Science of the Total Environment 782 (2021) 146933

Contents lists available at ScienceDirect

Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Silver nanowires kinetics and real-time imaging of in situ Ag ion

dissolution in Daphnia magna
Yiling Li, Wen-Xiong Wang ⁎
School of Energy and Environment and Hong Kong Branch of the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), State Key Laboratory of Marine Pollution, City
University of Hong Kong, Kowloon, Hong Kong, China
Research Centre for the Oceans and Human Health, City University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, China

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

• AgNWs were efficiently removed by

D. magna and difficult to be depurated.
• Diameter did not affect the toxicity,
uptake, depuration, and dissolution of
AgNWs.
• An aggregation-induced emission based
luminogen visualized the Ag+ in
D. magna.
• Ag+ release contributed less to AgNWs'
toxicity compared to AgNPs.
• Dissolved Ag+ in daphnid gut showed
the trends of anterior gut > midgut >
hindgut.

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

Article history: Silver nanowires (AgNWs) as high-aspect-ratio nanomaterials are extensively employed in industrial products,
Received 27 December 2020 and may pose potential risks of exposure to humans and other living organisms. Nevertheless, little is known
Received in revised form 2 March 2021 about their potential environmental fates and toxicological characteristic. Herein, the uptake and depuration ki-
Accepted 30 March 2021
netics of AgNWs in Daphnia magna at various exposure concentrations were comprehensively evaluated for the
Available online 6 April 2021
first time. Further, the distribution pattern of released Ag+ in the gut of D. magna as well as the total amount of
Editor: Daniel Wunderlin Ag+ were qualitatively and quantitatively investigated. Rapid accumulation of AgNWs in D. magna was observed,
and the steady-state concentration was obtained within 24-h. Depuration of AgNWs was limited, with 30–40% of
accumulated AgNWs being eliminated within 24-h. The released Ag+ from AgNWs in D. magna was monitored
Keywords: by real-time in situ imaging with the application of aggregation-induced emission luminogen. At equilibrium
Silver nanowires state of depuration, the released Ag+ reached 26.9 μg/g, equivalent to 5.4% of the total accumulated AgNWs in
D. magna D. magna exposed to 500 μg/L AgNWs for 12-h. Such in vivo dissolution of AgNWs was significantly higher
Ionic silver release than that in D. magna culture medium in vitro (2.6%). Compared with other Ag nanoparticles, Ag+ release from
In situ imaging
AgNWs in D. magna contributed much less to the observed AgNWs toxicity, and was able to transport to inner
Aggregation-induced emission
sides of daphnids, including gills and hemolymph. The slow depurations of both AgNWs and Ag+ in D. magna
may thus contribute to their joined chronic toxic effects.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction
⁎ Corresponding author at: School of Energy and Environment and Hong Kong Branch
of the Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou),
State Key Laboratory of Marine Pollution, City University of Hong Kong, Kowloon, Hong
The quantity and diversity of engineered nanomaterials (NMs) are
Kong, China. increasing rapidly in recent years. The high-aspect-ratio nanowires
E-mail address: wx.wang@cityu.edu.hk (W.-X. Wang). (NWs), especially those composing of metals, represent a promising

