Determination of Nanoparticle Uptake, Distribution, and

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Ecotoxicology and Human Environmental Health

Determination of nanoparticle uptake, distribution and characterization in plant root tissue after realistic long term exposure to sewage sludge using information from mass spectrometry Sandra Wagener, Harald Jungnickel, Nils Dommershausen, Thomas Fischer, Peter Laux, and Andreas Luch Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b07222 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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Environmental Science & Technology

Nanomaterials

Dispersion in Sewage Sludge

Distribution

Uptake & Dissolution in planta? ACS Paragon Plus Environment


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Determination of nanoparticle uptake, distribution and characterization in plant root tissue after realistic

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long term exposure to sewage sludge using information from mass spectrometry

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Sandra Wagener1*, Harald Jungnickel1, Nils Dommershausen1, Thomas Fischer1, Peter Laux1, Andreas Luch1

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1Department

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Straße 8-10, D-10589, Berlin, Germany

of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-Dohrn-

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*Corresponding author at: Department of Chemical and Product Safety, German Federal Institute for Risk

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Assessment (BfR), Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany.

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Phone: +49 30 18412 4164; fax: +49-(0)30-18412-47 41

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E-mail: [email protected]

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Abstract

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The use of nanoparticles (NPs) in numerous products and their potential accumulation causes major concern for

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humans and the environment. Until now, the uptake of NPs in plant tissue was mostly shown under greenhouse

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conditions at high doses and short exposure periods. Here we present results on the uptake of particulate silver

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(Ag) and cerium dioxide (CeO2) in the tissue of Triticum aestivum, Brassica napus and Hordeum vulgare, after

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exposure to sewage sludge treated with nano-Ag (NM300K at 1.8 and 7.0 mg/kg sludge per dm soil) and nano-

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CeO2 (NM212 at 10 and 50 mg/kg sludge per dm soil). All plants were cultivated in a rural area near the German

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town Schmallenberg according to the common regional crop rotation on outdoor lysimeters. The highest

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concentration measured was 86.4 mg/kg for Ag (Hordeum vulgare), and 94 mg/kg for Ce (Triticum sativum).

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Analysis of plant samples revealed the presence of Ag mainly in its ionic form. However, the occurrence of

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nano- and larger sized particles of Ag and CeO2 was observed as well. Quantitative shares of the particulate

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fraction of the total element concentration were estimated with up to 22.4% for Ag and with up to 85.1% for

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CeO2. A high abundance of particle agglomerates in the phloem suggests upward transport of the nanoparticles

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to other plant parts. A small number of agglomerates in the xylem suggests a downward transport, and

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subsequent accumulation in the root phloem. Exemplary investigations of Brassica napus root exposed to nano-

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CeO2 revealed no accumulation of the pristine material in the cell nucleus, however, CePO4 was found. The

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presence of this substance points to a dissolution of the low soluble CeO2 in planta and subsequent precipitation.

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Furthermore, for the first time, mixed NP-salt agglomerates, composed of Ca3PO4+ and K3SO4+ NPs, could be

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observed within Brassica napus root tissue.

33 34

Introduction

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In the past 15 years, the use of nanoparticles (NP) in daily life and medical products has constantly increased 1-3.

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As a consequence, NPs cause major concern as a potential contaminant of the environment, e.g. through waste

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water, deposition or precipitation.4-6 Furthermore, the uptake of NPs by agricultural plants is also relevant for

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human exposure.7 If not removed systematically after their use, NPs will enter the environment via numerous

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sources and may accumulate depending on their specific chemical and physical properties.8-9 In order to assess

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the potential effects of these small sized materials, numerous investigations concerning exposure scenarios,

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environmental fate and toxic effects of nanomaterials (NMs) have been conducted.10-13 Beside adverse effects

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including phytotoxicity, suppression of seed germination1 and oxidative stress leading to programmed cell

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death,14 other studies report improved seed germination and crop performance.15 While excess reactive oxygen

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species (ROS) formation and oxidative damage are considered main mechanisms of NP toxicity, some materials

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may also scavenge ROS.16 In a recent study by Tripathi et al.,17 silicon NPs at a concentration of 10 µM were

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shown to protect Zea mays seedlings against arsenic (As). While accumulation of As and reactive oxygen species

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was reduced, the levels of superoxide dismutase and gluthathione reductase were increased. Ion release was

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confirmed as a major factor of NP toxicity in plants. In a study with Glycine max, soluble ZnO- and Ag-NPs

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have reduced plant growth, rigidity and root cell viability. Simultaneously, normal seedling growth was observed

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in presence of the Al2O3-NPs, which has been attributed to their poor solubility12,18-19 Thus, the chemical identity

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needs to be considered with regard to potential effects of NPs on plants. This is of particular importance for the

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investigation of the food chain, which remains as one major research need.7, 14, 20

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So far, there is no evidence for significant human and environmental health effects that can be specifically

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attributed to the nano-size of materials.21-23 Instead, a number of other properties like surface area, solubility and

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morphology are being discussed as main determinants of

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guidelines for ecotoxicity and toxicity testing of chemicals are in principle considered suitable for NPs as well.

