Chemosphere 209 (2018) 815e822

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Chemosphere
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Analysis of gold and silver nanoparticles internalized by zebrafish

(Danio rerio) using single particle-inductively coupled plasma-mass
spectrometry
Hwa Kyung Sung a, b, Eunhye Jo a, Eunjeong Kim a, Sun-kyoung Yoo a, Jae-woo Lee a,
Pil-je Kim a, Younghun Kim b, *, Ig-Chun Eom a, **
a
Risk Assessment Division, Environmental Health Research Department, National Institute of Environmental Research, Incheon 22689, South Korea
b
Department of Chemical Engineering, Kwangwoon University, Seoul 01897, South Korea

h i g h l i g h t s g r a p h i c a l a b s t r a c t

SP-ICP-MS was applied to analyze the

exposure and uptake of NPs by living
zebrafish.

High recovery efficiency of NPs from
the liver of zebrafish was obtained by
alkaline extraction.

Uptake amounts of AgNPs in the liver
and intestine were significantly
higher than those of AuNPs.

SP-ICP-MS enabled an accurate anal-
ysis of NP features in an aquatic
organism.

a r t i c l e i n f o a b s t r a c t

Article history: With the increase in the application of nano-consumer products containing engineered nanoparticles
Received 31 January 2018 (NPs), the unintended environmental exposure to NPs has been inevitable. Because of the bio-
Received in revised form accumulation of NPs, concern about their potential cytotoxicity to aquatic organisms is also growing.
17 June 2018
Although measuring tools for analyzing particle size and/or concentration of NPs in intracellular uptake
Accepted 23 June 2018
Available online 26 June 2018
of tissues have been well developed, a simultaneous analysis of the two characteristics is difficult. The
objective of this study was to use single particle-inductively coupled plasma-mass spectrometry (sp-ICP-
Handling Editor: Petra Petra Krystek MS) to measure the bioaccumulation and particle size changes of NPs exposed to zebrafish (Danio rerio)
for 7 days. The uptake of NPs in the liver, intestine, and gill tissues was confirmed by electron microscopic
Keywords: (EM) analysis. However, the primary particle size of NPs in tissues could not be determined by the EM
Gold nanoparticles analysis. Therefore, sp-ICP-MS coupled with alkaline digestion was used for the easy extraction and
Silver nanoparticles immediate analysis of NPs from tissues. Zebrafish were exposed to four NPs (30 and 80 nm gold/silver
Zebrafish NPs; AuNPs/AgNPs). Uptake amounts of AgNPs in the liver and intestine were significantly higher than
sp-ICP-MS
those of AuNPs. Although larger NPs were finally accumulated in the liver and intestine tissues, most of
the smaller NPs were filtered in the gills. The sp-ICP-MS method coupled with alkaline digestion enabled
the accurate analysis of size, size distribution, and mass concentration of NPs in an aquatic organism.
© 2018 Elsevier Ltd. All rights reserved.

* Corresponding author.
** Corresponding author.
E-mail address: zorropro@gmail.com (Y. Kim).

