Supplemental data
Effect of 10 different TiO2 and ZrO2 (nano)materials on the soil invertebrate Enchytraeus
crypticus
Susana IL Gomes *†, Gianvito Caputo ‡, Nicola Pinna ‡, Janeck J Scott-Fordsmand §, Mónica
JB Amorim †
†Department
of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal
‡Department
of Chemistry & CICECO, University of Aveiro, 3810-193 Aveiro, Portugal and
Humboldt-Universität zu Berlin, Institut für Chemie, Brook-Taylor-Str. 2, 12489 Berlin,
Germany.
§Department of Bioscience, Aarhus University, Vejlsovej 25, PO BOX 314, DK-8600 Silkeborg,
Denmark
* Corresponding author: susana.gomes@ua.pt
* Corresponding author: Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro,
Portugal. Tel.:+351 234 370790. Fax: +351 234 372 587. E-mail address: susana.gomes@ua.pt
Experimental section
Synthesis and ligand exchange procedure
All the reagents were purchased form Sigma Aldrich and were used without further purification.
All the preparations were conducted in a glove box (H 2O and O2 below 1 ppm). Titanium dioxide (TiO2)
and Zirconium dioxide (ZrO2) nanoparticles have been synthesized according the following procedure. 500
mg of Titanium (IV) isopropoxide (99.9%) and 500 mg of Zirconium (IV) isopropoxide isopropanol
complex (99.9%) were added to 20 ml of benzyl alcohol (99%), respectively. The solutions were stirred
and transferred to a 45ml Teflon cup and inserted into a stainless steel autoclave and heated at 250 and 230
°C for 48 h, respectively. After the syntheses, the particles were purified by repeating three
purification/centrifugation cycles with ethanol. As obtained TiO2 and ZrO2 nanoparticles are denoted IHT
and IHZ, respectively.
In order to ensure colloidal dispersion in water, the particles were modified with glacial acetic acid using a
ligand exchange procedure. Briefly, 100 mg of preformed TiO 2 or ZrO2 nanocrystals were dispersed in 5
ml of chloroform. Then, 200 mg of glacial acetic acid were added to the dispersion. The obtained mixtures
were stirred overnight at room temperature. The particles were precipitated twice with ethanol to remove
the excess of acetic acid and the supernatant was discarded. To increase pH, 1-2 mg of sodium acetate was
added. The final pH of the solution was ca. 5. TiO2 and ZrO2 water dispersions are denoted IHTa and IHZa,
respectively.
Characterization
Structural characterization of the nanoparticles was carried out by X-ray powder diffraction (XRD) using a
Cu-Kα radiation operating at 45 kV and 40 mA on an X’Pert MPD Philips diffractometer. The patterns
were acquired in the 2θ range from 3 to 70° using a step size of 0.2°.
The size and shape of the particles prior and after surface modification were evaluated by transmission
electron microscopy (TEM). A Hitachi-9000 TEM operating at 300 kV was used. For TEM studies one or
two drops of the nanoparticle dispersions in ethanol and water solution were deposited on copper TEM
grids covered with amorphous carbon.
Fourier transform infrared (FTIR) spectra were acquired between 4000-600 cm-1, with a 4 cm-1 resolution
in Attenuated Total Reflectance (ATR) mode, using a Nicolet iS5 equipment.
Dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer Nano (Malvern
Instruments Ltd.)
The Brunauer-Emmett-Teller (BET) surface area was determined using a Micrometrics Gemini 2380
nitrogen adsorption apparatus. The samples were degassed at 150°C prior to measurements.
Discussion
Fig. S1 TEM micrographs of (a) pristine (IHT) and (b) surface modified TiO 2 (IHTA) nanoparticles
In Figure S1, two representative TEM images of TiO2 nanocrystals before and after surface modification
are shown. The nanoparticles possess a pseudo-spherical morphology. The average cristallite size,
determined from the XRD patterns (Fig. S4) using the Scherrer equation, is 8.7 nm and is in good agreement
with the size determined from TEM measurements (Fig. S3b). The as-synthesized nanoparticles are rather
aggregated (Fig. S1a). On the other hand, after surface modification the TiO 2 nanoparticles are better
dispersed on the carbon coated TEM grid (Fig. S2a).
1
Scherrer equation is a formula that relates the size of sub-micrometre particles, or crystallites, in a solid to the broadening of a peak
in a diffraction pattern (XRD). See also: The Scherrer Formula for X-Ray Particle Size Determination. (1939) A. L. Patterson Phys.
Rev. 56, 978
Fig. S2 TEM images of (a) pristine (IHZ) and (b) surface modified ZrO 2 (IHZa) nanoparticles
In Figure S2, TEM images related to ZrO2 nanocrystals are shown. After the ligand exchange procedure
(Fig. S2b), the particles show a lower degree of aggregation compared to the pristine ZrO 2 nanocrystals
(Fig. S2a). The particles possess a pseudo-spherical shape. Their crystallize size, evaluated from the XRD
patterns (Fig. S4) by the Scherrer equation, is 3.3 nm and is comparable to the size determined by TEM
measurements (Fig. S3a).