https://doi.org/10.1016/j.scitotenv.2021.146933
0048-9697/© 2021 Elsevier B.V. All rights reserved.
Y. Li and W.-X. Wang
and active field of nanoscience (Choi et al., 2016). Among these various
metal nanowires, silver nanowires (AgNWs) are currently garnering
great commercialization attention in conducting electrodes and me-
chanical equipment because of their comparable transparency and
high thermal conductivity (Langley et al., 2012; Sannicolo et al., 2016).
As a nanoscale platform, they also have various biological and medical
applications such as drug delivery, biomarker detection, and biosensors
(Lee et al., 2018; Mandal et al., 2017). During their manufacture, utiliza-
tion, and disposal processes, AgNWs may finally end up in aquatic eco-
systems and pose potential risks to aquatic organisms (Baalousha et al.,
2016; Dobias and Bernier-Latmani, 2013; Lazareva and Keller, 2014;
Maynard et al., 2006). Therefore, there are considerable needs to study
their environmental fates and toxicities in aquatic environments.
Works focusing on the environmental transformation of AgNWs and
their ecological or biological toxicity are limited. Sohn et al. (2015) in-
vestigated the ecotoxicological impacts of AgNWs on the freshwater en-
vironments, and classified AgNWs as “category acute 1” for D. magna,
“category acute 2” for fish, and “category acute 2” for algae. They sug-
gested that more attention should be paid to the toxicity of
nanomaterials on freshwater aquatic environments. There is now a gen-
eral consensus that the released Ag+ is the source of toxic effects of me-
tallic silver nanoparticles (AgNPs) (Li et al., 2015; Poynton et al., 2012;
Zhang and Wang, 2018), but this remains not well studied for AgNWs.
The increasing applications of AgNWs are not sufficiently accompanied
by the study of their potential impacts on living organisms, whereas
studies on spherical AgNPs are profuse (Toybou et al., 2019; Yang
et al., 2019). The contributions of AgNWs dissolution to their environ-
mental fate and biological effects have been rarely investigated and
remained uncertain (Zhang et al., 2018; Zhang et al., 2020). A few lim-
ited studies suggested that AgNWs toxicity could be linked to ionic
Ag+ release, while others indicated that the toxicity of AgNWs derived
from the combined contributions of both Ag+ release and particle ef-
fects of AgNWs. Visnapuu et al. (2013) found that the toxicity and bio-
availability of AgNWs to E. coli depended on the released Ag+ with no
shape-related impacts. Artal et al. (2013) investigated the toxicity of
Ag vanadate nanowire decorated with AgNPs to Daphnia similis, con-
cluding that the release of Ag+ to the test medium along with the re-
leased Ag+ from nanomaterial trapped in the gut were both
responsible for the toxicity. George et al. (2012) tested the toxic effects
of several AgNMs on fish epithelial cells and embryos, and found that
the effects may partially be due to the Ag+ release. Although the toxicity
of AgNWs was inconclusive, Ag+ release was at least partly responsible
for the toxicity of AgNWs. To further understand the AgNWs-related
hazardous impacts on living organisms, it is therefore critical to monitor
the dynamic processes of Ag+ released from AgNWs.
Localization and visualization of Ag+ released from AgNMs in bio-
logical organisms in situ remain a major challenge. Compared with
other Ag speciation techniques in biological systems, such as nanoscale
secondary ion mass spectrometry and synchrotron-based techniques,
fluorescence imaging appears to be more promising due to its conve-
nience, low cost, and easy operation (Li et al., 2017; Sekine et al.,
2017; Stegemeier et al., 2015; Wegner et al., 2010). The metallic fluores-
cent luminogen based on an aggregation-induced emission (AIE) pro-
cess showed excellent sensitivity and selectivity in metal detection
in vivo and in situ (Xie et al., 2018; Zhang and Wang, 2018). Such AIE-
based Ag+ probe can provide a vital approach to address the issues of
the dissolution of AgNWs in vivo in organisms, which may provide im-
portant insight into the toxicological mechanisms and ultimate risks of
AgNWs in aquatic environment.
In the present study, the qualitative as well as the quantitative Ag+
release from AgNWs at various exposure levels and time intervals in
Daphnia magna were systematically investigated. Daphnids were ex-
posed to polyvinylpyrrolidone (PVP)-coated AgNWs, and the uptake
and depuration of AgNWs at different exposure levels (5 μg/L, 50 μg/L,
and 500 μg/L) were assayed using inductively coupled plasma mass
spectrometry (ICP-MS). An AIE based Ag+ sensor, followed by confocal
2
Science of the Total Environment 782 (2021) 146933
laser scanning microscope (CLSM), was utilized to in situ image the dis-
solved Ag+ in D. magna. The findings of this work provided insight on
the biological behavior of AgNWs in D. magna for the first time.
2. Materials and methods
2.1. Ag nanowires characterization
Polyvinylpyrrolidone (PVP)-coated AgNWs were purchased from
Suzhou Lengshi Ltd (Suzhou, China). The morphology of AgNWs was in-
vestigated using optical microscope, scanning electron microscope
(SEM, Carl Zeiss EVO MA10, USA), and transmission electron micro-
scope (TEM, JEM, 2010). As shown in Fig. S1 and Table S1, the diameters
of the two AgNWs (named AgNWs-S and AgNWs-L) were 63.49