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However, specific adaptations in particular with regard to sample preparation, dosimetry and biokinetics are

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required.25-26 With regard to a possible accumulation of NP along the food chain, the influence of abiotic stress

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such as a variation of trace elements27 should be considered as a confounding factor. High salinity (100 mM

NP toxicity.24 In consequence, the established



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NaCl) for example, was shown to cause increased concentrations of Ce in roots and leaves of Brassica plants

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exposed to CeO2-NPs.28

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Thus, particle accumulation and fate at field conditions are of particular interest. In this context both, NP dose

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and exposure duration are important issues. Whereas several studies, mostly conducted in growth chambers,

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exist concerning fate and short time effects of NPs on plant germination,6, 20, 28-30 the long-term fate of these

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materials in the environment under real conditions has not yet been sufficiently addressed. Depending on their

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physical and chemical properties, NPs may dissolve in ions from which particles might be formed again during

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their transport through the environment. This may not only affect their size, but also significantly change their

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physical-chemical behaviour. In the case of CeO2, parameters like particle surface coating and presence of

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organic matter were demonstrated to affect the uptake by higher plants.31 Furthermore, chemical alteration of the

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particle itself, as e.g. the formation of CePO4 upon exposure of Cucumis sativus to CeO2,32 or changes of the

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plant protein profile, e.g. of Oryza sativa following treatment with Ag-NPs33 have been demonstrated. For a

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thorough assessment of NP fate within living organisms, methods are needed to allow for the following tasks: 1)

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detection and characterization of the particles within the organism, including concentration and size distribution,

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2) localization of the particles on tissue and cellular level 3) characterization of NP and aggregate composition

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including other substances that may have accumulated during their transport. Based on these data, conclusions

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on transformation and possible adverse effects could be drawn.

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Several methods for particle characterization have already been described in the literature, such as scanning

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electron microscopy (SEM),34 transmission electron microscopy (TEM)35 or X-ray Absorption Spectroscopy

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(XAS)30. However, for the reliable detection of relevant elements, images have to be strongly magnified, leading

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to either a limitation of the area of investigation or to a time consuming analysis. A method which allows for

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element-specific detection over a larger sample section is Time-of-Flight Secondary Ion Mass Spectrometry

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(ToF-SIMS).19,

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entities together with the generation of three dimensional (3D) information.38 By imaging the elemental

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constitution, not only information about the precise localisation of particles, but also on accumulation of other

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substances can be gathered.39-40 A further benefit of this technique is the possibility of an automated statistical

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agglomerate cluster analysis, allowing for the determination of particle or aggregate size distribution patterns.40

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This study is a part of the joint project on “Design criteria for sustainable nanomaterials – DENANA” and aims

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to investigate about the long-term fate of nano-Ag and nano-CeO2 in root tissues of Triticum aestivum, Brassica

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napus (Ag and CeO2) and Hordeum vulgare (only Ag). The plants were grown on different lysimeters according

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to their actual growth period and exposed to NPs via sewage sludge41 to mimic a real life situation.42 With nano-

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The major advantages of this technique are the area-wide detection of several chemical


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Ag and nano-CeO2, a soluble and a biopersistent NP species relevant for environmental exposure,43 have been

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chosen to elucidate the influence of solubility on NP fate. While nano-Ag is commonly used for its antimicrobial

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activity in numerous products such as medicinal applications and fabrics,44-46 nano-CeO2 mainly occurs in

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catalysts for vehicles,47-48 but is also used for industrial and construction purposes.49-50 Beside a quantification of

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Ag and Ce, the particulate occurrence of both materials and their systemic distribution are addressed. Single

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particle inductive coupled mass spectrometry (spICP-MS) and ToF-SIMS are used to quantify and characterize

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NPs within plant tissues, enabling not only information about size distribution, but also precisely localizing the

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particle deposition.

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Materials and methods

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Plant samples and nanomaterials

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Three different plant species, Triticum aestivum (Triticum aestivum ´Tybalt A‘ Saaten Union GmbH, Isernhagen,

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Germany), Brassica napus (Brassica napus ‘Treffer‘ KWS Saat SE, Einbeck, Germany) and Hordeum vulgare

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(Hordeum vulgare ‘SY Typee‘ Syngenta, Maintal, Germany, Table 1), were cultivated in outdoor lysimeters (0.9

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m × 0.9 m × 0.9 m, width x length x depth) in a rural area near the German town of Schmallenberg (North Rhine-

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Westphalia, 500 m above sea level). The chosen plant species are most relevant for sewage sludge fertilization in

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Germany.51

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http://www.refesol.de/english/analysedaten.shtml) and exposed to either nano-Ag (NM300K) or nano-CeO2

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(NM212) (see Table 1 for exposure concentrations, and supplement Table S1 for particle specification). Ag and

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CeO2-NPs were supplied from the Joint Research Center (JRC) Nanomaterials Repository and have been

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characterized within the OECD testing programme. Both NPs have been subject of numerous research

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projects.52-53 The materials were incorporated into sewage sludge of municipal sewage from the treatment plant

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of Schmallenberg, according to the German Sewage Sludge Ordinance,51 before application to lysimeters at

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given concentrations. For the incorporation of the NPs, the sewage sludge was dried, meshed to particles of a

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size smaller than 2 mm and then stored in a vessel under stable moderate stirring and aeration (2.5 mg O2/L).