https://doi.org/10.1016/j.chemosphere.2018.06.149
0045-6535/© 2018 Elsevier Ltd. All rights reserved.
816 H.K. Sung et al. / Chemosphere 209 (2018) 815e822
1. Introduction
With the rapid increase in the production and application of
engineered nanoparticles (NPs), there has also been significant
concern about human and environmental exposure to NPs. Because
the intracellular uptake of NPs depends on the shape, size, surface
charge, and functional moiety of the surface (Chithrani et al., 2006),
the cytotoxicity of NPs through environmental exposure is also
dependent on these characteristics (Park et al., 2017). The fate and
behavior of NPs in the environment and living organisms are poorly
understood because of the lack of analytical methods that are
applicable in environmental media (Merrifield et al., 2017). To
evaluate the transformation in environmental media and the up-
take of NPs in living organisms, it is important to analyze the
physicochemical properties of NPs in the actual exposed media.
Herein, single particleeinductively coupled plasmaemass spec-
trometry (sp-ICP-MS) was applied to obtain particle size distribu-
tion and concentration of NPs immediately within the biological
media.
Transmission electron microscopy (TEM), dynamic light scat-
tering (DLS), and sp-ICP-MS are considered representative tools for
analyzing particle size and size distribution. However, they have
some drawbacks, such as the overestimation of particle size
(Domingos et al., 2009) and dependence on the shape of NPs
(Khlebtsov and Khlebtsov, 2011). In TEM analysis, when the number
of particles used in the analysis is small, it is difficult to guarantee
the representativeness of the results. DLS is based on the mea-
surement of the autocorrelation function for fluctuations of scat-
tered light because of the random Brownian motion of particles. It
is suitable for analysis of spherical particles with a relatively high
concentration at the ppm level. However, if NPs are non-spherical
or the dispersibility of NPs in the medium is poor, error in size
evaluation can occur in DLS analysis. The measurement of NPs in
natural environments by DLS method is difficult because they
exhibit a duality of physical and chemical characteristics as they
switch from hydrophobic to polar forms upon exposure to water
(Satiner and Krar, 2011). For example, an uncritical application of
the DLS method without additional control using TEM may lead to
artifacts (Verleysen et al., 2015).
In contrast, sp-ICP-MS can be used as an alternative technique to
confirm TEM and DLS results (Verleysen et al., 2015; de la Calle
et al., 2017) because it enables researchers to determine the size
and size distribution of individual NPs, as well as the concentration
of particles (Kim et al., 2017). sp-ICP-MS can also be used to analyze
trace concentration at the ppt level with representative analysis
results, because the number of NPs used in the analysis increases
with the increase in acquisition time. Reliability assurance for
particle size analysis is recommended in the two guidance docu-
ments of the ISO and OECD (EFSA, 2011; Rasmussen et al., 2016).
The size of NPs is recommended to be measured by at least two
independent methods. Verleysen et al. independently performed
TEM and sp-ICP-MS analyses for silver nanoparticles (AgNPs)
contained in food to increase the reliability of particle size analysis
(Verleysen et al., 2015). Laborda et al. documented the feasibility of
using sp-ICP-MS as an alternative analysis tool for TEM. It has been
implemented in the European Commission definition of NPs by
analyzing different AgNPs and reference gold nanoparticles
(AuNPs) (Laborda et al., 2013). In sp-ICP-MS, the substance is
injected in cationic form and measured separately with atomic
weight. Therefore, the particle size is calculated by their mass-to-
charge ratios. For example, the signal intensity of a NP in sp-ICP-
MS depends on particle size, whereas the signal frequency is pro-
portional to particle concentration in samples (Degueldre et al.,
2006). Therefore, it generates a more accurate diameter than the
hydrodynamic diameter analyzed by DLS.
Although there have been some successful applications of sp-
ICP-MS for the analysis of size and concentration of NPs in envi-
ronmental and biological matrices (Dan et al., 2015), only a few
studies have reported the analysis of NPs in living organisms
following exposure. Gray et al. quantified NP properties in Daphnia
magna and Lumbriculus variegatus after exposure by sp-ICP-MS
(Gray et al., 2013). Dan et al. analyzed the uptake and accumula-
tion of NPs in tomatoes by sp-ICP-MS and TEM (Dan et al., 2015).
However, to the best of our knowledge, sp-ICP-MS has been rarely