(46.89–75.23 nm) and 80.52 (54.14–111.12 nm), respectively. There
was a statistically significant difference between the diameters of
these two AgNWs (p < 0.05). These two AgNWs showed similar lengths,
which were 7.75 ± 3.43 μm and 7.40 ± 3.14 μm for AgNWs-S and
AgNWs-L, respectively (Fig. S1 and Table S1). The surface areas for
AgNWs-S and AgNWs-L were determined by simplified geometric cal-
culations, assuming the AgNWs as perfect cylinder with a circular
base. The calculated surface area of AgNWs-S and AgNWs-L was 6.68
and 5.03 m2/g, respectively. The average hydrodynamic diameter and
zeta potential of AgNWs were characterized in D. magna culture me-
dium at the concentration of 500 μg/L using an dynamic light scattering
(DLS) on a Malvern Zetasizer Nano-ZSat (UK). The average hydrody-
namic diameter of AgNWs-S and AgNWs-L in SM7 medium was
1330 nm and 3831 nm, respectively (Table S1). The zeta potentials of
AgNWs-S and AgNWs-L in D. magna culture medium were −22.54 ±
1.11 and −22.28 ± 1.90 mV (Table S1). Total Ag concentration of
each stock solution was measured by ICP-MS (NexION 300X, Perkin-
Elmer, USA) in triplicate after acid digestion by concentrated nitric
acid (HNO3).
2.2. D. magna culture and toxicity test
Details of D. magna culture are given in our previous study (Tan et al.,
2012). The modified simplified M7 (SM7) medium (containing
0.04 mM NaHCO3, 0.35 mM CaSO4, 0.50 mM MgSO4, and 0.05 mM
KNO3) at pH 7.5 ± 0.3 was used as the test solution throughout the cur-
rent work. Neonates (<24-h old) were used in the present toxicity test.
D. magna were acclimated in clean SM7 medium without food for 2-h
before the toxicity test. For the toxicity test, ten individuals per treat-
ment were exposed in 100 mL of SM7 medium containing different con-
centrations of Ag+ (0.01, 0.05, 0.2, 0.5, 1, and 2 μg/L), AgNWs-S (0.1, 1, 2,
5, 8, 10, 20, 30, 40, and 50 μg/L), or AgNWs-L (0.1, 1, 2, 5, 10, 20, 30, 40,
and 50 μg/L). All the treatments were performed in triplicate. No food
was provided throughout the experiments. After 24-h, the concentra-
tions of Ag+, AgNWs-S, and AgNWs-L causing 50% immobility (EC50)
were calculated. Animals that were unable to swim within 15-s after
gentle agitation were considered to be immobile. To verify the back-
ground toxicity of AgNWs solution, AgNWs were filtrated via mem-
brane filters (pore size: 0.45 μm). The 24-h toxicity of the filtrate was
conducted using the same method for AgNWs-S and AgNWs-L. Results
showed that the filtrate did not present any toxic effects on D. magna.
2.3. Ionic Ag release into D. magna culture medium
AgNWs were diluted in D. magna culture medium (SM7 medium) to
a final concentration of 500 μg/L. Ag+ release from AgNWs-S and
AgNWs-L were quantified. After 24 h incubation, the dissolved Ag+
was separated from AgNWs by ultracentrifugation through 3 kD mem-
branes (Millipore) at 6000 rpm for 30-min. Then 0.5 mL of each super-
natant was subjected to HNO3 acid digestion followed by ICP-MS
determination. The percentages of dissolved Ag+ were calculated as
the released Ag+ in the SM7 medium divided by the total AgNWs dosed.
Y. Li and W.-X. Wang
2.4. Uptake and depuration of AgNWs in D. magna
For the uptake and depuration tests, the exposure conditions were
the same as those used for the toxicity test. Ten adult D. magna per treat-
ment were exposed to AgNWs in 100 mL SM7 medium. For all the treat-
ments, 1 μM cysteine was added to minimize the uptake caused by Ag+
dissolution from AgNWs in the exposure medium (Zhao and Wang,
2012). Before AgNWs exposure, D. magna were isolated and accommo-
dated in SM7 medium for 2 h to evacuate their gut contents. Then,
D. magna were exposed to AgNWs in the following three scenarios: 2-
h uptake period followed by 24-h depuration at the exposure levels of
mass concentration of 50 and 500 μg/L; 12-h uptake period followed
by 24-h depuration at the exposure levels of mass concentration of 50
and 500 μg/L; 2-d uptake period followed by 12-h depuration at the ex-
posure levels of mass concentration of 5 and 50 μg/L. For the last sce-
nario, the exposure medium were refreshed after 24-h of AgNWs
exposure. No food was added to the exposure medium during the ex-
periments. At depuration times of 0, 6, 12, and 24-h, D. magna were col-
lected and washed with pure water and fresh SM7 medium for three
times. They were then dried, weighted, and digested in 1 mL of 65%
HNO3 and 200 μL of hydrogen peroxide (H2O2) at 80 °C for 4 h. The con-
centrations of total Ag accumulated in D. magna were quantified by ICP-
MS.
2.5. In vivo quantitatively monitoring the dissolution of AgNWs in D. magna
An AIE fluorogen, tetrazole-functionalized tetraphenylethylene de-
rivative 1 (TEZ-TPE-1), was used to image the Ag+ released from
AgNWs in D. magna (Xie et al., 2018). Fluorescence spectra of TEZ-
TPE-1 (10 μM) in SM7 medium upon addition of various Ag+ amounts
were analyzed using a fluorescence spectrophotometer (RF5301 PC,
Shimadzu). In the case of real-time in vivo imaging of Ag+ release
from AgNWs in D. magna, the exposure conditions were the same as
those mentioned above. In short, D. magna were exposed to AgNWs
for different concentrations in SM7 medium. Cysteine was added to
minimize the contribution of medium-released Ag+ to the accumula-
tion of Ag+ in D. magna's gut. After 12-h exposure, D. magna from
