Nanomaterials in Biosolids Inhibit Nodulation, Shift Microbial

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Nanomaterials in biosolids inhibit nodulation, shift microbial community composition, and result in increased metal uptake relative to bulk/dissolved metals Jonathan D Judy, David H. Mcnear, Chun Chen, Ricky W. Lewis, Olga V. Tsyusko, Paul M Bertsch, William Rao, John P Stegemeier, Gregory Victor Lowry, Steve P. Mcgrath, Mark Durenkamp, and Jason M Unrine Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01208 • Publication Date (Web): 10 Jun 2015 Downloaded from http://pubs.acs.org on June 23, 2015

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Environmental Science & Technology

Nanomaterials in biosolids inhibit nodulation, shift microbial community composition, and result in increased metal uptake relative to bulk/dissolved metals Jonathan D. Judy1, 2, 3, 4*, David McNear1,2, Chun Chen1, 2,3, Ricky W. Lewis,1,2, Olga V. Tsyusko1, 2, 3

, Paul M. Bertsch1, 2, 3, 5, William Rao1, John Stegemeier2, 6, Gregory V. Lowry2 3, 6, Steve P. McGrath 2,7, Mark Durenkamp2,7 and Jason M. Unrine1, 2, 3* 1

Department of Plant and Soil Sciences, University of Kentucky, Lexington KY, 40546, USA 2

3

Transatlantic Initiative for Nanotechnology and the Environment (TINE)

Center for the Environmental Implications for Nanotechnology (CEINT), Duke University, Durham, NC 27708, USA 4 5 6

CSIRO Land and Water, Waite Campus, Urrbrae, South Australia, 5064, Australia

CSIRO Land and Water, 41 Boggo Road, Ecosciences Precinct, Dutton Park, Queensland, 4102, Australia

Department of Civil & Environmental Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA 7

Department of Sustainable Soils and Grassland Systems, Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom

*

To whom correspondence may be addressed

Jason M. Unrine, [email protected], 859-257-1657, 1100 S. Limestone St., Lexington , KY 40546

1 ACS Paragon Plus Environment


1

ABSTRACT

2

We examined the effects of amending soil with biosolids produced from a pilot-scale wastewater

3

treatment plant containing a mixture of metal-based engineered nanomaterials (ENMs) on the

4

growth of Medicago truncatula, its symbiosis with Sinorhizobium meliloti, and on soil microbial

5

community structure. Treatments consisted of soils amended with biosolids generated with (1)

6

Ag, ZnO, and TiO2 ENMs introduced into the influent wastewater (ENM biosolids), (2) AgNO3,

7

Zn(SO4)2, and micron-sized TiO2 (dissolved/bulk metal biosolids) introduced into the influent

8

wastewater stream, or with (3) no metal added to influent wastewater (control). Soils were

9

amended with biosolids at a rate intended to simulate 10 years of biosolids applications which

10

resulted in nominal metal concentrations of 1450, 100, and 2400 mg kg-1 Zn, Ag, and Ti,

11

respectively, in the dissolved/bulk and ENM treatments.

12

significantly higher in the plants grown in the ENM treatment (182 mg kg-1) compared to those

13

from the bulk treatment (103 mg kg-1). Large reductions in nodulation frequency and plant

14

growth, as well as soil microbial community composition were found ENM treatment compared

15

to the bulk/dissolved metal treatment. These results suggest differences in metal bioavailability

16

and toxicity between ENMs and bulk/dissolved metals at concentrations relevant to regulatory

17

limits.

Tissue Zn concentrations were

18 19


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20


INTRODUCTION

21

Engineered nanomaterials (ENMs) are entering waste streams in increasing quantities as the

22

result of their use in an increasing variety of consumer products employing nanotechnology.1

23

The majority of these ENMs have been shown to partition to the sludge within wastewater

24

treatment plants (WWTP) and there is a risk of environmental harm in agroecosystems where

25

biosolids are land-applied as fertilizer which has yet to be fully evaluated.2 While regulations

26

exist that limit the land application of biosolids that contain elevated concentrations of certain

27

metals, these regulations do not specifically consider the incorporation of metal-containing

28

nanomaterials.3

29

Ag, TiO2, and ZnO ENMs are some of the most commonly used ENMs in consumer products.

