24 and carbon black.21 Results from these

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studies have been mixed, indicating that CNM effects on soil bacterial communities might be

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tempered by CNM properties, soil conditions, exposure times, exposure doses, and CNM types.

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For example, Tong et al. (2007) reported limited effects of fullerenes on soil microbial biomass,

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community structure, respiration, and enzymatic activities.10 In contrast, Johansen et al. (2008)

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found 20–30% of the community changed in response to fullerenes, although soil respiration and

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microbial biomass were unaffected.9 The magnitude of effects seems to vary with soil conditions;

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for example, fresh, unmodified SWCNTs altered microbial communities and metabolic activity

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in low, but not high, organic matter soils.13 CNM effects also change with exposure time as

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evidenced by short-term (3-4 days)

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CNM type may also relate to the magnitude of effects. Shan et al. (2015) found that biochar had

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no effect on catechol mineralization; however, activated carbon at all amendment doses (0.2, 20,

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and long-term (1 year)

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studies. Exposure dose and

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and 2000 mg kg-1) and SWCNTs at 2000 mg kg-1 significantly reduced mineralization, whereas

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MWCNTs at 0.2 mg kg-1 significantly stimulated mineralization.17 Oyelami et al. (2015) found

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higher glucose mineralization in fullerene amended soils, compared to MWCNT-, SWCNT-, or

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fullerene soot-amended soils.11 In a 1-year exposure experiment where the effects of multiple

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CNMs were compared, some treatments (e.g., biochar, carbon black, narrow MWCNTs, and

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graphene) altered bacterial communities when compared to the no amendment control, but there

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were no significant differences across the amendment treatments.21 CNMs differ in size,

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morphology, surface chemistry and other physiochemical properties; thus, they may undergo

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agglomeration, sorption, migration, and surface modification differently when they are released

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into soils.26, 27 Such physicochemical changes to CNMs could alter their exposures to organisms

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and thus observed effects including to soil microbial communities.

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While multiple studies have examined CNM effects on microbial communities in unplanted

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soils, little is known about CNM effects on microbial communities in planted soils.28 Yet

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exposing soil microbial communities to CNMs in mesocosms with plant cultivation represents a

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societally-relevant situation, where the interactions between crop plant roots and soils may

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modify nanomaterial effects on soil microbial communities and their associated functions.29, 30

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For example, plants exude into the soil 5−10% of their fixed carbon in the forms of sugars,

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amino acids, organic acids, mucilage and organic chelators,31, 32 which may modify nanomaterial

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bioavailability and toxicity.33, 34 Also, the quantity and composition of root exudates change with

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plant growth,

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CNMs on soil microbial communities during plant growth, including as a consequence of plant

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growth stage, are mostly unknown.

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which may further temper nanomaterial effects. However, the effects of

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Here, we studied the effects of carbon black, MWCNTs, and graphene on soil prokaryotic

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communities during the course of soybean plant growth. This work builds on a prior

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publication37 from the same mesocosm study in which soybean plants were cultivated to maturity

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in soils amended with 0, 0.1, 100, or 1000 mg kg-1 of either carbon black, MWCNTs, or

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graphene. In the previous publication, Wang et al. (2017) reported that all three CNMs affected

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soybean growth, nodulation, and dinitrogen fixation potential, with stronger effects more

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frequently observed at lower CNM doses. Through separate studies on CNM concentration-

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dependent agglomeration in soil water extracts, the authors demonstrated that the greater CNM

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agglomeration at higher CNM concentrations likely decreased CNM dispersal and bioavailability

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in the soil, and thereby decreased CNM effects on soybean plants and dinitrogen fixing

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symbioses (Supporting Information).37 This preceding publication provides the foundation for

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the present study. Here we asked: How did the same three CNMs affect rhizosphere prokaryotic

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communities? How did the effects on bacterial communities change with time during the course

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of soybean plant growth which itself was affected by CNM exposure?37 How did the effects on

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bacterial communities vary with CNM concentration, including if—as observed for soybean

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plants by Wang et al. (2017)37—there were stronger effects at lower CNM concentrations? To