each treatment group were transferred to fresh SM7 medium for
depuration. At 6-h intervals, D. magna were collected, washed with
SM7 medium twice and exposed in SM7 medium containing 30 μM
TEZ-TPE-1 for 40 min. After rinsing D. magna, imaging was acquired
using a CLSM (LSM710, German). The instrumental conditions were as
follows: excitation wavelength: 405 nm and emission filters: 460–560
nm. The fluorescence intensities and Ag+ concentrations were then
corrected. The in vivo percentages of AgNWs dissolution in the gut of
D. magna were calculated as the total mass of Ag+ in the gut at the
end of depuration divided by the total accumulated AgNWs at the be-
ginning of depuration.
To establish the correlation of fluorescence intensity and Ag+ con-
centration in the gut of D. magna, daphnids were exposed in SM7 me-
dium containing different concentrations of AgNO3. At the end of
treatment, D. magna were collected and washed in pure water twice.
Their guts were removed from the bodies and collected with forceps.
Then, the guts were digested in 1 mL of 65% HNO3 and 200 μL H2O2 at
80 °C for 4-h and then submitted to ICP-MS for total Ag content determi-
nation. At the same time, the remaining D. magna were collected,
washed in pure water twice, and exposed in SM7 medium containing
30 μM TEZ-TPE-1 for 40-min. The fluorescence intensity of Ag+ in the
gut of D. magna was determined by CLSM as described above. Finally,
a linear correlation between fluorescence intensities from CLSM images
and Ag+ concentrations from ICP-MS was acquired.
2.6. Statistical analysis
All the data were expressed as mean ± standard deviation (n =
3) unless stated otherwise. Statistical analysis was carried out using
3
Science of the Total Environment 782 (2021) 146933
Student's unpaired t-test and p < 0.05 was considered as statistically
significant. All the statistical analyses were performed using Graph
Pad Prism version 8.4.
3. Results and discussion
3.1. Acute toxicity
The acute 24-h EC50 values of Ag+ (AgNO3), AgNWs-S, and AgNWs-L
for D. magna in SM7 medium are shown in Fig. S2A. The toxicities of
both AgNWs-S (24-h EC50 = 7.27 μg/L) and AgNWs-L (24-h EC50 =
6.22 μg/L) were much lower than that of Ag+ (24-h EC50 = 0.53 μg/L)
in the current tested conditions. The 24-h EC50 values indicated that
AgNWs were less toxic than Ag+ for D. magna, in accordance with pre-
vious works (Cui et al., 2017a; Scanlan et al., 2013). No major difference
was found in the EC50 values between AgNWs-S and AgNWs-L, suggest-
ing that diameters did not primarily affect the toxic effects of AgNWs to
D. magna under 24-h exposure conditions. The amounts of dissolved
ionic Ag+ in D. magna culture medium at EC50 concentrations of
AgNWs-S and AgNWs-L were investigated by ultracentrifugation and
ICP-MS measurements (Fig. S2B). At the 24-h EC50 (7 μg/L) values, the
released Ag+ was around 0.025 μg/L (equivalent to 0.36% of the expo-
sure concentration) for AgNWs-S and 0.024 μg/L (equivalent to 0.34%
of the exposure concentration) for AgNWs-L, respectively. No signifi-
cant difference was found in the amount of dissolved Ag+ between
AgNWs-S and AgNWs-L, again suggesting that for 24-h exposure parti-
cle diameters did not significantly affect dissolution of AgNWs. Dissolu-
tion of Ag+ was dependent on the surface area of Ag-based
nanomaterials (Sohn et al., 2015). In our study, comparable Ag+ release
ratios might due to the comparable surface area of AgNWs-S (6.68 m2/
g) and AgNWs-L (5.03 m2/g). These Ag+-based EC50 values
(0.024–0.025 μg/L) were only a fraction of the EC50 for Ag+ (0.53
μg/L), indicating that AgNWs themselves could also contribute to the
toxic effects to D. magna. These observations elucidated some previ-
ously uncharacterized aspects of the toxicity of AgNWs to D. magna,
which were at variance with those observed with AgNPs in previous
studies (Yan et al., 2018a; Zhao and Wang, 2012). For example, Zhao
and Wang (2012) suggested that the potential toxicity of AgNPs to
D. magna may be due to the release of Ag+. In their study, Zhao and
Wang (2012) examined the toxicity of three different surface coating
of AgNPs (lactate, sodium dodecylbenzene sulfonate, and PVP) to
D. magna. The quantified LC50 (48-h) was 28.7, 1.1, and 2.0 μg/L, for
AgNPs coated with lactate, sodium dodecylbenzene sulfonate, and
PVP, respectively. The calibrated LC50s based on the released Ag+ for
the treatments of AgNPs coated with lactate (1.1 μg/L) and PVP (0.57
μg/L) were however similar to that of Ag+ (as AgNO3, 0.88 μg/L), indi-
cating that the main toxicity of AgNPs to D. magna was caused by its re-
leased soluble Ag in the medium. Sohn et al. (2015) found that AgNPs
were more toxic (EC50 of AgNPs was 11.5-fold lower) than AgNWs to
D. magna, presumably due to the varying degrees of Ag+ dissolution.
The surface area of AgNPs was 2-fold higher than that of AgNWs,
which resulted in higher amount of Ag+ release (Sohn et al., 2015).
Based on our findings, the tested AgNWs presented toxicity to
D. magna through jointed mechanisms by Ag+ release to the medium
and interactions between AgNWs and D. magna following
internalization.
3.2. Uptake and depuration of AgNWs in D. magna
The bioaccumulation and depuration of different sized AgNWs
(AgNWs-S and AgNWs-L) in D. magna were investigated for the first
time. As shown in Fig. 1A, an increase of body burden of Ag was ob-