used for the analysis of exposure and uptake of NPs in living fish. To
evaluate biological uptake and environmental fate of NPs, the size
and concentration of NPs should be analyzed simultaneously in the
actual biological medium.
Therefore, the objective of this study was to use sp-ICP-MS to
measure the bioaccumulation and particle size changes of metallic
NPs (AgNPs and AuNPs) exposed to zebrafish (Danio rerio) for 7
days. Because the major challenge for NP analysis in biological
tissues is the extraction of NPs from tissues without compromising
their properties, two different extraction methods (alkaline and
enzymatic treatments) were applied in this study. After comparing
the recovery of NPs extracted from the liver of zebrafish, alkaline
extraction with a high recovery of NPs was used to extract NPs
spiked into the liver, intestine, and gills of zebrafish, followed by
analyses of their size and concentration by sp-ICP-MS.
2. Materials and methods
2.1. Materials
Ultrapure water (18.2 MU cm) and elemental standard solutions
for gold and silver were purchased from Alfa Aesar, USA. Alkaline
extraction was performed using tetramethylammonium hydroxide
(TMAH) purchased from Alfa Aesar. Proteinase K (Mbiotech, Korea)
and digestion buffer (2 mM calcium acetate and 1% sodium dodecyl
sulfate, Sigma-Aldrich, USA) were used for the enzymatic digestion
of NPs from the tissue. To analyze the total concentration of ele-
ments in tissue, acid digestion was performed using acidic chem-
icals (HCl, HNO3, and H2O2) purchased from Dongwoo Fine Chem,
Korea. Mono-dispersed suspensions of AuNPs (nominal diameter:
28 nm and 56 nm for RM 8012 and RM 8013, respectively) and
AgNPs (75 nm, RM 8017) with polyvinylpyrrolidone (PVP) coatings
used as calibration standards were purchased from NIST (Gai-
thersburg, MD, USA). AuNPs and AgNPs (with diameters of 30 and
80 nm) with PVP coatings used in spike recovery and uptake ex-
periments were obtained from nanoComposix (San Diego, CA,
USA). They were also used to verify the calibration in ICP-MS.
2.2. Digestion method for the extraction of NPs from tissues
Although traditional digestion using acidic solutions can readily
lead to the dissolution of NPs, nonacidic digestions such as those
using strong basic solutions and enzymes can successfully extract
NPs from tissues (Park and Kim, 2016). To select a suitable digestion
method to analyze the uptake of NPs in zebrafish, a quantitative
extraction of AuNPs (80 nm) spiked into the liver tissue of zebrafish
was performed. In alkaline digestion, the AuNPs-spikied liver tissue
added into 1 mL of 25 wt% TMAH, and the mixture was sonicated
for 5 min using a probe-type sonicator (10 W). The resulting colloid
solution was filtered through Advantac paper (0.45-mm pore, cel-
lulose acetate). In enzymatic digestion, the AuNPs-spiked liver
tissue was added to the digestion buffer followed by ultra-
sonication. Proteinase K was added into the resulting colloid solu-
tion at a final concentration of 1250 mg/L, followed by additional
sonication for 1 h at 37  C using a bath-type sonicator. The final
colloid solution was also filtered through Advantac filter paper. The
 H.K. Sung et al. / Chemosphere 209 (2018) 815e822
recovery efficiency and size change of NPs after digestion were
compared to their initial concentration and size. In biological up-
take experiments, intracellular particles in the liver, intestine, and
gills of zebrafish were extracted by alkaline digestion, resulting in a
relatively high recovery of NPs.
2.3. Uptake experiments of NPs spiked into zebrafish
Biological uptake experiments with exposed organisms were
conducted using zebrafish. Briefly, zebrafish (n ¼ 540) were ob-
tained from a commercial fish farm, Kangnam-Aquarium (Incheon,
Korea) and acclimated for 1 week in an experimental glass aquar-
ium (DO 5.8 mg/L, pH 7.6). Zebrafish (n ¼ 112) to be tested were
selected according to the OECD protocol (OECD, 1996) (mean length
of fish: 2 ± 1 cm). Certificated NPs (AuNPs and AgNPs with 30 and
80 nm diameters) were used for spiking NPs. Because no effect of
50 mg/L AgNPs on zebrafish was found after a short exposure
duration (24e48 h) (Griffitt et al., 2013), the concentration of
spiked NPs was fixed at 50 mg/L. These zebrafish were housed in de-
chlorinated and UV-sterilized tap water and fed with commercial
dry pellets (Topmeal, Tabia, Korea) at 1e2% of total body mass twice
daily and blood worms (Hikari, Japan) once every 2 days. Test
duration was 7 days with a 14:10 h light:dark photoperiod at 28  C.
After exposure for 2 and 7 days, zebrafish (n ¼ 8 fish/group) were
removed from exposure tanks, and tissue burdens of NPs were
assessed. Each sample was rinsed with deionized water several