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NMs were then spiked into the sewage. The sludge was spread over the first 20 cm of soil on the lysimeters,

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immediately after sowing. At harvest, the plants of each exposure group were divided into root, sprout and grain

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and frozen at -18 °C.

The

lysimeters

were

filled

with

reference

121 122


soil

01A

(RefeSol;


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Table 1: Overview of investigated plant species and exposure concentrations for Ag and CeO2 NP.

Plant species

Triticum aestivum Brassica napus Hordeum vulgare

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Triticum aestivum Brassica napus

Seeding

June 2014 October 2014 August 2015

April 2015 August 2015

Harvest

Control C0*

Exposure Group C1*

Exposure to Ag-NP (NM300K) September 0 2014

Exposure Group C2*

1.8

7.0

10

50

August 2015 July 2016 Exposure to CeO2-NP (NM212) 0 August 2015 July 2016

*Concentration is given in mg kg-1 dry matter sludge

125 126

Analysis and measurement techniques

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The characterization of particle fate and localization within plant tissue was conducted following the scheme

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described in

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. After harvest, the plants were divided into corn / shell, sprout and roots. The parts were then analyzed using

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ICP-MS for total Ce and Ag content. Only samples with a significant Ce or Ag amount were further analyzed

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with spICP-MS and ToF-SIMS.

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Quantification of the total Ag content with ICP-MS

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Approximately 100 mg of plant tissue was washed and dried in a drying stove for 24 hours. After cooling, the

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exact weight of the dry samples was determined. 2 ml of nitric acid (HNO3, 69%), 2 ml of hydrogen peroxide

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(H2O2) and 2.5 ml of water (H2O dest.) were then added to the sample. Samples were subsequently digested in a

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microwave oven (FA MLS, Ethos.start) following a temperature program suitable for biological material (step 1:

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heating up to 100° C in 2 min; step 2: heating up to 180° C in 6 min; step 3: heating up to 210° in 4,5 min: step

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4: holding at 210° C for 10 min). Samples were analyzed for their total Ag and Ce content using ICP-MS (X-

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Series II, FA Thermo). The limit of detection (LOD) was determined by taking the mean + 3 times the standard

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deviation of root blanks.

143 144

Single particle analysis using spICP-MS

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ICP-MS was additionally operated in the single particle (sp) mode, which enables the determination of particle

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mass, number and size distribution of the particulate fraction. A further feature is the elemental identification, ACS Paragon Plus Environment

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provided for the respective single particles of interest. Details about the calibration and the determination of

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single particle number, and dwell time can be found in Wagener et al.54

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Particle extraction and minimization of particle alteration is a crucial step of single particle determination in the

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plant matrix. All plant tissue samples were prepared according to an enzymatic digestion method published by

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Dan et al.29 Samples were cut with a ceramic knife and washed twice with MilliQ-water to remove remaining

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soil. The cleaned pieces were milled under liquid nitrogen in a mortar. Subsequently, 8 ml of citrate buffer (100

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mM) and 2 ml of Macerozyme R10 (50 mg/ml) were added at room temperature. Samples were incubated at

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1400 rpm and 37°C in a thermos shaker for 24 h. To determine the exact plant weight, the remaining water

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content was determined and subtracted. The supernatant was removed and diluted for subsequent spICP-MS

156

analysis.

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The LOD for spICP-MS is given by the frequency of single particles signals above the background, which is

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governed by dissolved element species.55 As no reference materials, i.e. plant materials with a known amount of

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particulate Ag or CeO2 exist, the recovery rate was determined by adding NPs to the root pieces before the

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enzymatic digestion. Similar to ICP-MS, spICP-MS measures only elements, but particle size and concentration

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can be calculated for molecules based on particle density and molar mass. As NM212 was used for these

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experiments and for determining the recovery rate; calculations with spICP-MS were conducted under the

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assumption that all particles are CeO2. For further interpretation of the results however, it is important to bear in

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mind that the Ce content measured with spICP-MS may actually be in another form. Similar issues must be

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considered for Ag. The calculation of pure Ag was used here because of the use of nano-Ag NM300K. Thus the

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sizes and concentrations presented below only apply to pure Ag particles and do not consider possible formation

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of larger particles containing other elements.

168 169

Characterization of particle fate using ToF-SIMS

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A ToF-SIMS5 (ION-TOF GmbH, Münster, Germany) instrument was used for the identification of the chemical

171

composition and distribution patterns of particles within plant tissue sections. ToF-SIMS uses a focused ion

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beam to sputter secondary ions arising from the sample surface. An image of the chemical composition of the

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sample can then be established. The instrument can be used in dual ion beam mode, where multiple surface

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layers are analysed and three-dimensional chemical information can be acquired. The lateral resolution of this

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method is ca. 80 nm.