served during the first 24-h exposure period followed by a slightly
levelling-off from 24-h to 48-h for both AgNWs-S and AgNWs-L at the
exposure concentration of 500 μg/L. This indicated that AgNWs accumu-
lation in D. magna reached a steady-state within 24 h. The slight
Y. Li and W.-X. Wang
Fig. 1. (A) Uptake of AgNWs-S and AgNWs-L by D. magna over 48-h at the exposure
concentration of 500 μg/L. (B) Uptake ratio of AgNWs with different sizes in D. magna at
different tested concentrations. (C) Ag accumulation in D. magna after 48-h exposure to
AgNWs-L at the exposure concentrations of 50 and 500 μg/L. Data are mean ± SD (n = 3).
decrease in Ag body burden of D. magna (24-h to 48-h) may be due to
the settling of AgNWs in the containers and the depuration of AgNWs
from D. magna. The similar decreasing trend was observed for the
4
Science of the Total Environment 782 (2021) 146933
uptake of other nanomaterials by D. magna, such as carbon nanotubes
and gold nanoparticles (Lovern et al., 2008; Petersen et al., 2009). Ag
concentrations in the control D. magna without AgNWs exposure
were below 0.01 μg/g, which were significantly lower than those of
AgNWs exposed D. magna. There was no statistically significant differ-
ence in AgNWs uptake between D. magna exposed to AgNWs-S and
AgNWs-L (p > 0.05), indicating that the diameter of AgNWs did not sig-
nificantly affect the uptake. Exposure to 500 μg/L for 24-h yielded Ag
body burdens of 47.1 ± 1.0 μg/mg and 44.8 ± 1.6 μg/mg of dry tissue
for AgNWs-S and AgNWs-L, respectively. These uptake results were
nearly identical to those reported in previous works with fullerenes
(56 ± 15 μg/mg) and carbon nanotubes (63 ± 15 μg/mg) at compara-
tive exposure levels (Petersen et al., 2009; Tervonen et al., 2010). As re-
vealed previously, exposure to 60 nm AgNPs at an exposure level of 500
μg/L for 48-h resulted in 9.41 μg/mg dry weight in D. magna (Yan et al.,
2018a), which was 5-times lower than AgNWs measured in our study.
As shown in Fig. 1B, about 18.9 ± 0.4% and 17.9 ± 0.7% of the total
mass of AgNWs-S and AgNWs-L added (500 μg/L) to the SM7 medium
were taken up by D. magna after 24-h exposure, respectively. For
AgNWs-L exposed at 50 μg/L, similar fraction of the total exposed
AgNWs (18.4 ± 0.7%) was accumulated in D. magna (Fig. 1B). The find-
ings indicated that the uptake ratio (%) of AgNWs was not dependent on
the size of AgNWs or their exposure concentrations. Yan and Wang
(2018a) found that the accumulated AgNPs in D. magna exposed to
20 nm AgNPs (15.4 μg/mg) was higher than that in 60 nm AgNPs
(9.41 μg/mg) group at the same treatment concentration. Similarly,
the bioaccumulation of citrate and tannic acid coated AgNPs in
D. magna decreased significantly with increasing nominal size (20 nm
> 50 nm > 100 nm, Zhao and Wang, 2012). Such contrary results indi-
cated that D. magna exhibited distinct uptake behavior for AgNWs and
AgNPs due to their different aspect-ratios. For D. magna exposed to
AgNWs-L at 50 μg/L, a body burden of 5.2 ± 0.2 μg/mg of dry tissue
was achieved (Fig. 1C). Under such condition, AgNWs accumulation in
D. magna reached the steady-state within 2-h, which was much faster
than that at 500 μg/L (Fig. 1C).
AgNWs-S was selected to investigate the depuration behavior of
AgNWs in D. magna after exposure to AgNWs-S for various times at var-
ious concentrations. To investigate the AgNWs depuration, D. magna
were exposed to AgNWs-S at different concentrations (5, 50, and 500
μg/L) for various times (2-h, 24-h, and 48-h) and then transferred to
clean SM7 medium. In the case of 2-h exposure to 500 μg/L of AgNWs-
S, the body burden of AgNWs in D. magna dropped from 18.5 μg/mg to
10.0 μg/mg, a decrease of 45.6% over 24-h depuration period (Fig. 2A).
A very similar depuration was observed for the case of 2-h exposure
to 50 μg/L dose, with a decrease of 44.8% (from 4.6 μg/mg to 2.5 μg/
mg) over 24-h (Fig. 2A). In the case of 12-h exposure, more decrease
was observed over 24-h depuration for both 50 μg/L (66%, from 5.0 to
1.7 μg/mg) and 500 μg/L (69%, from 42.3 to 13.3 μg/mg) AgNWs-S ex-
posed D. magna (Fig. 2B). For the long-term exposure experiment (48-
h, Fig. 2C), 65.1% (from 673.6 to 235.0 μg/g) and 59.8% (from 5199 and
2090 μg/g) of the accumulated AgNWs-S were depurated within 12-h
for the treatments of 5 and 50 μg/L AgNWs-S exposed D. magna, respec-
tively. These depuration behaviors of AgNWs were largely in accordance
with those observed for other high aspect ratio nanomaterials such as
nanotubes, whereas differed from those for AgNPs (Petersen et al.,
2009; Zhao and Wang, 2010). For AgNPs, around 80% of AgNPs was
lost after a depuration period of 24-h in D. magna exposed to 5 and
500 μg/L of AgNPs for 48-h (Zhao and Wang, 2010). On the contrary,
D. magna were nearly unable to purge carbon nanotubes from their bod-
ies during 24-h depuration (Petersen et al., 2009). Thus, it appeared that
the shape instead of compositions of nanomaterials would affect their
depuration behavior.
In all the above cases, the body burdens of D. magna decreased faster
within the first 6-h (42.9–74.5%) and then a much smaller (<20%) frac-
tion of AgNWs-S was removed in the following 6–24 h depuration. The
body burdens of Ag in D. magna remained nearly unchanged after 12-h
Y. Li and W.-X. Wang Science of the Total Environment 782 (2021) 146933

