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Environmental Aspects of Nanotechnology
Digestion Coupled with Programmed Thermal Analysis for Quantification of Multiwall Carbon Nanotubes in Plant Tissues Kamol K. Das, Lucas Bancroft, Xiaoliang Wang, Judith C Chow, Baoshan Xing, and Yu Yang Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.8b00287 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018
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Environmental Science & Technology Letters
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Digestion Coupled with Programmed Thermal Analysis for Quantification of
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Multiwall Carbon Nanotubes in Plant Tissues
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Kamol K. Das1, Lucas Bancroft2, Xiaoliang Wang2, Judith C. Chow2, Baoshan Xing3, and Yu
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Yang1* 1
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Department of Civil and Environmental Engineering, University of Nevada–Reno, 1664 N. Virginia Street, Reno, NV 89557, USA;
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2215 Raggio Parkway, Reno, NV 89512, USA;
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Division of Atmospheric Sciences, Desert Research Institute,
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Stockbridge School of Agriculture, University of Massachusetts–Amherst, 410 Paige
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Laboratory, Amherst, MA 01003, USA.
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*Corresponding author: Yu Yang, email at
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Abstract. Rapidly growing application of carbon nanotubes (CNTs) for industry and
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consumer products will inevitably lead to their accumulation in the environment. Protection of
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food safety from contamination by CNTs and best-practice management of agricultural
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application of CNTs require quantification of CNTs in agricultural plants. Herein, a novel
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method of digestion coupled with programmed thermal analysis (PTA) was developed for
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quantitative analysis of multiwall CNTs (MWCNTs) in plant (lettuce) tissues. MWCNT-bound
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carbon was linearly correlated with elemental carbon (EC) detected by PTA, including EC1
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(58.5%) (evolved at 580 ˚C) and EC2 (41.5%) (evolved at 740 ˚C) corresponding to less stable
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and stable carbon, respectively. The background plant materials could interfere with EC
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quantification of CNTs, as a substantial fraction of the plant biomass was charred during the
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thermal analysis. Sequential digestion with concentrated nitric acid (HNO3) and sulfuric acid
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(H2SO4) effectively minimized the interferences caused by the lettuce tissues, reducing the
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background EC generated from leaf tissues to 10.73 ± 10.26 µg C/g. By coupling digestion with
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PTA, a detection limit of 64.9 µg CNT-C/g plant tissues was achieved. This method can be
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applied for unambiguous quantification of CNTs in plant tissues at low concentrations and
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provide critical information for evaluating risk of CNTs exposure through crops and optimizing
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CNTs applications in agriculture.
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Environmental Science & Technology Letters
INTRODUCTION
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The increasing application of carbon nanotubes (CNTs) in consumer products and
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industry, such as polymeric composite materials (PCM), electronic devices, and others, will lead
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to their accumulation in soils as a result of release throughout the products’ life cycle.1, 2
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Previous studies showed that a substantial fraction (5.0-35.7%) of CNTs are released when PCM
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degrades, which can be affected by sunlight irradiation and other environmental processes.3, 4
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CNTs in soils can be taken up and translocated by agricultural crops, and consequently lead to
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potential human health risk through dietary exposure to CNTs.5, 6 To the other side, CNTs have
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been shown to enhance photosynthesis, seed germination, and yields of spinach, tomato,
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soybean, and corn with great potentials for agricultural application.7-9 Currently, there is rare
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quantitative information about the plant uptake of CNTs and their concentration in agricultural
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plants, because of challenges with efficient extraction and quantification of CNTs.
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CNTs in plant tissues have been examined by a variety of methods, such as scanning
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electron microscopy, transmission electron microscopy, Raman spectroscopy, microwave-
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induced heating, and near infrared analysis.6, 9-11 Most of these approaches are unable to quantify
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CNTs in plant tissues due to their low concentrations, heterogeneous distributions in plant
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tissues, and variations in the physicochemical properties of CNTs such as aggregation and
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components of surface functional groups.10 By using carbon-14 (14C)-labeled multiwall CNTs
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(MWCNTs), Zhao et al.5 demonstrated that 0.53–76.6 µg/g CNTs were accumulated in leaves,
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stems, and roots tissues of rice, maize, Arabidopsis thaliana, and soybean, noting that the applied
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concentrations of CNTs in the incubation media were much higher than those for naturally
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occurring waters or soils. Such radiocarbon-based analysis cannot be applied to natural samples,
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as 14C-labeled CNTs are not used in the real application. Limited methods, such as microwave
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irradiation-based analysis, have been developed for the quantification of non-isotope-labeled
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CNTs in environmental samples.12, 13 More efforts are warranted for quantifying CNTs in plant
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tissues to advance our understanding of CNTs toxicity and optimize their agricultural
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application.
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CNTs have similar physicochemical properties as naturally occurring pyrogenic carbon.
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Pyrogenic carbon in natural environment such as char/soot particles in soil and sediment have
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been quantified by programmed thermal analysis (PTA).14-16 The low detection limit of carbon in
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PTA allows the quantification of low-concentration pyrogenic carbon in natural environment.15,
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no studies that analyzed CNTs in plant tissues with PTA, which can be challenged by heat-
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derived charring process of plant tissues. The charring of leaf and its constituents such as
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hemicellulose, cellulose, and lignin during thermal treatments has been shown in previous
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studies.19, 20
Recently, PTA was used for analyzing CNTs in rat lung tissues.17, 18 However, there have been
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This study aimed to develop a PTA-based method for quantitative analysis of pristine
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MWCNTs (p-MWCNTs) in plant tissues. Digestion method was developed and optimized to
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minimize the interferences of plant tissue on the analysis of p-MWCNTs.