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Thin 70 µm layers of root tissue were prepared using a cryo microtome. Small pieces of roots were embedded in

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resin medium and cut at a temperature of -15°C (object holder) and -25°C (cutting chamber). In this study, a



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lateral resolution below 100 nm 56 and a depth resolution of approximately 10 nm per layer was achieved.57 Ion

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images and depth profiles were performed using a 30 keV nano-bismuth primary ion beam source and a 30kV

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argon cluster ion beam. 3D depth profiles were acquired in the dual beam analysing mode using a 30 keV nano-

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bismuth (Bi)x(y+)-cluster primary ion beam source with a BiMn emitter as analysing ion beam and a a 20 keV

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argon gas cluster ion source both mounted at 45° with respect to the sample surface as sputter ion beam and an

183

electron flood gun for charge compensation. The methodology has been demonstrated successfully for the

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reconstruction of 3D distribution patterns of NPs from living cell systems.19

185 186

Results and Discussion

187 188

Total concentration of Ag and Ce in planta

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The LOD for Ag was determined as 0.3 ppb, 0.1 ppb and 0.8 ppb for Triticum aestivum, Brassica napus and

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Hordeum sativum, corresponding to 0.04, 0.01 and 0.1 mg kg-1 plant tissue, respectively when using a dried plant

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sample of 75 mg. Correspondingly, the LOD for Ce in Triticum aestivum and Brassica napus were 127 ppb and

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40 ppb or 21 mg kg-1 and 8 mg kg-1 plant tissue, respectively. The increased LOD for Ce arises from the natural

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occurrence of Ce in soil. The recovery rate for the digestion of Ag was determined with 97% for a concentration

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range of 20 ppb and with 123% for a concentration range of 2 ppb. Due to the increased Ce levels in the roots, no

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reproducible recovery rate for Ce was achieved. These circumstances must be considered in the interpretation of

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the results.

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Analysis with ICP-MS revealed that plant roots absorb Ag and Ce in significant amounts after long-term

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exposure to nano-Ag and CeO2 containing soils (Table 2). In contrast, in the sprouts, corn and shells the

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concentrations for both, Ce and Ag, were below the LOD. For Ag, concentrations between 1.3 and 86.4 mg kg-1

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were observed in roots. For Ce, quantification was shown to be more difficult due to the large background levels

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in root tissue arising from naturally occurring Ce in the soil. However, when applying a larger amount of CeO2

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to the lysimeters (C2), a significant accumulation was revealed. The Ce content varied between below LOD and

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94 mg kg-1. The concentrations of Ag measured in Hordeum vulgare are significantly higher compared to the

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other plant species. The high standard deviation (SD) of the highest detected concentrations of Ag in Hordeum

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vulgare and of Ce in Triticum sativum demonstrate the inhomogeneous distribution of these elements in the

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roots. However, also an inhomogenous distribution of the NPs within the lysimeter soil after sludge application

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may lead to high variation of both elements.

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Concentrations of particulate Ce and Ag in plant roots measured with spICP-MS and ToF-SIMS

212

Ag and Ce are not only present in the plant root tissue in ionic form, but also as particulate matter (Figure 1,

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Figure S2, Table 2). The recovery rate of total Ag was 81.6% (±10%) with a particulate fraction of 12.3% (±2

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%), and for total CeO2 105.9 (±29%) with a particulate fraction of 70.7% (± 25%). A dissolution rate of ~10%

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was already determined for NM300K in pure water. The fact that in the recovery experiments the NMs are added

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to the surface of the roots instead of being incorporated within the matrix may lead to an overestimation of the

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recovery and therefore to an underestimation of the real particulate matter. Agglomeration may also contribute to

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the observed diameter for Ag-NP in plant extracts of 68 nm (± 1.1 nm) compared to a primary particle size of 15

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nm for NM300K. Similarly, for CeO2-NP, the median particle diameter in plant extracts was 89 nm (± 0.7 nm),

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compared to a primary particle size of 28 nm for the pristine material NM212. Corresponding analysis of both

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particles in pure water showed primary particle sizes approximating the sizes of the pristine materials. Analysis

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of roots revealed for plants exposed to Ag a significantly lower share of particulates compared to ions (Table 2).