Fig. 2. Retention of AgNWs-S in D. magna during 48-h depuration after being treated with different concentrations of AgNWs-S for different times. (A) Retention of AgNWs-S in D. magna
after being exposed to AgNWs-S for 2-h at the concentrations of 50 and 500 μg/L. (B) Retention of AgNWs-S in D. magna after being exposed to AgNWs-S for 12-h at the concentrations of
50 and 500 μg/L. (C) Retention of AgNWs-S in D. magna after being exposed to AgNWs-S for 2-d at the concentrations of 5 and 50 μg/L. Data are mean ± SD (n = 3). (D) Retained AgNWs-S
(% relative to those at the beginning of depuration) in 5, 50, and 500 μg/L AgNWs-S exposed D. magna at the end of depuration.

depuration. Roughly constant body burdens (1.7–2.5 μg/mg for 50 μg/L
AgNWs exposed D. magna and 10.0–13.3 μg/mg for 500 μg/L groups)
were obtained after 24-h depuration regardless of exposure time. The
depuration results indicated that final body burdens of AgNWs in
D. magna were determined by the exposure concentrations and had
no relationship with the exposure time during the uptake (Fig. 2D). It
was not surprising that Ag retention in high (500 μg/L) AgNWs-S ex-
posed D. magna was significantly higher than that of low (50 μg/L)
AgNWs-S treated individuals. The body burdens of D. magna remained
nearly unchanged after 12-h depuration. By comparison with AgNPs
(approximately 90% excretion) (Tervonen et al., 2010), AgNWs were
retained more efficiently during 24-h depuration. It could be speculated
that D. magna were unable to purge AgNWs completely from their bod-
ies. While AgNWs exposure did not cause acute toxic effects to
D. magna, the difficulty in depuration inevitably bring the concerns on
the chronic toxic effects of AgNWs and their potential long-term health
risks to aquatic organisms.
Considering that D. magna could not depurate AgNWs completely,
AgNWs may possibly be transferred along the aquatic food chains and
pose risks to higher trophic levels of aquatic organisms. Indeed, Chae
and An (2016) reported the transfer of PVP-coated AgNWs in a
laboratory-constructed aquatic food chain containing the alga
Chlamydomonas reinhardtii, D. magna, and the zebrafish (Chae and An,
2016). They found that AgNWs could be transferred through the inges-
tion of AgNWs-exposed algae by D. magna and subsequent ingestion of
D. magna by zebrafish. Besides AgNWs, other nanoparticles such as
AgNPs, gold nanoparticles, and fullerenols were also reported to show
dietary transfer potentials (Ferry et al., 2009; Holbrook et al., 2008;
Ribeiro et al., 2017). For example, Wang et al. (2018) found that 13C-
skeleton-labeled fullerenols underwent aquatic transfer from primary
producers to secondary consumers through dietary intake. Zhu et al.
(2010) documented a higher body burden of nanoscale TiO2 in the die-
tary D. magna exposed zebrafish than that in the aqueous exposed
zebrafish, suggesting that dietary intake constituted a major route of
nanomaterial exposure for a higher trophic level of aquatic organisms.
3.3. Imaging and quantitative analysis of time-dependent Ag+ release in
D. magna
In this study, an Ag+ fluorescent probe was applied to visualize the
+
Ag released from AgNWs in D. magna. This AIE-based probe showed
high selectivity and sensitivity to Ag+ in previous studies (Tervonen
et al., 2010; Xie et al., 2018; Zhang and Wang, 2018; Yan et al.,
2018b). Here, a good linear calibration curve was obtained over the con-
centration range of 2–200 μg/L with the detection limit of 1.96 μg/L
(Fig. S3). The in vivo Ag+ dissolution mapping in D. magna treated
with AgNWs were then assessed using the AIE-based fluogenic Ag+
probe and CLSM. As shown in Fig. 3, a large amount of dark materials
could be seen in the gut of AgNWs-S exposed D. magna, which were
confirmed to be AgNWs by reflectance signals. D. magna exposed to
both AgNWs-S and AgNWs-L displayed obvious fluorescence while no
obvious fluorescence was detected in the control D. magna without