30

Ag ENMs have widely documented anti-bacterial properties and have been incorporated into

31

many products such as bandages, paint, and clothing to inhibit the growth of bacteria.4-7 TiO2

32

and ZnO ENMs have broad UV-blocking properties and as a result, are widely used in sunblock

33

and sunscreen, as well as pharmaceuticals, paint, coatings and other products. 8, 9 TiO2 ENMs are

34

also photocatalytic and are being incorporated into building facades to degrade air pollutants. As

35

a result of these applications, Ag, TiO2, and ZnO ENMs are predicted to be among the most

36

prevalent ENMs within WWTP sludge.10

37

In the past few years, a number of studies have examined the phytotoxicity and

38

bioaccumulation of a variety of ENMs to different plant species.11, 12 The results of these studies

39

have been, in many cases, contradictory, likely at least in part due to differences in exposure

40

methods and in the biology between plant species.11 Also, the overwhelming majority of these

41

studies have been done in hydroponics and the literature examining phytotoxicity and/or

42

bioaccumulation of ENMs from soil is limited.13

As the degree to which plants can



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43

bioaccumulate ENMs has important implications for organisms at higher trophic levels in

44

terrestrial food webs including humans, a need exists to develop our understanding of the

45

bioavailability of ENMs to plants under environmentally realistic conditions.

46

There have also been a relatively large number of in vitro studies conducted examining the

47

anti-microbial activity of ENMs to various pathogenic bacteria and fungi.14, 15 Some of these

48

studies presumably formed the basis for the previously mentioned introduction of Ag ENMs into

49

consumer products.

50

beneficial soil microbes, such as nitrogen-fixing bacteria, arbuscular mycorrhizal fungi (AMF),

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or plant growth promoting rhizobacteria (PGPR), is limited. Considering the critical ecosystem

52

services that beneficial microorganisms deliver and the possibility that they may be more

53

sensitive to soil contaminants than higher organisms,16 a need exists to further examine how the

54

accumulation of ENMs in soil might affect these organisms.

However, the number of studies examining the impact of ENMs on

55

Furthermore, ENMs are transformed into end products fundamentally different from their

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original pristine state during processing within WWTPs.17-19 For example, the conversion of Ag

57

ENMs to Ag2S ENMs has been demonstrated in several studies and Ag2S ENMs have even been

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found within municipal WWTP sludge.20, 21 More recently, Ma et al. (2013) demonstrated that

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Ag ENMs transform to Ag2S under a wide variety of WWTP conditions and biosolid treatments

60

(e.g. liming, heat treatment, and composting).19 In the same study, ZnO ENMs were found to

61

transform into a combination of ZnS, Zn-ferrihydrite, and Zn3(PO4)2.

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overwhelming majority of studies to date, including those currently published on beneficial soil

63

microorganisms, have been conducted using as-manufactured, pristine ENMs, and very little

64

information is available with which to evaluate the risk to beneficial soil microorganisms or

65

terrestrial ecosystems posed by ENMs under more realistic exposure scenarios.


However, the

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In this study, we used total metals analysis of plant tissues coupled with X-ray fluorescence

67

mapping to examine the bioaccumulation of Ag, TiO2, and ZnO ENMs by Medicago truncatula

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grown in biosolid amended soil.

69

containing ENMs affects legume-rhizobium symbiosis and soil microbial communities. In a

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companion paper, we explore possible toxicity mechanisms by describing gene expression

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signatures and their associated biological pathways identified from the whole genome microarray

72

analyses using the plant tissues.22 These experiments were conducted using biosolids generated

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by introducing ENMs into influent of a pilot scale WWTP, thus subjecting the ENMs to similar

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chemical and physical processing as would occur in a municipal scale WWTP, resulting in test

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organisms being exposed to representative ENM transformation end products. The design and

76

operation of the model WWTPs, metal dosing method and characterization of the resulting

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biosolids have been previously described.19 The experiment was designed to simulate a worst

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case exposure scenario of a mixture of these three common ENMs, dosed based on the current U.