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Table 2: Total and particulate Ce1 and Ag concentration in mg kg-1 root tissue after exposure to sewage sludge tissue ± standard deviation

Plant species

Analyte

Total C1

Total C2

Particulate C0

Particulate C2

%Particulate C2

Triticum aestivum

Ag

2.3 ± 0.12

9.1 ± 4.13

not determined

not determined

not determined

Brassica napus

Ag

1.3 ± 1.94

7.6 ± 2.35

< LOD

1.7 ± 0.2

22.4

Hordeum vulgare

Ag

12.7 ± 10.1

86.4 ± 45.1

< LOD

0.6 ± 0.1

0.7

Triticum aestivum

Ce/CeO2

24.0 ± 7.0

94 ± 77.6

13.7 ± 5.8

80 ± 6.2

85.1

Brassica napus

Ce/CeO2

< LOD

8.9 ± 1.3

3.5 ± 0.9

3.7 ± 1.3

41.6

225 226 227

1

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Ag NP were detected in exposed plants of Brassica napus (1.7 mg kg-1) and to a smaller amount in Hordeum

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vulgare (0.6 mg kg-1) by spICP-MS. The detection of both Ag isotopes, 106.9 u and 108.9 u, by ToF-SIMS

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measurements of Brassica napus roots confirms the occurrence of particulate Ag (Figure 2, Figure S3). In

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Triticum aestivum, which was not measured with spICP-MS, ToF-SIMS measurements also verify an

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accumulation of Ag NPs inside the root tissue (Figure 3). No Ag-NP were detected in control plants with spICP-

233

MS and ToF-SIMS. The low concentration of Ag-NPs measured in Hordeum vulgare is noticeable when

: Total concentrations are given for Ce, particulate concentrations for CeO2. Concentrations revealed for plant samples from the same lysimeter published in Schlich et al.41 were as follows: 2: 2.6 mg kg-1, 3: 10.9 mg kg-1, 4: 3.4 mg kg-1, 5: 8.4 mg kg-1.



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compared with the high total concentration of the element. Our results achieved with spICP-MS point to a

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significant uptake of ionic Ag or to a partial dissolution of Ag particles during or following their uptake. In

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Triticum aestivum, which was not measured with spICP-MS, the detection of both Ag isotopes, 106.9 u and

237

108.9 u, by ToF-SIMS (Figure 3) confirms an accumulation of Ag-NPs inside the root tissue. For plants exposed

238

to CeO2, the precise quantification for particulate matter, as already described for the measurement of the total

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content, was more difficult due to the large natural background. The concentration of particulate Ce varied

240

between 3.5 to 13.7 mg kg-1 for the control samples (C0) and between 3.7 and 80 mg kg-1 for the exposed

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samples (Table 2). The detection of Ce containing particles in the control samples demonstrates their natural

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occurrence in root tissue. Our findings of a significant total Ce accumulation in Triticum aestivum, however,

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together with the low solubility of CeO2 indicate that at least some of the supplied CeO2-NPs are taken up in

244

particulate form. However, in case of Brassica napus, no significant discrimination between control and samples

245

exposed to CeO2 is possible. Total and particulate concentrations are closer in Brassica napus compared to the

246

cereals tested. In contrast to Brassica napus, Ce containing particles in Triticum aestivum account for the largest

247

portion of their total Ce-content with 13.7 and 80 mg kg-1 respectively in control and exposed plants. Thus, the

248

spICP-MS results for control and exposed samples give hints of an uptake of particulate Ce in Triticum aestivum,

249

but not in Brassica napus. However, ToF-SIMS analysis suggests an uptake of CeO2 by both, Brassica napus

250

and Triticum aestivum. The CeO2-specific ion CeO+ m/z 156 was detected in exposed, but not in control plants

251

(Figures 4, S4 and S5). The results demonstrate that the measurement of elements of a high abundance in the

252

environment is challenging. Uptake studies should therefore apply at least one different technique. Previously,

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Lopez-Moreno et al.58 proved an uptake of CeO2-NPs by soybean using synchrotron X-ray absorption

254

spectroscopy. While in the case of Ag there was no significant difference in NP uptake by the different plant

255

species, the particulate fraction of CeO2 was significantly higher in Triticum aestivum compared to Brassica

256

napus. These differences are possibly due to the different solubility of Ag and CeO2 which seemingly holds also

257

true within the investigated plant root tissue sections. The results indicate material specific transformation,

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bioavailability and toxicity of NPs. Furthermore, plant species and growth substrate were described to influence

259

these characters.59-60 The influence of physicochemical variations among plant species, e.g. hydraulic

260

conductivity or pore size of the cell wall on transport and accumulation of NPs has been described previously by

261

Judy and Bertsch.61 In addition to the plant species, environmental conditions may alter NPs stability, oxidation

262

state and precipitation and thus affect their reactivity and translocation inside the plant.2 Thus, the same NPs may

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provoke different plant responses, depending on the species and conditions. The different particle accumulation

264

pattern of plant species observed in this study may therefore be explained by different physiological


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environments that impact on substance transportation. This might explain why there is a larger difference

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between the uptake of the less soluble CeO2 in Triticum aestivum and Brassica napus , than for the more soluble

267

Ag.

268 269

Size distribution of CeO2- and Ag-NPs in planta

270

SpICP-MS and ToF-SIMS provide complementary information on different particle agglomerate sizes. Whereas

271

spICP-MS detects particles in the nm-range, ToF-SIMS gives evidence of the occurrence of larger agglomerates.