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MATERIALS AND METHODS
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Programmed thermal analysis (PTA). The IMPROVE-A protocol was applied to
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determine organic carbon (OC) and elemental carbon (EC) with DRI Model 2001
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Thermal/Optical Carbon Analyzer.15-18, 21-24 The volatilized carbon was oxidized to carbon
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dioxide (CO2), followed by reduction to methane (CH4) for analysis with a flame ionization 4 ACS Paragon Plus Environment
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detector (FID). A helium/neon (He/Ne) laser light (633 nm) was reflected and transmitted
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through the quartz filter for continuous measurement of laser reflectance and transmittance.
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Following the widely used IMPROVE-A protocol, the OC evolved under He atmosphere as the
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sample was heated stepwise to 140, 280, 480, and 580 ˚C were defined as OC1, OC2, OC3, and
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OC4, respectively. 14, 21, 22 A mixture of 2% O2 and 98% He atmosphere was used to oxidize EC
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at 580, 740, and 840 ˚C to quantify EC1, EC2, and EC3, respectively. 14, 21, 22 EC from samples
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was calculated based on the obtained EC1 and EC2 (EC = EC1 + EC2), when EC3 was not
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observed in our samples. At the end of each run, 1 mL of 5% CH4 in He was injected as an
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internal standard. p-MWCNT was suspended with a nonionic surfactant Triton X-100 and
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analyzed with PTA. The details of the reagents and MWCNTs suspension preparation are
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provided in the Text S1 and S2 of the Supporting Information (SI). The thermal stability of
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MWCNTs and TX-100 was studied with thermogravitational analysis (TGA) (SI, Text S3).
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Digestion and extraction of p-MWCNTs from plant tissues. Research-grade p-
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MWCNTs, with 95% C and average diameter of 9.5 nm, were purchased from Nanocyl products
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(SI, Text S1, Table S1). Eight-week-old lettuce (Lactuca sativa, Bionda Ricciolina), a
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commodity plant previously used for studying the toxicity of CNTs,25-27 was purchased from a
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local nursery (Sparks, Nevada). The plants were washed with doubly deionized water (DDW)
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(18.3 MΩˑcm) and separated into leaves, stems, and roots, and dried in an oven at 80 ˚C for 12
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hours. The dried leaf tissues were ground and sieved with a 60-mesh (< 0.25 mm) sieve,
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followed by HNO3-digestion with 125-600 µg MWCNTs/g leaf (directly spiked with dried leaf
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tissues) or without p-MWCNTs. 1.0 mL HNO3 (15.8 M) was added to ~20.0 mg of leaf tissues in
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a 15.0 mL Corex glass centrifuge tube. The centrifuge tube was placed inside the Corex
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digestion tube containing 15.0 mL DDW in a chamber for 12 hr digestion at 60 ˚C. The digested 5 ACS Paragon Plus Environment
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samples were centrifuged at 3000 rpm for 15 minutes and washed with DDW. The precipitates
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were re-suspended with about 20 µL DDW and collected with a glass pipette, deposited to a
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quartz fiber filter punch (0.51 cm2) for PTA.
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To further remove the background leaf tissues and minimize their influences on EC
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analysis, double digestion method was applied to the final p-MWCNTs extraction process.
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Residues from HNO3 digestion were transferred to micro-centrifuge tubes and centrifuged at
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14,500 rpm for 15 minutes. The precipitates were suspended with 0.3 mL secondary digestion
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reagent (H2SO4 (18.4 M), or HCl (12 M), or HNO3 (15.8 M), or NH4OH (14.8 M)) and
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transferred to glass centrifuge tubes for 3 hr digestion at 60 ˚C. 5.0 mL DDW was added to the
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extract and centrifuged at 3000 rpm for 15 minutes. This process was repeated for additional
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three times to neutralize the extract with DDW. The final sample slurry was transferred onto a
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quartz fiber filter punch for PTA. An aliquot of p-MWCNTs suspension only was also digested
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in the same way and prepared for PTA. The obtained EC1 and EC2 from the digested p-
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MWCNTs were compared with those of original p-MWCNTs. The protocol for additional
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treatment was described in Text S4 (SI).
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RESULTS AND DISCUSSION Thermal stability of original p-MWCNTs and PTA. Following the thermal stability
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analysis for original (as received) p-MWCNTs and TX-100 with TGA (SI, Text S5, Figure S1-
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2), PTA was used for studying thermal decomposition of p-MWCNTs suspension in TX-100
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(Figure 1A, SI, Figure S3). The FID signals of TX-100 showed that the surfactant mainly
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evolved as OC (SI, Figure S3), while p-MWCNTs decomposed at high temperature under
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oxidative atmosphere as EC (Figure 1A). The decomposition of TX-100 at low temperature was
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also observed by TGA (SI, Text S5, Figure S2). Same amount of OC was detected for solutions
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(0-20 µL) with and without p-MWCNTs (t-test, p>0.05, relative difference