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S. EPA cumulative pollutant loading limit for Zn in biosolids-amended soils, as defined in the

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Guide to the Biosolids Risk Assessments for the EPA, Part 503 document and known

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concentrations of these metals in sewage sludge in the United States. This cumulative pollutant

82

loading limit for Zn corresponded to approximately the 97th percentile of Zn concentrations in

83

the U.S. Targeted National Sewage Sludge survey (TNSS).3

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concentrations had similar concentration percentiles from the TNSS since these materials are not

85

regulated in biosolids in the U.S.

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MATERIALS AND METHODS

87 88

Soil-sludge mixtures.

We also investigated how amending soil with biosolids

The chosen Ag and TiO2

Woburn sandy soil (Arenosol, FAO; Typic Udipsamment, US),

previously described by McGrath et al. was sieved 0.33, p 8 fold decrease in nodulation frequency observed in the ENM treatment compared to the

251

bulk/dissolved metal treatment was striking given that the concentrations and XAS-based

252

speciation of the metals in the bulk/dissolved metal and ENM treatments were similar.19

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Considering the role that nodulation plays in plant N acquisition, the reduced growth observed in

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the plants exposed to the ENM treatment is not surprising. We initially hypothesized that the

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reduction in nodulation was the result of either direct toxicity of the ENMs to S. meliloti or

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possibly differences in N species concentrations between the different treatments, as higher N

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content in the ENM treatment may have reduced the plant demand for N resulting in reduced

258

nodulation.

259

bulk/dissolved metal and ENM media (Table 1, Table S2). Also, although we inoculated the

260

plants with S. meliloti, it is possible that the nodulation observed in the control and

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bulk/dissolved treatment was the result of native N-fixing bacteria which were inhibited by the

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ENMs.

However, concentrations of S. meliloti, NH4+, and NO3- were similar in the

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Alternatively, we hypothesize that the inhibition may be the result of metals, specifically Zn,

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in the ENM media being more bioavailable than those in the bulk/dissolved metal media.

265

However, estimates of operationally defined bioavailable metal fractions from extracts of the

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media suggest that metals were not more available in the ENM treatment compared to the

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bulk/dissolved metal treatment (see supporting information; Table S1). These extractants also

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did not reveal a difference in the metal dissolution in the media since the differences between the

269

colloidal and dissolved fractions of the extracts did not differ by treatment. Despite this, total Zn

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concentrations in the plant shoots were significantly higher in plants from the ENM treatment



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compared to those from the bulk/dissolved metal treatment, suggesting that the operationally

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defined extraction methods used were unable to capture differences in bioavailability between

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the ENM and bulk/dissolved metal treatments and highlighting the importance of pairing

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biological uptake to media extractions to adequately assess the bioavailability of a substance.

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Results from the companion study indicate that the decrease in nodulation in the ENM versus

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the control and bulk/dissolved treatment coincides with down regulation of several genes related

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to nodulation and up regulation of genes related to flavonoid biosynthesis (involved in signaling

278

of rhizobia for nodulation) and oxidative stress and metal tolerance.22 Taken together, the

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increased bioaccumulation of Zn in the ENM treatment relative to the bulk/dissolved treatment

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and changes in gene expression suggest that increased uptake of Zn may have caused adverse

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effects in the plant that resulted in suppression of nodulation rather than an impact on the

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viability of the S. meliloti in the soil. Additionally, previous studies have demonstrated that M.

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truncatula nodulation is particularly sensitive to Zn exposure.31, 32

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This study is not the first to report inhibition of nitrogen-fixing bacteria by ENMs. Kumar et

285

al. (2011) reported that Bradyrhizobium canariense growth was reduced by exposure to pristine

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Ag ENMs.33

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with Bradyrhizobium japonicum was not affected by ZnO or CeO2 ENMs at concentrations up to

288

500 and 1000 mg kg-1, respectively, the presence of CeO2 ENMs in concentrations greater than

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500 mg kg-1 greatly inhibited the N fixation potential of root nodules.34

Priester et al. (2012) reported that while nodulation rate in soybeans inoculated

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Reports have also indicated that pristine ENMs may alter soil microbial communities. Ge et

291

al. (2011) reported that the presence of pristine TiO2 and ZnO ENMs reduced soil extractable

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DNA and shifted soil microbial communities.35

293

and TiO2 ENMs affect microbial communities in flooded paddy soils.36 Shah et al. (2014)

Recently, Xu et al. (2015) reported that CuO


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reported that while ZnO and Cu0 ENMs did not affect soil microbial communities, a mixture of

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Ag and TiO2 did and that the microbial community response varied with time.37 Simonen et al.