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answer these questions, soil prokaryotic communities were analyzed at day 0, and at the

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vegetative (day 20), and reproductive (day 39) plant growth stages to compare the temporal

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variation of treatment effects. Prokaryotic taxa were mapped to metabolic or other ecologically

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relevant functional groups using the Functional Annotation of Prokaryotic Taxa (FAPROTAX)

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database, which allows for comparing CNM effects on individual functional groups at different

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soybean growth stages. The findings of this study newly show that CNMs have higher effects on

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soybean rhizosphere prokaryotic communities with associated functional groups during the plant

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reproductive growth phase relative to vegetative growth. Among the effects are changes to taxa

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associated with C, N, and S biogeochemical cycling. That such effects are more pronounced

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when soybean plants are actively directing resources to seed production is suggestive of the

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potential for ecosystem consequences of CNM exposure at late plant growth stages.

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Materials and Methods

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Soil. Surface soil (0-20 cm depth) was collected from the University of California Sedgwick

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Reserve (34°40’32”N, 120°2’27”W), sieved (4 mm) and stored (4 oC) for less than two weeks

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before the exposure experiment. Soil properties, including texture, pH, saturation, cation

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exchange capacity, soluble salts, organic matter, total nutrients (C, Cu, Fe, Mn, N, Zn),

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extractable nutrients (B, Ca, Cl, Cu, Fe, Mg, Mn, Na, P, Zn, HCO3-, CO32-, NH4+, NO3-), and

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exchangeable nutrients (Ca, K, Mg, Na), were characterized by the UC Davis Analytical

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Laboratory (Davis, CA; http://anlab.ucdavis.edu/) and reported previously.37 The soil is a Pachic

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Argiustoll in the Botella series, with a sandy clay loam texture (50% sand, 25% silt, and 20%

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clay) containing 3.03% organic matter, 1.53% total C, and 0.15% total N; the pH was 7.38.37

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Carbonaceous Nanomaterials (CNMs). The three CNMs used in this study had distinct

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morphologies and sizes.37, 38 Carbon black (Printex 30, Orion Engineered Carbons, Kingwood,

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TX) was spherical, 36.6 ± 8.3 nm diameter; MWCNTs (Cheap Tubes, Grafton, VT) had outer

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diameters of 18.8 ± 4.1 nm; graphene (Cheap Tubes, Grafton, VT) was a two-dimensional sheet

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with an average diameter of 350 ± 320 nm and a thickness of 8-12 nm.37, 38 The three CNMs had

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high purities, as reflected by low non-carbon impurities (< 2.2% for all) and low metal contents

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(< 0.1% for all metals measured, except 0.9% of nickel in the MWCNTs).37 The primary

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oxidation temperatures (an indicator of thermal stability) for CB, MWCNTs, and GNPs differed

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and were approximately 620, 585, and 623 °C, respectively.37

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Soybean Seedlings. As described before,37 to cultivate soybean seedlings, soybean seeds

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(Glycine max, Midori Giant variety, Lot No. WA15060001, Park Seed Co., Hodges, SC) were

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soaked in a Bradyrhizobium japonicum USDA 110 inoculum (1.0 optical density at 600 nm) for

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10 min. Then, the inoculated seeds were sowed into rehydrated peat-filled seed starter pellets

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(Park Seed Co., Hodges, SC), with an additional 100 µL of the B. japonicum inoculum dispensed

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onto the sowed seeds. The pellets were watered daily and incubated on a heating mat (23 °C) for

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ten days before seedling transplantation.37

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Exposure Experiment. To homogenously distribute CNMs into soils, a previously reported 39

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10-fold dilution method

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mg kg-1 for either carbon black, MWCNTs, or graphene). The exposure doses were chosen,

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based on the evaluation of exposure concentrations in previous publications, to represent a range

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of possible environmental exposures: the predicted environmental concentration, the possible

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presence of CNM hotspots in soil, and a relatively high exposure concentration to explore

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potential toxicity.3, 40, 41 In brief, powder nanomaterials were initially added to soil in doses of

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0.01, 10, and 100 g kg-1, and mechanically mixed for 10 min with handheld kitchen mixers. Then

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each mixture was diluted 10-fold twice using unspiked soils, and mechanically mixed each time

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as described above. Soil without CNMs was used as the control. After mixing, triplicate soil

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samples of each treatment were immediately subsampled, denoted as 0-day samples, and frozen

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at -80°C for later DNA-based analyses.

