Supplementary Material for:
Multiwalled carbon nanotubes in alfalfa and wheat:
toxicology and uptake
Pola Miralles1, Errin Johnson2, Tamara L. Church1, Andrew T. Harris1*
Item
Page
1. Exposure-media preparation
S2
2. Carbon nanotube (CNT) characterization
S2
3. Elongation of alfalfa and wheat seedlings
S9
4. Optical microscope images of semi-thin plant sections
S13
5. References
S15
S1
1. Exposure-media preparation
Carbon nanotubes (CNTs) were ultrasonicated in deionized water and mixed by
vigorous shaking with the agar substrate. Prior to imaging, CNT-agar was oven dried
for 24 h. The CNT-agar matrix contained both isolated CNTs and some aggregates,
the former facilitating CNT availability to the plants.
Figure S1. Scanning electron microscope images of carbon nanotubes dispersed in
agar. Image D shows the outlined area of image B at higher magnification.
2. Carbon nanotube (CNT) characterization
CNTs were characterized by thermogravimetric analysis (TGA; Figure S2). The
samples were heated at 10 C/min to 1000 C under a flow of 60 mL air and 40
mL/min N2. A threefold increase in CNT content was observed following purification,
with no significant change in material crystallinity.
S2
.
Figure S2. Thermogravimetric analysis of as-synthesized, purified and Fe3O4functionalized carbon nanotubes (CNTs) A) Weight-loss profiles. B) Derivativeweight-loss profiles.
S3
Thermogravimetric analysis residues (≥4 samples for each CNT type) were digested
in aqua regia to extract the Fe, which was quantitatively analyzed by inductively
coupled plasma-atomic emission spectroscopy (Figure S3). Catalytic impurities in
purified CNTs were composed of 5.3 ± 0.3 wt% Fe and 19.7 ± 0.8 wt% Al2O3.
Figure S3. Composition of catalytic impurities present in the as-synthesized and
purified carbon nanotube (CNT) samples.
The surface characteristics of the CNTs were determined by N2 adsorptiondesorption at 77 K. Specific surface areas were calculated from five adsorption
points using the Brunauer-Emmett-Teller method over P/P0 = 0.05–0.25. All
materials displayed type III isotherms with hysteresis loops. Pore-size distributions
were calculated from the adsorption branch using the Barret-Joyner-Halenda
method. The majority of pores were mesopores according to IUPAC classification.
S4
Figure S4. N2 adsorption/desorption data for carbon nanotube (CNT) materials. A) N2
adsorption/desorption isotherms. B) Pore-size distributions.
S5
CNT samples were also analyzed by Raman (Figure S5) and Fourier-transform
infrared (FTIR) spectroscopy (Figure S6).
Figure S5. Raman spectra of as-synthesized, purified and Fe3O4-functionalized
carbon nanotubes (CNTs). The D band (1349 cm-1), G band (1585 cm-1) and D* band
(2700 cm-1) are clearly visible in the CNT samples. The lack of peak shifts and the
similarity in full widths at half height (refer to Table 1) confirm that the quality of
CNTs was not modified by purification or Fe3O4 functionalization.
S6
Figure S6. Fourier-transformed infrared spectra of as-synthesized, purified and
Fe3O4-functionalized carbon nanotubes (CNTs). The three materials had peaks in very
similar positions. The as-synthesized material also contained functional groups.
The FTIR spectra of the CNT samples showed evidence of multiple functional groups.
The peak detected at ~2952 cm-1 is assigned to the asymmetric C-H stretching of
methyl groups (as C-H CH3), and the peak at ~2922 cm-1 corresponds to the
asymmetric C-H stretching of methyl groups bound to aromatics or to methylene
groups (as C-H CH3-Ar, -CH2-alkane); these are associated with defect sites on the
walls of functionalized CNTs[1-3]. The bands at ~2870 cm-1 and 2850 cm-1
correspond to the symmetric C-H stretching of the methyl and methylene groups,
respectively (s C-H CH3 and CH2)[1-4]. Analysis of the fingerprint region also
indicated the presence of these groups, with bands at ~1448 cm-1 and ~1370 cm-1
S7
being characteristic of the asymmetric and symmetric C-H deformations,
respectively, of CH3 groups[4, 5].
