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APPENDIX. SUPPLEMENTARY DATA
Bioavailability of cobalt and iron from citric-acid-adsorbed CoFe2O4 nanoparticles in
the terrestrial isopod Porcellio scaber
Tea Romiha,*, Barbara Drašlera, Anita Jemeca, Damjana Drobnea, Sara Novaka, Miha
Golobiča, Darko Makovecb, Robert Susičc, Ksenija Kogejc
aDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000
Ljubljana, Slovenia
bJožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
cFaculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva cesta 5,
1000 Ljubljana, Slovenia
*Corresponding author: Tea Romih
Department of Biology
Biotechnical Faculty
University of Ljubljana
Večna pot 111
1000 Ljubljana, Slovenia
Tel: +386-1-3203375
Fax: +386-1-2573390
Email: tea.romih@student.uni-lj.si
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The Supplementary Data comprises:
Figure S1. Photograph of the acidified and non-acidified supernatants of CoFe2O4
nanoparticle suspensions and CoCl2 solutions.
Figure S2. Distributions of the hydrodynamic particle size, Rh, in the water-diluted
supernatants of bare and citric-acid-adsorbed CoFe2O4 nanoparticles.
Figure S3. Feeding rate (mg consumed leaf per mg of isopod mass) of the isopods fed for 14
days on food dosed with CoCl2, Fe3+ salt, bare CoFe2O4 nanoparticles or citric-acid-adsorbed
CoFe2O4 nanoparticles.
Table S1. Co and Fe concentrations in supernatants of ultracentrifuged suspensions of bare
CoFe2O4 nanoparticles, citric-acid-adsorbed CoFe2O4 nanoparticles, and solutions of CoCl2
and Fe3+ salt, as determined with flame atomic absorption spectrometry.
Supplementary methods:
Dynamic light scattering measurements of acidified and non-acidified supernatants of
the bare and citric-acid-adsorbed CoFe2O4 nanoparticles.
Equations used.
Supplementary Data references.
3
Supplementary Figure S1. Photograph of the acidified and non-acidified supernatants of CoFe2O4 nanoparticle
suspensions and CoCl2 solutions at concentrations of 2000 µg and 5000 µg Co/mL. (a) Supernatant of CoFe2O4
nanoparticles (2000 µg Co/mL) diluted with deionised water (1:1, v/v); (b) the same supernatant as for (a),
acidified with 1 M HCl (1:1, v/v). (c) Supernatant of CoFe2O4 nanoparticles (5000 µg Co/mL) diluted with
deionised water (1:1, v/v); (d) the same supernatant as for (c), acidified with 1 M HCl (1:1, v/v). (e)
Ultracentrifuged solution of CoCl2 (2000 µg Co/mL) diluted with deionised water (1:1, v/v); (f) the same
solution as for (e), acidified with 1 M HCl (1:1. v/v). (g) Ultracentrifuged solution of CoCl2 (5000 µg Co/mL)
diluted with deionised water (1:1, v/v); (h) the same solution as for (g), acidified with 1 M HCl (1:1,v/v).
a) b) c) d) e) f) g) h)
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Supplementary Table S1. Co and Fe concentrations in supernatants of ultracentrifuged suspensions of bare CoFe2O4 nanoparticles, citric-acid-adsorbed CoFe2O4
nanoparticles, and solutions of CoCl2 and Fe3+ salt, as measured with flame atomic absorption spectrometry.
Condition Nominal Co/Fe
concentration
(mg/L)
Co concentration (µg/mL);
mean ±SD (n = 3)
Fe concentration (µg/mL);
mean ±SD (n = 3)
Supernatant diluted with
deionised water (1:1)
Supernatant diluted with
1 M HCl (1:1)
Supernatant diluted with
deionised water (1:1)
Supernatant diluted
with 1 M HCl (1:1)
Bare CoFe2O4 nanoparticles 2000 (Co) 0.16 ±0.03 0.22 ±0.04 0.24 ±0.12 0.32 ±0.12
5000 (Co) < LOD
a
< LOD
a
< LOD
a
0.10 ±0.03
Citric-acid-adsorbed CoFe2O4
nanoparticles
2000 (Co) 111 ±5 111 ±3 224 ±6 224 ±1
5000 (Co) 52.4 ±0.5 51.7 ±0.1 46.0 ±0.8 46.2 ±0.3
CoCl2 2000 (Co) 2096 ±14 2127 ±48 Not measured Not measured
5000 (Co) 4451 ±151 4904 ±88 Not measured Not measured
C6H8O7 ·xFe
3+
·yNH3 3800 (Fe) Not measured Not measured 4047 ±156 3957 ±266
9500 (Fe) Not measured Not measured 8843 ±1368 9583 ±696
a Our estimation of the limit of detection of Co for the instrument used for the current analysis (Perkin Elmer AAnalyst 100) was 0.017 µg/mL, following the approach of
Armbruster & Pry (2008).