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was used to achieve the targeted exposure doses (0.1, 100, and 1000

For each of the ten treatments (control, and three doses of each CNM), eight experimental pots were prepared following the procedures reported previously.37, 39 In each experimental pot (2.84

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L container with bottom perforations, high density polyethylene) there was a layer of

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polyethylene WeedBlock fabric (Easy Gardener Products, Waco, TX) at the bottom, and then 0.4

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kg of washed gravel on top to maintain drainage. Then, 2.3 kg of soil in a perforated

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polyethylene bag (40 holes) was overlain on the gravel. One soybean seedling was transplanted

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into each experimental pot. Each seedling was inoculated with B. japonicum (10 mL, prepared as

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above) during transplantation to ensure effective inoculation.37

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The planted pots were placed in a greenhouse for up to 39 days wherein the soybean plants had

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grown to full maturity (before senescence). The greenhouse was under full sunlight, with daily

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temperatures ranging from 15oC to 34oC, and photosynthetically active radiation (PAR)

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fluctuating between 21 and 930 µmol m-2 s-1. The soil water content averaged 0.25 m3 m-3 by

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watering the experimental pots with tap water.37

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Soil Sampling. During the exposure experiment, soil and plant samples were destructively

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harvested twice based on soybean plant growth stages: vegetative (20 days post transplantation,

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before flowering) or reproductive (39 days post transplantation, full seed production, before

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senescence).37 For each of the ten treatments, three and five replicates were sacrificed at the

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vegetative and reproductive stages, respectively. This sampling strategy minimized the

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possibility of insufficient experimental replicates (i.e., less than 3) at the second sampling time,

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in case of plant death. As reported previously, plant samples were examined for CNM effects.37

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For this study, soil samples were preprocessed by manually removing most main roots and

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sieving (2 mm) out any remaining fine roots. The sieved soils were stored at -80o C for later

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DNA extraction to characterize soil prokaryotic communities. Because the plant roots extended

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throughout the pots and were of similar masses at the intermediate and reproductive growth

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stages,37 all soils were rhizospheric.

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Soil DNA Extraction and Illumina Sequencing. Total DNA was extracted from 0.3 g of

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subsampled soil from each of the replicates, for each treatment and sampling time, using the

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Powersoil DNA Isolation Kit (Mo Bio, Carlsbad, CA, USA). The sequencing library was

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prepared by the UC Santa Barbara Biological NanoStructures Lab

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(https://www.cnsi.ucsb.edu/resources/facilities/bnl/ngs-core) following a standard protocol. In

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brief, prokaryotic primers 341F (CCT ACG GGN GGC WGC AG) and 805R (GAC TAC HVG

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GGT ATC TAA TCC) were used to amplify the V3-V4 region of genes encoding 16S rRNA for

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each DNA sample, with each primer set containing a unique barcode. The unique barcodes were

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used to assign sequences to samples post-sequencing. After size and quality verification by

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TapeStation (Agilent, Santa Clara, CA), PCR products from each sample were quantified using a

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Qubit Fluorometer (Invitrogen, Eugene, OR) and equally pooled by mass. The pooled PCR

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products were shipped to the UC Riverside IIGB Genomics Core facility

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(http://illumina.ucr.edu/ht/) on dry ice for paired-end sequencing with 300 cycles (PE300) on a

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MiSeq platform (Illumina, San Diego, CA). There were four sequencing outliers defined as

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having a low sequencing depth (< 8500 sequence counts): two were from the medium and high

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doses of the MWCNT treatment at day 0, and the other two were from the medium and high

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doses of the carbon black treatment at the reproductive stage (day 39). These outliers were not

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included in the statistical analyses (Table S1).