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(2015) examined the effects of TiO2 ENMs on soil microbial communities in six different soils.38

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These researchers only observed toxicity in one of the soils, a sandy clay loam, and suggested

298

that the toxicity was largely driven by pH and soil organic matter.

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demonstrated that sulfidized Ag ENMs introduced into wetland mesocosms via biosolids

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amendment had significant impacts on microbial community composition and function at

301

environmentally relevant (sub mg kg-1) concentrations.39

Colman et al. (2013)

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As previously mentioned, the number of studies examining the effects of ENMs on beneficial

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soil bacteria is small compared to the number examining the effects of ENMs on pathogenic

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microorganisms. Calder et al. (2012) reported that the presence of humic acid in soil pore water

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reduced the toxicity of pristine Ag ENMs to the PGPR Pseudomonas chlororaphis. Toxicity

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was otherwise largely governed by the presence of Ag ions.40 Another study examining the

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transport of quantum dots by soil mycorrhiza into Poa annua provided evidence that mycorrhiza

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may be involved with trafficking ENMs into plant roots.41 Bandyopadhyay et al. (2012) reported

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that pristine ZnO and, to a lesser degree, CeO2 ENMs were toxic to the Sinorhizobium meliloti in

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vitro.42

311

However, unlike the present study, except for the work by Colman et al., the above studies

312

were conducted using pristine ENMs added directly to soil or through hydroponic exposures.

313

Considering that pristine ENMs will be transformed into greatly different end products by the

314

time they are introduced into terrestrial ecosystems, it is unclear how effects observed in those

315

studies translate to transformed materials.43

316

lacked appropriate bulk/dissolved controls and/or have not attempted to determine the

Furthermore, many of the previous studies have



317

contribution to toxicity of ions from ENM dissolution. By introducing ENMs into pilot WWTP

318

influent and using sludge generated during a pilot-scale WWTP process for our exposures, we

319

have generated treatments containing ENMs that closely represent the ENM transformation end

320

products likely to be introduced into terrestrial ecosystems via land application of biosolids. It is

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also important to note that the microbial communities in the biosolids may have differed due to

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ENM or bulk/dissolved treatment prior mixing with soil.

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It has been suggested that because the speciation of transformation products from

324

bulk/dissolved metals is similar to nano-metals in sewage sludge, the toxicity should be

325

equivalent.18,

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determined by XAS spectra or by operationally defined extractant methods may not adequately

327

predict toxicity of ENMs. XAS, as conducted here, measures the oxidation state and average

328

coordination environment of the metal centers in the sample and as such is an Angstrom scale

329

characterization. There are many other aspects of the structure of the transformation products on

330

the nano/micro scale that it does not measure. For example, Zn3(PO4)2 has been identified as a

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major transformation product of ZnO during wastewater treatment.18, 19 The Zn3(PO4)2 particles

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formed during transformation in the sludge may have had different sizes between the different

333

treatments. Such differences could arise from the fact that ionic Zn directly precipitates as

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Zn3(PO4)2 while ZnO ENMs must first either undergo dissolution and subsequent reprecipitation

335

or undergo a surface mediated transformation of the particle surface in towards the core of the

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particle without undergoing dissolution.17 This surface mediated transformation can lead to

337

transformation products that themselves are nanoparticles while dissolution and re-precipitation

338

tends to yield micron scale particles, although this is difficult to characterize in complex media

339

such as soil.17 The Zn3(PO4)2 products formed may have different properties, distribution within

44

However, this study unequivocally demonstrates that metal speciation


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the soils, dissolution rates, and subsequent bioavailability of Zn in the rhizosphere.

The

341

extraction test is designed to assess bioavailability, and presumably accounts for the different

342

spatial distribution and morphological differences in the formed Zn products. However, the

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extraction tests used here were not adequate to account for these differences.

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potential modification to these procedures or alternative procedures (e.g. diffusive gradient thin

345

films) to better predict metal bioavailability observed in plant uptake studies is a potential area

346

for future study.