times. The liver (circulatory system), gill (respiratory system), and
intestine (digestive system) of zebrafish were dissected to evaluate
the intracellular uptake and the average particle size of NPs in
tissues. All the exposures were repeated three times per test group.
2.4. Characterizations of NPs in aquatic medium and tissues
All the samples were analyzed using an Agilent 7900 quadru-
pole sp-ICP-MS (Agilent Technologies) instrument with shorter
dwell times (below 1 ms). As a reference material, NIST 8012 AuNP
(nominal diameter 30 nm) was used for the determination of
nebulization efficiency. Data were processed in time-resolved
analysis mode (TRA) using Mass Hunter 4.1 software (Wilbur
et al., 2015; Adeleye et al., 2016) provided by Agilent technology.
Morphology and sizes of NPs exposed to aquatic medium were
obtained by TEM (JEM-2010, JEOL, Japan). For microscopy analysis
of NPs in tissues using energy-filtering TEM (EF-TEM, LIBRA 120,
Carl Zesis, Germany), specimens from exposure groups were fixed
in 2.5% glutaraldehyde solution for 2 h at 4  C and then post-fixed in
1% osmium tetroxide in the same buffer at 4  C for 2 h (Lee et al.,
2014a). Each specimen was dehydrated in graded ethanol series,
followed by two changes of propylene oxide (30 min for each
change). All the specimens were then permeated in a 1:1 mixture of
epoxy resin (epone-812) and propylene oxide for 90 min. Ultrathin
sections (100 nm in thickness) prepared by ultramicrotome
(EMUC7, Leica Mircosystems, Germany) were collected on Cu grids.
Structural changes and subcellular localization of NPs were
analyzed by EF-TEM. Particle size was measured using ImageJ™
software (NIH, USA).
3. Results and discussion
3.1. General setting for sp-ICP-MS analysis
In sp-ICP-MS, a sufficiently diluted NP suspension was intro-
duced into the plasma with adequate data acquisition frequency to
ensure that one NP was measured during each reading period
(Degueldre et al., 2006). The frequency of pulse and signal intensity
of counts of each pulse were correlated with the concentration of
817
NPs and particle size, respectively. Therefore, it was crucial to set a
short dwell time suitable for the diameter of NPs with an appro-
priate concentration of NPs so that a single particle could be
detected. Recommended dwell time used in previous studies was
typically in the range of 1e20 ms per point (Degueldre et al., 2006;
Gray et al., 2013; Lee et al., 2014b; Olesik and Gray, 2012). With the
latest generation of ICP-MS systems, such as the Agilent 7900 used
in this study, shorter dwell times (below 1 ms) can be used, and the
settling time between measurements has been eliminated, thus
enabling the measurement of several data points across the signal
pulse from the ion cloud created as a single NP passes through the
plasma. Each NP peak was integrated for each set of data. Larger
particle size and longer dwell time can ensure that one particle is
not detected as multiple particles (Olesik and Gray, 2012). However,
with a longer dwell time, several small particles could be simul-
taneously detected as one large particle. Therefore, an appropriate
dwell time should be chosen. In this study, it was set as 1 ms, which
was enough to analyze 30- and 80-nm-sized NPs. The instrument
was calibrated using three standard NIST-NPs ranging from 0.1 to
100 mg/L. Although a larger number of particles could increase the
probability of coincidence, a smaller number of particles might
reduce the reliability of the data analysis (Kim et al., 2017).
Therefore, the injected particle concentration was set to 102 and 103
particles per mL for 80-nm and 30-nm NPs, respectively.
In addition, the flow rate of carrier gas and sample depth were
adjusted using a standard tuning solution (Agilent 5185e5959) to
enhance the signal sensitivity of 89Y and 205Tl elements, which were
close to the target element in NPs. General parameters used for the
sp-ICP-MS are listed in Table 1. As shown in Table 2, the reliability of
the analysis under the conditions set out above was confirmed by
the estimation of precision, accuracy, method detection limit
(MDL), and limit of quantitation (LOQ). The precision and accuracy
of the assay were within acceptable limits. For these assessments,
the analysis of each reference material was replicated six times and
estimated by relative standard deviation (RSD) for precision, MDL,
and LOQ. Accuracy was assessed for each analyzed diameter and
concentration from the nominal diameter and concentration by the
percentage.
3.2. Stability of NPs in aquatic medium
It is known that PVP-coated NPs stabilized by steric hindrance
have a good dispersion stability, compared to bare- or citrate-
coated NPs stabilized by electrostatic repulsion (Roh et al., 2013).