Fig. S3 Statistical analysis performed on ZrO2 (IHZa) and TiO2 (IHTa) nanoparticles, respectively
In Figure S3, the size distribution (TEM based) related to IHZa and IHTa particles investigated in the
present work is presented. For the statistical analysis, 250 ZrO 2 and 300 TiO2 particles have been counted
using an imaging processing software (ImageJ ®). In particular, IHZa nanocrystals possess a unimodal
distribution (Fig. S7a), and their calculated average size is 4.0±0.6 nm.
The IHTa nanoparticles present a broad range of sizes (Fig. S7b). The average size determined is 9.0±2.8
nm.
Fig. S4 XRD patterns of (1) TiO2 (IHT) and (2) ZrO2 (IHZ) nanoparticles and corresponding (a-b) JPCDS
cards, respectively.
The powder X-ray diffractograms related to the nanoparticles used in this work are shown in Figure S4.
TiO2 nanoparticles (1, a) crystallize in anatase polymorph (JPCDS card 004-0477). As-synthesized ZrO2
nanocrystals (2, b) are present in the tetragonal modification (JPCDS card 049-1642) with a small
percentage of monoclinic phase. In agreement with a structural study recently reported. [3]
Fig. S5 XRD pattern of bulk ZrO2 powder and relative JPCDS reference card, respectively
In Figure S5, the diffractrogram related to bulk ZrO 2 is shown. The material has a crystallite size of 55.8
nm as determined from the Scherrer equation. It crystallizes in the monoclinic polymorph.
Fig. S6 (a) Overall and (b-c) detailed FT-IR spectra of as-synthesized (black trace) and modified (red trace)
TiO2 nanocrystals, respectively
The FTIR spectra of TiO2 nanoparticles before and after surface modification are shown in Figure S6. In
the spectral region between 3600 and 3000 cm-1, the stretching band ascribable to –OH groups bound to the
nanocrystal surface is visible (Fig. S6b). This is further confirmed by the –OH bending signal situated at
1630 cm-1 for the pristine and modified particles, respectively.
Moreover, a small peak due to the stretching of the –CH band of phenyl rings is detectable at 3080 cm-1
(Fig. S6b, black line). This feature disappears after the ligand exchange procedure. In fact, the typical
signals due the stretching vibrations of the methyl group of the acetic acid are present between 2900-3000
cm-1 (Fig. S6b, red line).
Interestingly, the region between 1800 and 1000 cm-1 is strongly affected after the surface modification
step (Fig. S6c). The presence of benzoate species can be evidenced in the initial spectrum of the pristine
nanoparticles (Fig. S6a, black trace). In fact, the asymmetric (1540 cm-1) and symmetric (1420 cm-1)
stretching bands can be detected, in addition to the frequencies related to the C-C of the phenyl rings. The
difference between the two bands is ca. 120 cm-1. Benzoate species attached to the surface of the
nanoparticles are often detected upon reaction of metal alkoxides in benzyl alcohol. [1-3] Upon surface
modification, the carboxylate symmetric band is shifted (Fig. S6, red trace) and the contribution of the
phenyl rings disappear. The difference is ca. 110 cm-1, pointing out the presence of a bidentate coordination
of the surface acetate species.[4] Below 1000 cm-1, very intense absorption tails due to the stretching of TiO bonds are present.
Fig. S7 (a) Overall and (b-c) detailed FT-IR spectra of as-synthesized (black trace) and modified (red trace)
ZrO2 nanocrystals, respectively
In Figure S7, the FTIR spectra of ZrO2 nanoparticles before and after the surface modification procedure
are shown.
Similarly to TiO2 spectra (cf. Fig. S6), the signals in the region between 4000 and 2500 cm -1 are modified
(Fig. S7b). It can be noted the presence of the typical –CH3 stretching bands and the absence of the –CH
vibrations of the phenyl rings after the treatment with acetic acid. This is confirmed by the lack of features
between 1200 and 1000 cm-1 ascribable to the bending vibrations of the –CH bonds and to the stretching
vibrations of the C-C bonds in the phenyl ring at 1600 cm-1 (Fig. S7c). This proves that the benzoate species
at the surface of the pristine nanoparticles are removed after ligand exchange process. [1-3] The carboxylate
vibrations of the acetate species have a different signature compared to the ones of the benzoates. Indeed,
the asymmetric and symmetric vibrations for the as-synthesized ZrO2 nanoparticles are found at 1545 cm1
and 1430 cm-1, respectively. After the treatment with acetic acid they are centered at 1560 cm-1 and 1410
cm-1 (Fig. S7c, red trace). The calculated difference between these two bands is ca. 150 cm-1, revealing the
possible presence of a bridging coordination.[4] The sharp absorption features below 1000 cm-1 can be
attributed to the stretching of the Zr-O bonds.
Fig. S8 DLS plots related to NM104: as prepared in ultra-pure water (red line); in the test media, ISO water
(green line); in the test media after 5 days of exposure under standard illumination (black line); and in test
media after 5 days of exposure under UV radiation (blue line)
In Figure S8, four representative DLS distributions related to NM104 samples are shown. It can be noticed
the increase of the nanoparticle size during the test duration (5 days) and higher increase in agglomeration
size after the exposure under UV radiation (blue line).
Fig. S9 N2 adsorption-desorption isotherm of bulk ZrO2.
In Figure S9, a representative N2 adsorption-desorption isotherm plot of bulk ZrO 2 is displayed. The
calculated BET surface area is 8.4 m2/g. Similar analysis was performed for the IHT and IHZ.
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