5
Y. Li and W.-X. Wang Science of the Total Environment 782 (2021) 146933

AgNWs exposure (Fig. 4). Merged images of fluorescence and bright-

Fig. 3. Representative CLSM images of AgNWs in D. magna, including the scattering
imaging and bright-field images of control D. magna and D. magna exposed to AgNWs-S
for 2-h at a concentration of 500 μg/L.
field images indicated that Ag+ fluorescence signals were mainly accu-
mulated in the gut. This finding indicated that gut was the major site for
AgNWs deposition and the Ag+ was subsequently released, consistent
with previous studies on other nanomaterials (Tervonen et al., 2010;
Zhao and Wang, 2010; Zhao and Wang, 2012). As D. magna's gut was
the major site for AgNWs accumulation, only the gut was retained for
better comparison among different exposure conditions. Fluorescent
images of guts were then transformed into pseudocolor heat maps.
The above AIE probe and CLSM method could provide qualitative infor-
mation on Ag+ dissolution behavior of AgNWs in D. magna. To obtain
the quantitative information on AgNWs dissolution information, the
total fluorescence intensity of Ag+ detected by CLSM and the total
amount of Ag+ quantified by ICP-MS were correlated as described in
Methods (Fig. S4). A good linear relationship was obtained (y =
0.5215x − 7.366, R2 = 0.98, Fig. S4). Hence, it was possible to both qual-
itatively and quantitatively monitor the profile of dissolved Ag+ in
D. magna by the intensity of CLSM.
The in vivo Ag+ dissolution of AgNWs in D. magna gut during the
depuration processes was monitored and quantified using the AIE-
based fluogenic Ag+ probe and CLSM. D. magna were exposed to
AgNWs for various times at different concentrations and then allowed
to depurate as described above. AgNWs-S was selected for the real-
time imaging of Ag+ dissolution in D. magna during AgNWs depuration
process. Representative time-course images of Ag+ fluorescent signals
in D. magna's gut are shown in Fig. 5. After 2-h exposure at the concen-
trations of 50 and 500 μg/L, the total dissolved Ag+ was 19.9 μg/g and

Fig. 4. Confocal microscope images of control D. magna (exposed to fluorogenic Ag+ probe, TEZ-TPE-1 only) and D. magna treated with AgNWs-S (500 μg/L) or AgNWs-L (500 μg/L) for 6-h.