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Sequence

Preprocessing.

Sequences

were

preprocessed

using

QIIME

software

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(http://qiime.org/).42 Briefly, all sequences that passed the quality controls of the MiSeq platform

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were assigned to samples by examining the unique barcodes, and the primers were trimmed.

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Then, the forward and reverse sequences from the same sample were merged based on the

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overlap between them (10 bp of minimum overlap). The merged sequences were further screened

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to remove low quality sequences that had an average quality score of < 20 and contained any

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ambiguous characters. After initial trimming and screening, the PCR chimeras were removed,

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and similar sequences were clustered into operational taxonomic units (OTUs, a cutoff

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dissimilarity of 0.03) using the “usearch” method. Because the number of final qualified

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sequences varied according to the sample, the sequence counts of all samples (i.e., 15000

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sequence counts) were similarly rarefied through a random subsampling process to increase the

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reliability of community comparisons across samples. The rarefied sample-OTU matrix was used

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for the OTU-based community analysis. The OTU abundance was counted as the number of

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sequences that clustered into a specific OTU. The community richness was estimated by the

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number of observed OTUs per sample.

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The OTUs were assigned to a set of hierarchical taxa (phylum, class, order, family, and genus)

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using the Ribosomal Database Project (RDP) Classifier. Then the taxa were classified to

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different functional groups using the Functional Annotation of Prokaryotic Taxa (FAPROTAX)

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database (http://www.zoology.ubc.ca/louca/FAPROTAX/). The FAPROTAX is a software tool

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designed to map prokaryotic taxa (e.g., genera or species) to established metabolic or other

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ecologically relevant functions based on the functional descriptions of cultured strains in the

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peer-reviewed literature.43 In FAPROTAX, functional groups may be nested since a taxon may

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be affiliated with multiple functions; for example, all taxa associated with nitrate denitrification

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are also associated with nitrate respiration and nitrate reduction. Currently, the FAPROTAX

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includes 90 functional groups covering the main processes of C, N, and S cycling (e.g.,

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cellulolysis, methanotrophy, methanogenesis, dinitrogen fixation, nitrification, denitrification,

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sulfur oxidation and respiration; Table S2).

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Statistical Analysis. After testing the normality and variance homogeneity, one-way analysis

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of variance (ANOVA) was performed to test the global effect of exposure dose on measured

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variables at each sampling time. Where the global ANOVA was significant (P < 0.05), a post-

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hoc least significant difference (LSD) test was further conducted to test the significance (P
0.18 for all pairs, Figure 1a), by the

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convergence of 0-day treatments in the PCoA graph (Figure 2a), and by the PERMANOVA test

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of community shifts (P > 0.09, Table S3). These results corroborate other studies,21, 45 indicating

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the negligible effects of CNM exposure on DNA extraction and bacterial community analysis

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methods.

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The CNM effects became more substantial with soybean growth (exposure time). At the

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vegetative stage (day 20), only the low dose of MWCNTs significantly changed soil prokaryotic

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communities (P < 0.05, Figure 1b). At the reproductive stage (day 39), almost all CNM-amended

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treatments significantly affected soil prokaryotic communities (P < 0.05 for all except the high

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dose of MWCNTs, Figure 1c). This was also supported by the PCoA graphs (Figure 2) and the

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associated PERMANOVA test (Table S3), showing the greater separation of CNM-amended

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treatments from the control at the reproductive stage when compared to day 0 and the vegetative

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stage.

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A similar time-dependent trend was observed for community richness. The community

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richness was not significantly affected by any of the CNM treatments at either day 0 (P > 0.39)

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or the vegetative stage (20 days, P > 0.12), but the CNM effects tended to be more distinct after

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39 days at the soybean reproductive stage (Figure 3). At that later sampling, community richness

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was significantly reduced by the medium and high doses of carbon black (10.7% and 9.6%), as

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well as the medium dose of MWCNTs (12.2%, P < 0.05, Figure 3c). These three treatments were

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also the treatments that caused the most distinct community shifts (Figure 1c), indicating that the

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CNM-induced richness reduction might explain the community shifts.