Because NPs used in the spiking test were coated with PVP, their
dispersion stability in biological media was good. To evaluate the
dispersion stability of NPs in breeding water, NPs were exposed to
water medium for 2 days. Changes in particle size and concentra-
tion of NPs were then measured by sp-ICP-MS and compared to
their initial characteristics. AuNPs exposed to water (Fig. 1a) were
also analyzed after 1 day. Concentrations of both 30- and 80-nm
AuNPs decreased to 83% and 87% of their initial concentrations,
Table 1
Instrumental parameters for sp-ICP-MS data acquisition.
Parameter Value
RF generator power 1550 W
Carrier gas 1.14 L/min
Nebulizer pump 0.1 rps
Nebulization pump rate 0.346 mL/min
Integration time 1 ms
Acquisition time 60e90 s
107
Mass monitored (m/z) Ag, 197Au
Sample depth 8.0 mm
818 H.K. Sung et al. / Chemosphere 209 (2018) 815e822
Table 2
Accuracy, precision, MDL and LOQ of the Au and Ag reference nanomaterials.
Samples Mean diameter Concentration
Accuracy (%) Precision (%) Accuracy (%)
27 nm- AuNPs 94.0 0.75 97.0
75 nm-AgNPs 98.4 2.6 94.7
respectively. After 2 days, their concentrations further decreased to
73e79% of their initial spiking values. The initial size of AuNPs was
maintained for 1 day but slightly decreased on day 2, as shown in
Fig. 1b. To directly observe the aggregation of AuNPs after 24 h, NPs
exposed to the water medium for 24 h were analyzed by TEM. As
shown in Fig. 2, AuNPs aggregated between neighboring NPs. Their
secondary particle size also increased. The size of 30-nm AuNPs
with an initial size of 31.69 ± 3.00 nm (n ¼ 135) increased to
40.46 ± 14.63 nm (n ¼ 145) after 24 h, while that of 80-nm AuNPs
increased from 88.49 ± 14.34 nm (n ¼ 209) at 0 h to
90.13 ± 23.56 nm (n ¼ 141) at 24 h. This size-dependent instability
leads to Ostwald ripening, which is the transfer of atoms over time
from smaller, less stable particles to larger ones (Gentry et al., 2011).
Long-term exposed NPs to liquid phase broadened the particle size
distribution. In other words, the standard deviation of particle size
increased. As the concentration of NPs in the supernatant
decreased, their average size might also decrease because of the
Fig. 1. Changes in (a) concentration and (b) particle size of AuNPs exposed in aquatic
medium for 24 h and 48 h.
Method detection limit (ng/L) Limit of quantitation (ng/L)
Precision (%)
3.8 0.5 1.6
5.3 3.2 10.1
Fig. 2. TEM images of 30-nm AuNPs exposed in water at (a) 0 h and (b) 24 h.
precipitation of relatively larger particles. Therefore, maintaining
the initial concentration of NPs during long-term exposure in water
is difficult. Thus, the semi-static toxicity test was performed by
replacing the test colloid solution in test tanks with a new test
colloid solution, such as a batch system at 24-h intervals.
3.3. Extraction of NPs from tissues
sp-ICP-MS as an alternative tool for analyzing particle size and
size distribution of NPs exposed in environmental and biological
media is recommended by international organizations (ISO and
OECD) (OECD, 2016; ISO, 2017). However, existing studies are
mainly focused on NPs suspended in aqueous matrices (Lee et al.,
2014b; Olesik and Gray, 2012; Yang et al., 2016). To apply sp-ICP-
MS to more complex matrices such as biological samples, it is
essential to solubilize the sample matrix in a way so that NPs are
released into suspension without self-dissolution. Alkaline and
enzymatic digestions have been proven to yield high recoveries of
NPs from tissues in terms of both particle size and total mass. To
find a more suitable pre-treatment method for zebrafish, the
quantitative extraction of AuNPs spiked into the liver tissue of
zebrafish was performed by these two methods.
As shown in Fig. 3a, recoveries of the mass concentration of
AuNPs by alkaline and enzymatic methods were 102% and 74%,
respectively. The average particle diameter of NPs after the treat-
ments was almost maintained with a maximum SD of 1.49%
compared to their initial average diameter (Fig. 3b). Loeschner et al.
reported that alkaline digestion is more appropriate than enzy-
matic digestion for the extraction of NPs from spleen samples of
rats, leading to higher recoveries of AuNPs (Loeschner et al., 2014).
In addition, this method was considered a promising sample
preparation method for the extraction of metallic NPs from
D. magna (Gray et al., 2013), activated sludge (Park and Kim, 2016),
and sprout plants (Park et al., 2016). In contrast, Dan et al. used the
enzymatic digestion method with Macerozyme R-10 enzyme for
the extraction of AuNPs from tomato plants (Dan et al., 2015). The
extraction of AgNPs from chicken meat was also successful using
 H.K. Sung et al. / Chemosphere 209 (2018) 815e822 819