LOD, limit of detection
Note: Metal content in the supernatant is presented as in the original suspensions before the dilution (i.e. measured values of diluted samples were multiplied by a factor of 2).
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Dynamic light scattering measurements of suspensions of the bare and citric-acid-adsorbed
CoFe2O4 nanoparticles
To confirm the data from the dissolution measurements, both water-diluted and acid-diluted
supernatants of ultracentrifugated bare and citric-acid-adsorbed CoFe2O4 NPs (2000 µg
Co/mL only) were examined by dynamic light scattering using a 3D-DLS-SLS spectrometer
(LS Instruments GmbH, Fribourg, Switzerland) at the Faculty of Chemistry and Chemical
Technology, to determine whether all NPs had been removed from the supernatant.
The spectrometer was operated in 3D cross-correlation mode (Urban &
Schurtenberger, 1998). In this geometry, two parallel beams of the coherent incident light
(one above the other) are obtained using a beam splitter positioned between the laser and the
sample. The beams were focused in the centre of the studied sample, and the scattered light
was collected with two detectors (also one above the other). The 3D cross-correlation was
especially developed for studying strongly scattering samples, as CoFe2O4 NP dispersions in
our case, to suppress multiple scattering (Urban & Schurtenberger, 1998). Here, two coherent
incident light beams were generated with a 20 mV He-Ne laser (Uniphase JDL 1145P),
operating at λo = 632.8 nm. The instrument was equipped with a laser attenuation system,
combined with an online incident laser intensity measurement that allowed for the
normalisation of the static LS data. The correlation functions of the scattered light intensity,
G2(t), were recorded at an angle of 90°.
G2(t) was then converted into a correlation function of the scattered electric field, g1(t),
using the Siegert relationship (Brown, 1993; Schärtl, 2007):
|()|=
()(∞)
(∞)
(S1)
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where G2(∞) is the experimentally determined baseline and β is the coherence factor
determined by the geometry of the detection.
For monodispersed particles of small diameter compared to the wavelength of light,
and also for hard spheres of any size, the relaxation (or decay) time of g1(t), τ, was related to
the relaxation rate of g1(t), Γ, and the translation diffusion coefficient, D, by the relationships:
|()|=
= = (S2)
and
= = (S3)
The hydrodynamic radii, Rh, of the colloidal particles were then obtained from the diffusion
coefficient D, via the Stokes-Einstein equation:
=
(S4)
where k is the Boltzmann constant, T is the absolute temperature, and is the solvent (water)
viscosity.
Equation (S4) is strictly valid for monodispersed hard spheres. For polydispersed
samples, the correlation functions were analysed using the inverse Laplace transform
programme CONTIN (Provencher, 2011), by fitting the correlation curves with a sum of a
limited number of exponents (in our case, up to 50).
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()=∑()
(S5)
where A(τi) is the weight of the corresponding exponent with decay time τi. The weight A(τi) is
proportional to the LS intensity scattered by the ith-particle. Decay times τi are converted into
Rh using Equations (S3) and (S4), and are presented as distributions over the hydrodynamic
radii. Each scattering species of radius Rh,i is represented in this distribution by its relative
contribution; i.e., by the amplitude Ai(Ri), to the total intensity of scattered light.
We collected five correlation functions with a 60-s scan length. Each correlation curve
was analysed independently and compared with the averaged curve, to ensure that the
mathematical solution provided by CONTIN was correct.
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Supplementary Figure S2. Unweighted (black line), mass-weighted (magenta line), and number-weighted
(green line) distributions of the hydrodynamic particle size, Rh, in the water-diluted supernatant of bare CoFe2O4
nanoparticles from the suspension with a concentration of 2000 µg Co/mL (a), and from the water-diluted
supernatant of citric-acid-adsorbed CoFe2O4 nanoparticles from the suspension with a concentration 2000 µg
Co/mL (b).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1 10 100 1000 10000
Relative intensity, Ai
Hydrodynamic radius, Rh(nm)
Unweighted relative intensity
Mass-weighted relative intensity
Number-weighted relative intensity
Σ(Ai)=1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1 10 100 1000 10000
Relative intensity, Ai
Hydrodynamic radius, Rh(nm)
Unweighted relative intensity
Mass-weighted relative intensity
Number-weighted relative intensity
Σ(Ai)=1
a)
b)
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Feeding rate of isopods
Figure S3 shows the data obtained from two separate experiments. The first set of isopods
was exposed to citric-acid-adsorbed CoFe2O4 NPs, and the second one was exposed to Fe(III)
salt. Their corresponding control groups are marked as Control 1 and Control 2, respectively.