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Exposure time has been found to be an important factor in modulating nanomaterial effects on

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soil microbial communities in unplanted soil microcosms. For example, functionalized SWCNTs

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and graphene have been found to cause more distinct effects on soil bacterial communities at the

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early stages of exposure (3-4 days),14, 22 while another study reported the effects of carbon black,

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narrow MWCNTs, and graphene on soil bacterial communities even after 1-year exposure.21

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Previous studies focusing on metal oxide nanoparticles also found that the effects on soil

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bacterial communities increased with exposure time.45,

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studies may be partially attributed to differences in nanomaterial types, soil properties, associated

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communities, and experimental conditions. In this study, because soybean plants were cultivated

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in the exposure experiment, it was also possible that the observed time-dependent CNM effects

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on soil bacterial communities were due to soybean plant growth effects. Plants exude into the

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soil 5−10% of their fixed carbon which may modify nanomaterial bioavailability and toxicity.33,

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34

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growth,35, 36 further altering nanomaterial effects. Besides the natural variations of root exudates

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with plant growth, CNMs may have affected the quantity and composition of root exudates by

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affecting plant growth,37 which in turn caused the time-dependent effects on soil prokaryotic

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communities. In addition, the CNMs, e.g., carbon black, may be oxidized after deposition,47

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which may also contribute to the effects. Although it is difficult to differentiate whether the

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cause is purely time—independent of live plants—versus plant mediation, our results showed

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that CNMs had higher effects on soybean rhizosphere prokaryotic communities during the

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reproductive growth phase (day 39)—the most crucial stage of plant development in terms of

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seed yield, when compared to the vegetative growth period (day 20).

46

The different time effects between

Also, the quantity and composition of root exudates may have been changed with soybean

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CNM Effects Vary with CNM type. Besides the time-dependent CNM effects, we also found

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that the three CNMs had different effects on soil prokaryotic communities. For example, only the

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low dose of MWCNTs induced significant shifts of soil prokaryotic communities at the soybean

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vegetative stage (day 20, P < 0.05, Figures 1b). At the soybean reproductive stage (day 39),

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although almost all CNM treatments clearly affected soil prokaryotic communities (P < 0.05

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except the high dose of MWCNTs), the effects differed in magnitude, as reflected by varied

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community dissimilarities induced by different CNM treatments (Figure 1c). For example, the

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community dissimilarities between the control and carbon black or MWCNT treatments (except

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the high dose of MWCNTs) were around 1.9 times of the within-control community dissimilarity,

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while the community dissimilarities between the control and graphene treatments were around

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1.2 times of the within-control community dissimilarity (Figure 1c). This was also illustrated by

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the PCoA graphs: the samples exposed to carbon black or MWCNTs (except the high dose of

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MWCNTs) separated from the control more clearly than those from the graphene treatments

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(Figure 2c, d, e).

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These results indicate that the effects of CNMs on soil microbial communities are driven by

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the type of material. In monocultures, metal oxide nanomaterials have been demonstrated to be

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different in their toxicity to cell lines and microorganisms, yet nano-ZnO and nano-CuO appear

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to be more toxic than other metal oxide nanoparticles.48-50 Herein, the differences in CNM effects

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among different types of CNMs could derive from differing toxicity mechanisms. The toxic

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mechanisms include membrane disorganization, surface coating-related photocatalytic oxidation

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and associated cell damage, toxic ion release, and cell-damaging reactive oxygen species (ROS)

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accumulation.51, 52 The specific toxicity mechanisms could be related to CNM physicochemical

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properties (e.g., size and shape), which could vary with CNM type. For example, MWCNTs

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were more toxic to Bacillus subtilis than graphene,53 which is consistent with our observation

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that MWCNTs were more effective on soil prokaryotic communities than graphene. Even when

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comparing within each type of CNM, the differences in particle size have been shown to alter the

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toxicity to microorganisms. For example, the antimicrobial activities of graphene oxide sheets

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were reported to vary with sheet size.54 Meanwhile, the variation in CNM effects could also be

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attributed to different CNM exposures due to differing CNM agglomeration behaviors in soil,

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and thus, differing bioavailabilities. In a prior publication

from the same mesocosm study,

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Wang et al. (2017) examined the relationships between CNM concentrations and agglomeration

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in soil extracts (Supporting Information). Graphene was found to be significantly less stable in

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soil water extracts than carbon black at the same concentration (Table S4).37 Therefore, it is

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possible that more extensive agglomeration of graphene significantly decreased its

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bioavailability in soil and mitigated toxicity-related effects on soil prokaryotic communities.