272

The size distributions for Ag- and CeO2-particles in Brassica napus roots measured with spICP-MS are given in

273

Figure 1. In case of CeO2-NPs, the size distribution is comparable between the control and exposed samples. In

274

Triticum aestivum, however, a clear shift towards larger particles could be observed in the exposed sample as

275

well as an overall increase in concentration. Considering a primary particle size of 28 nm for NM 212, our

276

results provide evidence for an agglomeration of the applied CeO2-NPs, either before or after uptake into the

277

root. Most of the Ag amount in the root samples appears to be ionic. However, some particles could also be

278

detected in the exposed sample (Table 2, Figure 1). While the tested material Ag NM300K owns a primary

279

particle size of 15 nm, a mean size of 60 nm was determined in the root samples (Figure 1), pointing to an

280

agglomeration as observed for CeO2. No difference was observed between the investigated plant species.

281

282 283

Figure 1: Detected spICP-MS signals and size distribution of Ag- and CeO2-NPs in Brassica napus root sections. a) 7.0 mg Ag-NP

284

(NM300K) /kg sludge per dm soil, Ag exposure group C2 and unexposed control, Ag exposure group C0, b) 50.0 mg CeO2-NP (NM212) /kg

285

sludge per dm soil, CeO2 exposure group C2 and unexposed control samples, CeO2 exposure group C0.



286

A three dimensional distribution of Ag within a root section of Triticum aestivum was revealed by ToF-SIMS

287

analysis of a 100 µm x 100 µm tissue section of 3.5 µm thickness and is presented in Figure 3-f. The density of

288

the spots indicates agglomerates in the tissue, whereas a more refined Ag layer of lower intensity points to the

289

presence of ionic Ag. Analysis of agglomerates with regard to their size distribution revealed a number of 280

290

agglomerates in the total size range between 2 to > 281 µm². The highest number of agglomerates (126) was

291

found in a size range between 23 and 32 µm² (Figure 3-g). Based on these results, a presence of Ag in

292

particulate form is confirmed for the investigated tissue.

293

Based on our results it can be concluded that the particles cover a broad size range, possibly in a bi-modal

294

distribution with the 1st mode at 40 or 60 nm, for CeO2 and Ag respectively (Figure 1), and the 2nd mode in the

295

µm range (Table 1 and Figure 3). The particles in the roots were all larger than the pristine NPs. It also has to be

296

considered that the sizes given here are assuming pure Ag or CeO2 particles. If the particles accumulate other

297

substances, the particle sizes would be under- or overestimated. Reasons for the increase of the particles may be

298

different and can most probably be explained by their solubility, but also by their natural occurrence in the soil

299

or root, respectively. For both, Ag and CeO2, an increase in particle size has already been observed in the

300

recovery samples. Thus it is conceivable that this increase is at least partly a consequence of the enzymatic

301

digestion. However, the detection of large Ag and CeO+ agglomerates within the root tissue with ToF-SIMS

302

provides proof that larger particles already occur in the root, as ToF-SIMS requires no digestion procedure. In

303

the case of Ag, a de novo formation of particles from soluble Ag is also very likely to occur after uptake. De

304

novo formation of Ag agglomerates from ionic suspensions has been shown in other contexts, e.g. the formation

305

of particulate Ag species during migration experiments with Ag containing textiles in artificial sweat.62 For the

306

less soluble CeO2, agglomeration inside the root seems to represent the most probable mechanism for particle

307

size increase. When particles interact with plants, they have to cross the cell wall as the first barrier. The wall,

308

which is composed of cellulose, permits the entry of small particles and restricts passage of the larger ones.63

309

According to Dietz and Herth,64 the size exclusion limit for the plant cell wall is between 5 and 20 nm.

310

Therefore, it can be assumed that the formation of most of the larger agglomerates occurred after uptake into the

311

root. However, some of the nano-sized particles have been reported to induce the formation of larger pores in the

312

cell wall which would enable the entry of larger particles.65

313 314

Localization, characterization and distribution of CeO2- and Ag-NPs in roots

315

Analyses of rhizodermis and exodermis were conducted with Ag as a case study using ToF-SIMS. The Ag

316

signal distribution pattern was recorded with ToF-SIMS within a root tissue section of Brassica napus (Figure


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317

2). While in the left hand side of the picture the rhizodermis was still present, it was removed in the right hand

318

side, showing Ag distribution patterns within the exodermis. Ag was not detected in or on the rhizodermis, but

319

was present in the exodermis. As ToF-SIMS is able to detect isotopes up to several µm in depth, this proves that

320

the Ag content detected with ICP-MS and spICP-MS arises from an uptake of Ag into the tissue and not from a

321

deposition outside the root. NPs have been found to penetrate the outer root cell layers, and accumulate later in

322

the membrane of the root epidermis from where they enter the xylem, and further translocated to the leaves.67

323

Even though in our study the NPs could be detected throughout the whole vascular system in roots, they were

324

not equally distributed along the central cylinder of the root. As shown for Ag and CeO2 in Triticum aestivum