Exploring

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While the metal concentrations used in this study are high relative to near-term predicted soil

348

ENM concentrations, we have clearly demonstrated that there is a distinct plant and

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microorganism response as a result of exposure to biosolids containing ENMs compared to

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biosolids containing bulk/dissolved metal of the same composition. This result suggests that soil

351

accumulation of ENMs could potentially affect critical ecosystem services, agricultural

352

productivity, and ultimately human well-being. However, while spiking Ag, ZnO, and TiO2

353

ENMs into the sludge together generates a realistic scenario considering that these three ENMs

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will occur in the waste stream together, it remains unclear which ENM or combination of ENMs

355

is responsible for the effects observed in this study. Though available data from this study and

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the companion study22 suggest that Zn is the most likely cause of differences observed in

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phytotoxicity, further investigation will be required to clarify the causative agent(s) of the

358

observed effects in response to individual ENMs.

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single concentration in this study due to the limited supply of biosolids generated by the pilot

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WWTP and as a result, the concentration dependence and the effect threshold of the effects

361

observed here remain unknown.

Furthermore, we were only able to test a



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Supporting information available.

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Results of media extractions, media extraction

363

methodology, details of S. meliloti quantification methods, results of qPCR quantification of S.

364

meliloti, information regarding interpretation of MRPP results, details of PLFA extractions and

365

analysis, detail of the configuration of BL 4-1 at Stanford Synchrotron Radiation Lightsource,

366

results from X-ray fluorescence mapping of leaves and X-ray absorption near edge spectroscopy

367

(XANES) of leaf tissue and biosolids amended soil are provided. This material is available free

368

of charge at http://pubs.acs.org.

369


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TABLES

371

Table 1.

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deviation calculated from n=3. BDL=below detection limit. ENM= engineered nanomaterial.

Media characterization data. Concentrations expressed as mean ± one standard

373 pH WHC (ml/100g) F- (mg kg-1) Cl- (mg kg-1) SO42- (mg kg-1) NO3- (mg kg-1) PO43- (mg kg-1) NH4+ (mg kg-1) Zn (mg kg-1) Ag (mg kg-1) Ti (mg kg-1)

Control 6.7 ± 0.01 90.7 ± 1.2 BDL 33.7 ± 1.6 603.9 ± 24.9 BDL 112.7 ± 2.6 184.2 ± 5.5 471.2 ± 27.3 2.0 ± 0.2 1180.4 ± 32.7

Bulk/dissolved 6.5 ± 0.09 90.9 ± 1.9 BDL 34.8 ± 1.6 426.5 ± 5.5 BDL 97.4 ± 0.0 173.2 ± 3.8 1484.7 ± 98.8 102.9 ± 7.4 2364.9 ± 61.8

ENM 6.7 ± 0.06 92.3 ± 1.5 BDL 33.4 ± 1.5 309.0 ± 16.8 BDL 75.7 ± 5.7 163.5 ± 2.2 1434.0 ± 87.1 98.2 ± 10.1 2466.8 ± 181.8

374 375 376

Table 2. Fresh shoot biomass, dried root biomass, and shoot length measurements. Each value

377

represents mean ± one standard deviation. Means with the same superscript are not significantly

378

different at α=0.05. ENM= engineered nanomaterial.

379

Treatment

Fresh shoot biomass (mg)

Control Bulk/dissolved ENM

85.1 ± 16.4a 74.8 ± 19.3a 57.8 ± 12.6b

Dried root biomass (mg) 1.7 ± 0.8a 1.8 ± 0.6a 1.1 ± 0.5a

Shoot Length (cm) 7.0 ± 0.9a 6.7 ± 0.8a 5.7 ± 0.8b

380 381 382



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Table 3. Concentrations (mean ± 1 SD; nmol g-1) of various components of the soil microbial

384

community. Means with the same superscript are not significantly different at α=0.05. ENM=

385

engineered nanomaterial.