The stretching of C=O bonds are responsible for the sharp peak detected at ~1730
cm-1 (C=O) and the weaker stretch (C=O) at 1832 cm-1. As there is no band for O-H
vibration (usually observed as very broad band at 3500–2500 cm-1, these C=O groups
could be assigned to aliphatic ketone, aldehyde and/or ester groups[3, 4]. However,
the strong bands at 1230 cm-1 (asC-O-C), 1167 cm-1 (C-O) and 1124 cm-1 (sC-O-C)
are evidence of the last group. The strong line detected at ~1560 cm-1 is the cause of
some controversy in the literature: some authors have assigned it to the stretching
mode of the inherent structure of CNTs[3, 6], whereas others report that the band
corresponds to carboxyl and carbonyl groups[7, 8]. We detect this peak in all of the
as-synthesized, purified and Fe3O4-functionalized CNT samples, confirming that it
belongs to the C=C stretching vibration from the inherent structure of graphitic CNT
walls[4].
S8
3. Elongation of Alfalfa and Wheat Seedlings
Seeds from each test species were germinated in deionized water and, once the
radicle was 20 mm long, transplanted to the CNT-agar (40–2560 mg/L CNTs). As the
CNTs contained 25 wt% catalytic impurities, agar containing only these impurities at
one-quarter of the nominal CNT concentration was also used. The elongations of
each seedling were measured after 6 d.
Figure S7. Shoot elongation of alfalfa seedlings. The first leaf emerged in some
seedlings, but no relevant differences were observed in this regard. Abscissae
represent nominal concentrations. († = statistically significant difference between
CNT and catalyst treatments).
S9
Figure S8. Cotyledon elongation of alfalfa seedlings. No significant differences were
observed between treatments. Abscissae represent nominal concentrations.
S10
Figure S9. Shoot elongation of wheat seedlings. No significant differences were
observed between treatments. Abscissae represent nominal concentrations.
S11
Figure S10. Leaf elongation of wheat seedlings. No significant differences were
observed between treatments. Abscissae represent nominal concentrations.
S12
Figure S11. Coleoptile elongation of wheat seedlings. No significant differences were
observed between treatments. Abscissae represent nominal concentrations.
4. Optical microscope images of semi-thin plant sections
Semi-thin sections of the root tips of alfalfa and wheat were examined by optical
microscopy. There were no obvious differences between the control and exposed
plants, with normal macrostructure being observed or both alfalfa and wheat. No
apparent tissue damage or holes were detected.
S13
Figure S12. Semi-thin sections of root tips (5 mm from the main root tip) of alfalfa
and wheat grown in agar without carbon nanotubes (CNTs) (Control), with 2560
mg/L CNT and with 2560 mg/L Fe3O4-functionalized CNT.
S14
5. References
1.
2.
3.
4.
5.
6.
7.
8.
Chen C, Liang B, Ogino A, Wang X, Nagatsu M. 2009 Oxygen functionalization
of multiwall carbon nanotubes by microwave-excited surface-wave plasma
treatment. J. Phys. Chem. C 113, 7659-7665. (DOI: 10.1021/jp9012015.)
Scheibe B, Borowiak-Palen E, Kalenczuk RJ. 2010 Oxidation and reduction of
multiwalled carbon nanotubes - preparation and characterization. Mater.
Charact. 61, 185-191. (DOI: 10.1016/j.matchar.2009.11.008.)
Zhao C, Ji L, Liu H, Hu G, Zhang S, Yang M, et al. 2004 Functionalized carbon
nanotubes containing isocyanate groups. J. Solid State Chem. 177, 4394-4398.
(DOI: http://dx.doi.org/10.1016/j.jssc.2004.09.036.)
Socrates G. Infrared and Raman characteristic group frequencies: Tables and
charts. 3rd ed. New York: Wiley; 2001.
Peng H, Alemany LB, Margrave JL, Khabashesku VN. 2003 Sidewall carboxylic
acid functionalization of single-walled carbon nanotubes. J. Am. Chem. Soc.
125, 15174-15182. (DOI: 10.1021/ja037746s.)
Vesali Naseh M, Khodadadi AA, Mortazavi Y, Alizadeh Sahraei O, Pourfayaz F,
Mosadegh Sedghi S. 2009 Functionalization of carbon nanotubes using nitric
acid oxidation and DBD plasma. World Acad. Sci. Eng. Technol. 49, 177-179.
Osswald S, Havel M, Gogotsi Y. 2007 Monitoring oxidation of multiwalled
carbon nanotubes by Raman spectroscopy. J. Raman Spectrosc. 38, 728-736.
(DOI: 10.1002/jrs.168.)
Ovejero G, Sotelo JL, Romero MD, Rodríguez A, Ocaña MA, Rodríguez G, et al.
2006 Multiwalled carbon nanotubes for liquid-phase oxidation.
Functionalization, characterization, and catalytic activity. Ind. Eng. Chem. Res.
45, 2206-2212. (DOI: 10.1021/ie051079p.)
S15