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Consequently, both the colloidal stability of CNMs in soil water and their differing toxicity

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mechanisms (likely related to their physicochemical properties) are critical to the bioavailability

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and effects of CNMs in soils.

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CNM Effects Vary with Exposure Dose. We found that the low or medium doses of CNMs

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tended to cause the most distinct effects on soil prokaryotic communities. For example, at the

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vegetative stage, only the low dose of MWCNTs significantly altered soil prokaryotic

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communities (P < 0.05, Figure 1b). At the reproductive stage, although all doses of carbon black

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and graphene significantly affected soil prokaryotic communities (P < 0.05), the medium dose of

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carbon black and low dose of graphene showed the highest effects (Figure 1c). In addition, with

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MWCNTs, the lower doses significantly changed soil prokaryotic communities (P < 0.05), while

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the high dose did not significantly affect soil prokaryotic communities (P = 0.79, Figure 1c).

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These results indicate that the CNM effects on soil prokaryotic communities did not follow the

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typical dose-response relationship in which the response increases with toxicant dose.

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Previously, Wang et al. (2017) showed that the low dose of MWCNTs inhibited soybean

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growth, as reflected by shorter plants, slower leaf cover expansion, and less final leaf area.37

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Further, CNMs negatively affected nodulation and dinitrogen fixation potential, with stronger

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effects at lower CNM doses.37 To explain the observed inverse dose-response relationships,

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Wang et al. (2017) conducted separate experiments to examine the effect of CNM concentration

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on CNM dispersal in soil water, and thus on CNM bioavailability in soil.37 Specifically, they

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studied CNM stability at two CNM concentrations (10 and 300 mg L-1) in water extracts of the

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control soil (Supporting Information).37 The two CNM concentrations studied were relevant to

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the CNM concentrations in the soil solution of the mesocosm experiment (Supporting

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Information). By measuring CNM agglomeration and sedimentation dynamics, and imaging

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CNMs using environmental scanning electron microscopy, Wang et al. (2017) found that CNM

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agglomeration increased with increasing CNM concentration (Table S4).37 The strength of CNM

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effects depends partly on CNM bioavailability (defined as “the accessibility of a chemical for

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assimilation and possible toxicity” 55), which in turn is affected by CNM colloidal stability in soil

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pore water.56 CNM stability in soil water could determine the amount (i.e., available dose) and

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physicochemical characteristics (e.g., size and shape) of CNMs that would disperse in soil water

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and directly interact with plant roots and soil microorganisms. At higher CNM concentrations,

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greater CNM agglomeration resulted in increased average sizes of CNM agglomerates (shown as

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the larger hydrodynamic diameters) and decreased CNM concentrations (reflected by the lower

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derived count rates) remaining in soil water (Table S4).37 Therefore, CNM bioavailability and

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observed effects were reduced at higher CNM concentrations.37

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In the present study, focused on soil prokaryotic communities, we found that lower doses of

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CNMs caused more distinct effects on soil prokaryotic communities. This result may be

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explained by higher dispersion-mediated bioavailability of inherently toxic CNMs, similarly to

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the effects on soybean plants as was concluded in the previous study.37 The trend was better

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evidenced at the reproductive plant growth stage, i.e. after 39 days of plant growth (Figures 1c

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and 3c). If this was an effect of growth stage, rather than merely time, then the effect could

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derive from plants exerting a differential influence on rhizosphere prokaryotic communities if

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plants changed their root physiology while adjusting resource allocation during pod filling.

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Functional Implications of Prokaryotic Community Shifts Responding to CNM Exposure.