386 387 Microbial Group Gram positive bacteria Gram negative bacteria Anaerobic bacteria Actinomycetes Fungi AM Fungi Eukaryotes Total

Control 207.3 ± 39.8a 531.2 ± 115.8a 4.7 ± 1.6a 64.2 ± 12.7a 66.5 ± 13.9a 33.9 ± 6.7a 55.5 ± 12.5a 963.3 ± 187.4a

Bulk/dissolved 181.4 ± 20.7b 567.4 ± 75.0a 3.9 ± 0.7b 53.7 ± 7.4b 101.4 ± 17.8b 50.8 ± 7.5b 79.5 ± 13.5b 1038.1 ± 122.9a

388 389 390 391 392 393 394


ENM 148.9 ± 24.1c 362.3 ± 65.4b 4.1 ± 1.1b 38.7 ± 7.3c 41.8 ± 4.6c 15.9 ± 4.8c 57.5 ± 12.5a 669.0 ± 109.2b

Page 21 of 28

FIGURES FIGURE 1

mg Ti /kg dry mass

10 8

Control Bulk/ dissolved ENM

6 4 2

A

80

A ng Ti accumulated

395 396 397


A

0

60


40

A

20

A

A

0

Ti

Ti

398 3000

200


150 100

C

B A

50

ng Zn accumulated

mg Zn/kg dry mass

250


2500 2000

B

1500

A

1000 500 0

0

Zn

Zn

399

15


1

B 0.5

A

B

ng Ag accumulated

mg Ag /kg dry mass

2 1.5

10


B 5

A

0

0

Ag

Ag

400

C

401 402 403 21 ACS Paragon Plus Environment

B


404

FIGURE 2

Nodules per plant

10

Control

8 6

Bulk/dissolved

A A

4

B

2 0

Treatment 405 406 407

FIGURE 3

408 22 ACS Paragon Plus Environment

ENM

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Page 23 of 28


409 410

FIGURE CAPTIONS

411

Figure 1. Concentrations (left) of Ti (top), Zn (middle), and Ag (bottom) in Medicago truncatula

412

shoot tissue and (right) accumulated mass of Ti (top), Zn (middle), and Ag (bottom). Treatments

413

with the same letter are not significantly different from one another at α=0.05. Error bars =

414

standard error. ENM= engineered nanomaterial.

415 416 417

Figure 2. Nodulation frequency (number of nodules per plant). Treatments with the same letter

418

are not significantly different from one another at α=0.05. Error bars = standard error. ENM=

419

engineered nanomaterial.

420 421

Figure 3. PC-ORD non-metric multidimensional scaling (NMDS) ordination plot using the

422

relative abundance of measured PLFAs. Treatments (TRT) 1=bulk/dissolved metal; 2=control;

423

3=engineered nanomaterial (ENM). Comparisons of all treatments are significantly different (A

424

0.33-0.45 and p0.300) with

425

axis scores are shown as bioplot vectors the direction and length of which indicate the direction

426

(positive or negative) and strength (longer = stronger) of the correlation. The angle between the

427

vectors indicates the correlation between the biomarker group concentrations (small angles =

428

higher correlation).

429 430 431

Acknowledgments. The authors acknowledge the advice and assistance of J. Kupper, Z. Elhaj Baddar, J.

432

Kirby, and R. Tammer. This research was funded by a grant from the U.S. Environmental Protection



Page 24 of 28

433

Agency's Science to Achieve Results (STAR) program (RD834574), by the UK Natural Environment

434

Research Council grant NE/H013679/1 and Biotechnology and Biological Sciences Research Council

435

grant BBS/E/C/00005094. J. Unrine and O. Tsyusko were also supported by the National Science

436

Foundation (NSF) and the Environmental Protection Agency (EPA) under NSF Cooperative Agreement

437

EF-0830093, Center for the Environmental Implications of NanoTechnology (CEINT). Any opinions,

438

findings, conclusions or recommendations expressed in this material are those of the author(s) and do not

439

necessarily reflect the views of the NSF or the EPA. This work has not been subjected to NSF or EPA

440

review and no official endorsement should be inferred. Portions of this work were performed at Beamline

441

X26A, National Synchrotron Light Source (NSLS), Brookhaven National Laboratory. X26A is supported

442

by the Department of Energy (DOE) - Geosciences (DE-FG02-92ER14244 to The University of Chicago

443

- CARS). Use of the NSLS was supported by DOE under Contract No. DE-AC02-98CH10886. Use of

444

the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by

445

the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No.

446

DE-AC02-76SF00515.

447 448

TOC ART

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Nanomaterials in Biosolids Inhibit Nodulation, Shift Microbial

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