325

(Figures 3 and 4) and Brassica napus (Figures S3 and S4 and S5), NP-agglomerates mainly accumulate in the

326

phloem layers around the central cylinder. Together with the presence of only few particles in the xylem, this

327

indicates an upward transport of absorbed NPs via the xylem, followed by a downward transport from the

328

vegetative parts of the plant via the phloem with subsequent accumulation. The transport via the xylem, which,

329

according to Aslani et al.66, serves as the most important vehicle in the distribution and translocation of NPs, has

330

already been proven in cucumber plants.67 Xylem and phloem mediated uptake, translocation and distribution

331

from root to shoot through the xylem and its reverse transport to root through the phloem was additionally

332

demonstrated by Wang et al.68 for nano copper oxide in Zea mays.

333

334 335

Figure 2: Reconstructed ion pictures showing the Ag signal from the Ag isotopes 106.9 u and 108.9 u: a) on the rhizodermis and b) on the

336

exodermis of a Brassica napus root exposed to 7.0 mg/kg sludge per dm soil. The white arrows indicate that Ag was not detected on the

337

surface of the rhizodermis. The upper panels show the Ag distribution of a root, where the rhizodermis (a, depicted with an arrow) was partly

338

removed (exodermis depicted in the picture as b). The lower panel shows a root, where the rhizodermis was removed completely and shows

339

the exodermis (b).

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341

342 343

Figure 3: Ag distribution in a longitudinal tissue section of Triticum aestivum exposed to Ag NP, ToF-SIMS total ion reconstruction from a

344

root tip, exposure group C2, 7.0 mg/kg sludge per dm soil: a) total ion picture of a root section (1500 µm x 500 µm), b) spectra from tissue

345

sections of an exposed root (upper panel, shown are both silver isotopes) and an untreated control root (lower panel, no silver isotope peaks

346

are present). c) ion reconstruction (500 µm x 500 µm) of the red square, marked in a. d) distribution of the reconstructed silver signal (in

347

green) from c. e) overlay of c and d, silver particles are visualized in green. f) 3D distribution of the silver nanoparticles, depicted in blue

348

(100 µm x 100 µm x 3.6 µm, length x width x depth) of the white square in e. g) size distribution of silver particles from f, size groups

349

depicted: Group I: 2 µm Ø , Group II: 3 - 12 µm Ø, Group III: 13 - 22 µm Ø, Group IV: 23 - 32 µm Ø, Group V: 33 - 42 µm Ø, Group VI:

350

43 - 62 µm Ø, Group VII: 63 - 82 µm Ø, Group VIII: 83 - 102 µm Ø, Group IX: 103 - 122 µm Ø, Group X: 123 - 142 µm Ø, Group XI >

351

142 µm Ø.

352 353 354 355 356 357 358 359 360 361 362 363 364 365 366


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367

368

e 369

Figure 4: CeO distribution in a longitudinal tissue section of Triticum aestivum, exposed to CeO2 NP, exposure group C2, 50 mg/kg sludge

370

per dm soil: a) ToF-SIMS total ion reconstruction of a root section (3.5 mm x 1.5 mm), b) spectra from tissue sections of an exposed root

371

(upper panel, the CeO+ peak, m/z 156, is present) and an untreated control root (lower panel, no CeO+ peaks are present. c) ion reconstruction

372

(500 µm x 500 µm) of the red square marked in a. d) 3D distribution of CeO2 NPs, depicted in red from the 3D ToF SIMS ion reconstruction

373

(100 µm x 100 µm x 3.6 µm, length x width x depth) of the red square in c). e) 2D enlarged section (20 µm x 20 µm) from the 3D

374

reconstruction, CeO2 particles are depicted in red.

+

375 376

Occurrence of NPs in cell nucleus and cytosol

377

ToF-SIMS analysis and 3D ion reconstruction were used to investigate the presence of CeO2-NPs in the

378

cytosolic fraction and the nucleus of Brassica napus root cells. The masses of adenine and guanine, m/z 136 and

379

152, were employed for identification of the nucleus shown in yellow in Figure 5a. The mass of m/z 58

380

(C3H8N+) was used as an indicator for the cytosolic fraction (Figure 5). CeO2-NPs were identified in the cytosol



381

(Figure 5-a-3, small red circles left of the nucleus) but not in the nucleus (Figure 5-a-2). However, cerium

382

phosphate (CePO4+, ion m/z 235) agglomerates were identified in the nucleus (Figure 5-a-3, green encircled).

383 384

385 386 387

Figure 5: Reconstructed 3D ToF-SIMS image (15 µm x 15 µm x 4 µm) of a single cell from a Brassica napus root, exposed to CeO2,

388

exposure group C2, 50 mg/kg sludge per dm soil. 5a: Cell nucleus is shown in yellow as the sum of adenine (m/z 136) and guanine (m/z 152)

389

ions. Cytosol is shown in blue (m/z 58). CeO+ is depicted by black dots in 2 and red encircled in 3. CePO4+ is green encircled in 3.