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We used Functional Annotation of Prokaryotic Taxa (FAPROTAX) to map prokaryotic clades

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(e.g. genera or species) to metabolic or other ecologically-relevant functions. Among the 90

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functional groups in the FAPROTAX database, 69 groups (76.7%) were present in at least one of

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the samples (Table S2). These functional groups included taxa (Table S5) associated with

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processes in the biogeochemical cycling of C (e.g., cellulolysis, ligninolysis, methanotrophy,

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methanogenesis, hydrocarbon degradation), N (e.g., dinitrogen fixation, nitrification,

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denitrification), S (e.g. the oxidation and reduction of sulfur, sulfite, and sulfate), and other

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elements (e.g., the oxidation and reduction of iron, manganese, and arsenic, Table S2).

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Pearson correlations between prokaryotic community dissimilarities, and the relative

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abundance of each functional group for each CNM type at each sampling time, were examined.

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This allowed determining the functional groups that significantly (adjusted P < 0.05) contributed

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to the community shifts. We further examined how these functional groups were altered by CNM

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exposure using ANOVA in conjunction with an LSD test. None of the 69 functional groups

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represented in the samples was significantly correlated with bacterial community shifts for all

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three CNM types at either day 0 or the vegetative stage (adjusted P > 0.05 for all, Table 1).

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However, at the reproductive stage, 18, 25, and 5 functional groups, whose relative abundances

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were significantly correlated to the community shifts (adjusted P < 0.05, Table S6), were also

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significantly altered by carbon black, MWCNTs, and graphene, respectively (P < 0.05, Figures

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4-6). Therefore, these functional groups were defined as sensitive functional groups (Table 1).

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These results (i.e., more sensitive functional groups at the reproductive stage) mirrored the

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community analysis results, showing that key functional groups affected by CNMs were changed

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more at the soybean reproductive stage (Table 1). Further, the significant correlations (Table S6)

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suggested a linkage between community shifts induced by CNM exposure and functional shifts.

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This result may appear intuitive, given that affected functional groups are determined from

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analyzing the phylogenetic data using the FAPROTAX database. However, given that less than 1%

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of all microbial community taxa have been studied in culture,57 the functional implications in the

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prokaryotic community shifts herein could be larger than what can be currently inferred using the

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FAPROTAX database.

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In this study, sensitive functional groups were not related linearly to NM dose, differently from 29

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previous studies of metal oxide ENM effects on bacterial communities in soils with

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without 46 plants. Herein, the medium doses of carbon black and MWCNTs, and the low dose of

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graphene, tended to cause the highest effects on soil prokaryotic communities at the soybean

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reproductive stage (Figures 1 and 2). Consistently, almost all of the sensitive functional groups

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were significantly (P < 0.05) affected by the medium dose of carbon black (100% of the

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sensitive functional groups, Figure 4) and MWCNTs (96%, Figure 5), and the low dose of

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graphene (100%, Figure 6), while fewer functional groups were significantly altered by other

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treatments (Table 1).

and

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Interestingly, some of the functional groups associated with C, N, and S cycling were

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significantly changed at the reproductive stage with some groups increasing while others

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decreasing. For example, with carbon black exposure, 9 of the 18 sensitive functional groups,

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such as those associated with methanogenesis, chitinolysis, aerobic nitrite oxidation, and nitrate

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reduction, were significantly decreased (by 27-74%, P < 0.05), while functional groups

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associated with cellulolysis, xylanolysis and aerobic ammonia oxidation were significantly

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increased (P < 0.05, Figure 4). Of the 25 sensitive functional groups in the MWCNT treatments,

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15 functional groups—including those associated with nitrification (aerobic nitrite oxidation),

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denitrification (nitrate, nitrite, and nitrous oxide denitrification), nitrogen respiration (nitrate and

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nitrite respiration), and anoxygenic photoautotrophic S oxidation—were significantly decreased

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due to exposure to a certain dose (by 22-70%, P < 0.05, Figure S1), while the functional groups

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associated with chitinolysis, xylanolysis and sulfur respiration were significantly increased (P