Fig. 4. Changes in (a) concentration and (b) particle size of 30- and 80-nm AuNPs
Fig. 3. Changes in (a) concentration and (b) particle size of AuNPs according to alkaline exposed in aquatic breeding medium.
digestion or enzymatic digestion.

(Fig. 5a and b). Sizes of primary particle constituting these black

proteinase K (Lakani et al., 2016). These studies showed that pre- granules were measured by ImageJ program. They were found to be
treatment methods should be selected with the target matrix. In 28.2 and 68.2 nm, matching the initial particle size of 30- and 80-
this study, the alkaline method using TMAH was found to be more nm AuNPs, respectively. It was noted that NPs penetrated into he-
suitable to extract NPs from zebrafish than the enzymatic method. patocytes and accumulated, consistent with the result of decreasing
mass concentration in the water medium (Fig. 4a). This feature has
also been reported in the liver of common carp (Cyprinus carpio)
3.4. Uptake comparisons of NPs in the liver, intestine, and gill
after exposure to zinc oxide NPs (Lee et al., 2014a). In other TEM
images, AuNPs attaching villi (Fig. 5c) and near the nucleus (Fig. 5d)
Semi-static toxicity test of NPs spiked into zebrafish was per-
were observed for intestinal tissue. Sizes of NPs accumulated in the
formed for 7 days. The average particle size and mass concentration
intestinal tissue were measured to be 36.2 and 64.7 nm, matching
of NPs in breeding water were then analyzed. Although zebrafish
the initial size of 30-nm AuNPs and 80-nm AgNPs, respectively.
were exposed to 50 mg/L of NPs, no repression effect on body weight
AgNPs with 30 nm exposed in the gill were found in gill lamella
or length was found during the testing period. However, as shown
(Fig. 5e), showing a primary particle size of 36.3 nm. Although the
in Fig. 4a, mass concentrations of NPs in the aquatic medium after 3
gill acts as an uptake route for environmental toxicants that can be
and 7 days reached ca. 60% and 25% of their initial value, respec-
trapped in the mucous layer, it is difficult to find NPs in gill tissues
tively. The average particle size of NPs for 7 days maintained its
by TEM analysis. It was also noted that the intracellular uptake of
initial size (Fig. 4b), similar to the results of stability test in the
NPs in the gill was relatively low compared to that in the liver or
water medium. Some NPs might have been ingested by zebrafish or
intestine. Collapse or deconstruction of tissues was not observed by
precipitated because of the disturbance caused by dissolved
TEM analysis because the spiking concentration of NPs was below
organic material in the water. Therefore, the bioaccumulation of
LC50, clearly indicating that NPs accumulated in various intracel-
NPs in zebrafish might be time dependent.
lular tissues. Overall, primary particle sizes seemed to be retained.
The intracellular uptake of NPs was confirmed by EF-TEM
However, the aggregation of intracellular particles was severe.
(Fig. 5). When zebrafish were exposed to AuNPs, NPs were inter-
TEM analysis has some advantages, such as the direct observa-
nalized into cells. Black granules were easily observed in the liver
tion of NPs at the intracellular accumulation site. However,
tissues, near the rough endoplasmic reticulum and cytoplasm
820 H.K. Sung et al. / Chemosphere 209 (2018) 815e822
Fig. 5. EF-TEM images of intracellular uptake NPs in zebrafish after 7 days of exposure.
(a) 30-nm AuNPs in the liver, (b) 80-nm AuNPs in the liver; (c) 30-nm AuNPs in the
intestine, (d) 80-nm AgNPs in the intestine; (e) 30-nm AgNPs in the gill.
ultrastructural observation of NPs in tissues is labor intensive; thus,
it is unsuitable for quantitative analysis, such as particle size and