For comparison, we included also the (adapted) data from experiments on bare CoFe2O4 NPs
and CoCl2 with permission from Novak et al. (2013) (Cellular internalisation of dissolved
cobalt ions from ingested CoFe2O4 nanoparticles: In-vivo experimental evidence.
Environmental Science and Technology, 47 (10), 5400-5408; Copyright American Chemical
Society, 2014). There was a statistically significant difference (p < 0.001) in the feeding rates
between the control group and both of the groups exposed to CoCl2, as shown in the Figure
S3. There were also statistically significant differences (p < 0.05) in comparison to control in
the group of isopods exposed to bare CoFe2O4 NPs at 2000 µg Co/g food, and in the group
exposed to citric-acid-adsorbed CoFe2O4 NPs at 5000 µg Co/g food (Figure S3). The
decrease in the feeding rate was not concentration dependent. There was no significant
difference in the feeding rates between the isopods from the two control groups. There were
significant differences in the feeding rates among the two concentrations of the CoCl2-treated
samples (p < 0.01, not marked), whereas there were no significant differences among the two
concentrations in the case of bare or citric-acid-adsorbed CoFe2O4 NPs. There were no
significant differences in the feeding rates observed among isopods exposed to Fe(III) salt and
the control group, neither between the two concentrations of Fe(III) salt or between the same
concentration of iron originating from salt or from both types of bare and citric-acid-adsorbed
CoFe2O4 NPs.
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Supplementary Figure S3. The feeding rate (mg consumed leaf per mg of isopod mass) of P. scaber fed for 14
days on food dosed with CoCl2 (Co2+), Fe(III) salt (Fe3+), bare CoFe2O4 nanoparticles (CF) or citric-acid-
adsorbed CoFe2O4 nanoparticles (CF-CA). Symbols on the box plot represent the minimum and maximum data
values (whiskers), mean value (□), 75th percentile (upper edge of box), 25th percentile (lower edge of box),
median (line in box) and max and min values ( - ). Statistically significant differences between exposed and
control isopods (C1, C2; both controls were generated in this study) are indicated by *** (p < 0.001), ** (p <
0.01) and * (p < 0.05). Data for groups of isopods exposed to CoCl2 and both nanoparticles were compared to
Control 1, while isopods exposed to the Fe(III) salt were compared to Control 2. There were no significant
differences in the feeding rate between the isopods in the control groups from the two experiments. Nominal
exposure concentrations (2000 µg or 5000 µg Co/g of leaf, or 3800 µg or 9500 µg Fe/g of leaf; originated from
Co or Fe salts or from suspensions of bare CoFe2O4 or citric-acid-adsorbed CoFe2O4 nanoparticles) are provided
on the x-axis. N, number of isopods that were analysed in each group (from 15 per group exposed at the
beginning of the feeding). For the purposes of comparison, the unshaded box plots are reprinted (adapted) with
permission from Novak et al. (2013) (Cellular internalisation of dissolved cobalt ions from ingested CoFe2O4
nanoparticles: In-vivo experimental evidence. Environmental Science and Technology, 47 (10), 5400–5408).
Copyright (2014) American Chemical Society.
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Equations used
(%)=
(µ
)
(µ
)×100 (S6)
(µ)=
Actual measured concentration of metal on leaves after the experiment µ
× consumed leaves mass (g) (S7)
(µ)= (%)
×
Total consumed metal with food (µg) (S8)
(µ/ ) =
(µ)
() (S9)
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REFERENCES
Armbruster, D.A., Pry, T., 2008. Limit of blank, limit of detection and limit of quantitation.
Clin. Biochem. Rev., 29, Suppl. 1: S49-S52.
Brown, W. Ed.: Dynamic Light Scattering: The Method and Some Applications; Clarendon
Press, Oxford, UK, 1993.
Novak, S., Drobne, D., Golobič, M., Zupanc, J., Romih, T., Gianoncelli, A., Kiskinova, M.,
Kaulich, B., Pelicon, P., Vavpetič, P., Jeromel, L., Ogrinc, N., Makovec, D., 2013.
Cellular internalization of dissolved cobalt ions from ingested CoFe2O4 nanoparticles: In-
vivo experimental evidence. Environ. Sci. Technol., 47, 5400–5408.
Provencher, S.W. CONTIN: A general purpose constrained regularization program for
inverting noisy linear algebraic and integral equations; accessed 2011;
http://s-provencher.com/pages/contin.shtml
Schärtl, W. Light Scattering from Polymer Solutions and Nanoparticle Dispersions, Springer
Verlag, Berlin Heidelberg, Germany, 2007.
Urban, C., Schurtenberger, P.J., 1998. Characterization of turbid colloidal suspensions using
light scattering techniques combined with cross-correlation methods. Colloid Interface
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