390

5b: Mixed NP agglomerates identified in the cytosol, red: CeO2, light blue: Ca3PO4+, yellow: K3SO4+ NP-agglomerate (Ion m/z 213).

391 392

As CePO4 was only found in exposed roots but not in the controls, it is assumed that the formation of CePO4

393

results from ion release of CeO2-NPs with subsequent formation of CePO4 within the cell nucleus. This would

394

support the theory of partial solubility of CeO2 within plant tissue similar to processes observed within animal

395

tissue.13, 72 This may indicate that NPs, even if they are not absorbed directly into the cell nucleus, can dissolve

396

and reform particles with different chemical entities in such a way that the heavy metal reaches the cell nucleus.

397

Similar findings were reported by Zhang et al.73,35 in vacuoles of romaine lettuce and cucumber roots. They

398

provided proof of flocculent CePO4 particles in the intercellular spaces and vacuoles using scanning transmission

399

X-ray and electron microscopy and explained the function of the vacuole as a “phosphorus pool”. The storage of

400

heavy metals in vacuoles represents a detoxification mechanism of plants.74 Small amounts of Ce3+ were shown

401

to be released from CeO2 at the presence of organic acids and reducing substances in root exudates.75 From a

402

study investigating Lactuca plants, the authors suggested transformation processes to occur at the root surface.

403

The interaction between the NPs and root exudates at the nano-bio interface was assumed to be necessary for the

404

transformation of nano-CeO2 in plants. It is hypothesized, that a part of the Ce3+ released at the nano-root

405

interface is immobilized by the formation of CePO4 precipitates on the root surface, in intercellular spaces and

406

vacuoles.3−35 ACS Paragon Plus Environment

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407 408

Identification of mixed agglomerates in Brassica napus root samples

409

Mixed agglomerates of CeO2 with either calcium phosphate (Ca3PO4) or potassium sulphate (K3SO4) were

410

identified by ToF-SIMS in Brassica napus root samples (Figure 5-b). These results point to the presence of

411

hetero agglomerates in plant roots and indicate a common crystallization point for both agglomerates, with a

412

mixed identity of CeO2 and the corresponding salt material. It is assumed that each agglomerate grows from its

413

crystallization point in one specific direction only. This illustrates that NPs are altered during their life cycle in

414

plant tissue. The presence of mixed agglomerates may indicate different solubility rates of different

415

agglomerates. This is probably due to the fact that compounds generally dissolved in the plant vascular system

416

may associate with NPs. Therefore, on the one hand they may move to plant parts where they normally do not

417

occur or, on the other hand, they may dissolve differently than agglomerates of pristine NPs.

418

This alteration may further influence the composition of the plant tissue, which in turn may have adverse, but

419

possibly even positive effects on the plant´s metabolism. For example, Rossi et al.28 were able to confirm their

420

hypothesis that CeO2-NPs modify the formation of plant apoplastic root barriers, with the consequence of an

421

enhanced salt stress tolerance of Brassica napus following exposure to CeO2-NPs in combination with NaCl in a

422

growth chamber. An enhanced transport of Na+ to the shoots and a decreased accumulation of the element in the

423

roots were identified to advance physiological performance.

424 425

Whereas particle uptake has already been shown in laboratory experiments, mostly during their germination

426

process, our study proved that particle uptake in plant roots is an important issue also under realistic exposure

427

conditions. Even though no significant concentrations could be detected within the edible parts of the

428

investigated plants, the presented data are relevant for human exposure by consumption of root crops.

429

Characterization of NP fate remains an urgent need for the depiction of processes in biological tissues. The

430

understanding of these mechanisms represents the basis for further hazard and risk assessment considering

431

realistic environmental conditions. SpICP-MS and ToF-SIMS were found to be suitable instruments for

432

providing the necessary information. However, it became also clear, that in case of Ce, an element that occurs

433

frequently in the environment, ICP-MS and spICP-MS measurements are challenging due to the high

434

background. Imaging techniques such as ToF-SIMS that are able to record the specific chemical composition of

435

particulate materials provide a suitable complementation. Beyond the characterization of particulate matter as

436

such, they are able to analyze the alteration of other substances in the vicinity of NPs. Such an analysis may give

437

indications of possible metabolic activities.



438 439

Acknowledgement

440

This study was funded by the Federal Ministry of Education and Research within the project DENANA

441

(03X0152). We thank Aaron Katz and Ajay Vikram Singh for proofreading the manuscript.

442 443

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Supporting information

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Scheme for the analysis of plant tissues with ICP-MS, spICP-MS and ToF-SIMS

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Detection and characterization of CeO2-NPs in Triticum aestivum root

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Ag distribution in a longitudinal tissue section of Brassica napus exposed to Ag-NP

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CeO+ distribution in a longitudinal Brassica napus tissue, exposed to CeO2-NP

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Particle size and crystallinity data of the tested nanomaterials


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Determination of Nanoparticle Uptake, Distribution, and

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