total concentration of NPs. The analysis of primary particle size
using image analyzing software can also lead to errors. Therefore,
for the quantitative analysis, TEM analysis should be combined
with sp-ICP-MS analysis. TEM results have been verified by sp-ICP-
MS in a few reports (Verleysen et al., 2015; Degueldre et al., 2006).
In this study, after exposure of zebrafish to NPs, the NPs were
extracted by alkaline digestion and quantified by sp-ICP-MS. The
amount of accumulated NPs in tissues was found to depend on the
type and size of NPs (Figs. 6a and 7a). As described above, the
bioaccumulation of NPs in zebrafish showed time dependence.
Intracellular uptake in all tissues was found to be increased from
day 2 to day 7. The movement and uptake of AuNPs in the intestine
increased with time, whereas those of AgNPs increased in the liver
with time. The average particle size of AuNPs analyzed by sp-ICP-
MS slightly increased (Fig. 6b), while that of AgNPs decreased
(Fig. 7b). Because AgNPs could be easily dissolved in ionic form
when they had contact with an acidic medium, the particle size of
AgNPs in tissues slightly decreased compared to their initial size.
For further quantitative comparisons in terms of the
Fig. 6. (a) Intracellular uptake and (b) size change of 30- and 80-nm AuNPs in the liver,
intestine, and gill of zebrafish (ZF).
bioaccumulation of NPs, the bioconcentration factor (BCF) was
calculated. The BCF was defined as the ratio of the concentration
measured in the organism to that in water (Gray et al., 2013). Re-
sults of BCF value in target tissue are summarized in Table 3. As the
particle size of NPs is smaller, the uptake of NPs is lower. BCF values
of 80-nm AuNPs in the liver and intestine were 12.1 and 2.3 times
larger than those of 30-nm AuNPs. Among these three organs, NPs
appeared to accumulate predominantly in the intestine, whereas
smaller particles were more likely to be accumulated in the gills. In
other words, larger NPs were filtered by the gills. Therefore, they
were not accumulated in the gill filaments. Uptake amounts of 30-
and 80-nm AuNPs in the intestine were found to be 27.4 and 5.2
times larger, respectively, than those in the liver. However, uptake
amounts of 30- and 80-nm AgNPs in the intestine were 12.4 and 1.9
times larger, respectively, than those in the liver. Similar features
have been reported in the literature. For example, copper NPs have
been reported to accumulate in the gill, liver, and intestine of Si-
berian sturgeon (Acipenser baerii) juveniles (Lakani et al., 2016).
Overall, BCF values of AgNPs in these organs were larger than those
of AuNPs. When breathing, zebrafish ingests water including NPs.
However, NPs are mostly removed through gills. These ingested
NPs appear to migrate primarily to the intestine and accumulate in
the liver. As shown in our results, a quantitative analysis of intra-
cellular uptake and particle size of NPs in living organisms could be
easily and quickly determined by sp-ICP-MS.
 H.K. Sung et al. / Chemosphere 209 (2018) 815e822 821

results both showed that larger NPs were predominantly accumu-

lated in the intestine, whereas smaller NPs were filtered and
attached in the gill lamella. In addition, the BCF of AgNPs was found
to be higher than that of AuNPs. Moreover, this study showed that
sp-ICP-MS coupled with alkaline digestion could be used for the
direct observation of bioaccumlation of NPs in exposed aquatic
organisms such as zebrafish.

Acknowledgments

This study was supported by the National Institute of Environ-

mental Research (NIER-2016) and the Research Grant of Kwang-
woon University (2018 to Y. Kim).

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