6
Y. Li and W.-X. Wang
Fig. 5. Heatmap images showing the distribution of dissolved Ag+ in the gut of D. magna during depuration after being exposed to AgNWs-S at the concentrations of 50 μg/L (A) and 5 μg/L
(B) for 2-d. White arrows represent the anterior gut of D. magna. (C) Quantitative analysis of dissolved Ag+ in the gut of D. magna.
17.5 μg/g, respectively (Fig. S5). The dissolved Ag+ in D. magna's gut
were then reduced to around 10 μg/g after 6-h depuration for both the
50 and 500 μg/L AgNWs treatments and remained unchanged as
depuration time further increased to 24-h depuration. These results
suggested that, after short time AgNWs exposure, the accumulated
Ag+ in the gut of D. magna was continuously reduced over time. The
net amount of Ag+ in the gut was the balance between the dissolution
of accumulated AgNWs in the gut and the excretion of Ag+ to the me-
dium. The decrease in the amount of released Ag+ with increasing
depuration time could be caused by the reduction of AgNW concentra-
tion in the gut as well as the excretion of Ag+. On the contrary, following
12-h of AgNWs exposure, the fluorescence intensity of Ag+ derived
from AgNWs dissolution increased continuously over the depuration
period and reached a maximum value at 12 h (Fig. S6). After 12-h, the
mass of dissolved Ag+ remained relatively steady during the rest of ex-
perimental time. At the end of the experiment, the dissolved Ag+ in the
50 and 500 μg/L AgNWs exposed D. magna's gut was 28.1 and 26.9 μg/g,
respectively. Time sequential mapping of Ag+ in D. magna exposed to 5
and 50 μg/L AgNWs for 2-d indicated that Ag+ amount in the gut in-
creased over the test depuration period of 12 h (Fig. 5). The dissolved
Ag+ was 1.8 and 1.7 μg/g in the 5 and 50 μg/L AgNWs exposed
D. magna initially after depuration and increased to 2.5 and 7.2 μg/g
after 12-h depuration (Fig. 5C). The amount of Ag+ derived from
AgNWs dissolution in D. magna at 500 μg/L exposure level was nearly
equivalent to those exposed to 50 μg/L AgNWs initially after depuration.
The dissolution may vary with the surrounding environment and the
related dispersed chemicals to which AgNWs react with. At the high ex-
posure dose, AgNWs in D. magna gut formed more compact and larger
aggregates compared to the low exposure level, which were more diffi-
cult to dissolve and owned lower dissolution rate. The dissolved Ag+ in
D. magna's gut showed a constant value after 12-h of depuration, which
indicated that a rough state of equilibrium of Ag+ dissolution was ob-
tained at the end of the depuration. It can be concluded that both the
AgNWs in the gut of D. magna and their dissolved Ag+ reached roughly
constant body burdens after 24-h depuration. The final released Ag+ in
the gut was dependent on the initially accumulated AgNWs in D. magna.
The in vivo percentage of dissolved Ag+ in the gut of D. magna (5.4%)
was much higher than that occurred in vitro in SM7 medium (2.6%). The
dissolution of AgNWs-S in SM7 medium at the concentration of 500 μg/L
was also investigated. At 24 h, 12.2 μg/L (2.6%) of Ag+ was released from
AgNWs-S. The in vitro dissolution of AgNWs in SM7 medium was much
lower than that of AgNPs (10.5%) as reported previously (Yan et al.,
2018b). Such lower dissolution ratio of Ag+ from AgNWs than AgNPs
was at least partly due to the lower surface area of AgNWs than that
of AgNPs (Sohn et al., 2015). In the case of in vivo dissolution, after 24-
h depuration, a total amount of 26.9 μg/g (5.4%) of Ag+ was detected
in D. magna exposed to 500 μg/L AgNWs-S. The dissolution of
7
Science of the Total Environment 782 (2021) 146933
nanomaterials varies vastly depending on the experimental conditions,
e.g., ionic strength, pH, and salinity (Zhang et al., 2011). The microenvi-
ronment in D. magna's gut was more complex than SM7 medium and
the complex biological matrices could possibly facilitate AgNWs disso-
lution. Yan et al. (2018a) reported an 8.3–9.7% dissolution of AgNPs to
Ag+ in the gut of D. magna exposed to 500 μg/L of AgNPs. Our present
study suggested that the dissolved Ag+ from AgNWs in the gut of
D. magna was slightly lower than that of AgNPs. Thus, Ag+ release
would contribute less to AgNWs' toxicity to D. magna than AgNPs did.
As revealed by the microscopic picture, the anterior gut and midgut
showed slightly stronger Ag+ signal than the hindgut initially after
depuration (Fig. 5A and B). However, the majority of Ag+ was accumu-
lated in the tail of the gut at the end of depuration (Fig. 5A and B). The
heterogeneous distribution of Ag+ in the gut was partly derived from
the heterogeneous distribution of AgNWs. Indeed, the dissolved Ag+
was highly concentrated in specific part of the gut where incompact
AgNWs were accumulated (Fig. S7). At the beginning of depuration,
the whole gut was filled with compact and large aggregate of AgNWs.
AgNWs as well as their dissolved Ag+ gradually diffused from the ante-
rior gut to the hindgut. Thus, when the fluorescence imaging was con-
ducted (40 min after depuration), the anterior gut and midgut
showed incompact AgNWs stacks that could have full contact with the
gut microenvironment. Additionally, the pH of the gut could influence
the dissolution of AgNWs significantly. Yan et al. (2018a) investigated
the relative pH of the digestive tract of D. magna using a fluorescence
dye, with a pH trend of hindgut > midgut > anterior gut (Yan et al.,
2018a). The AgNPs dissolution was consistent with the distribution of
pH condition, and AgNPs at a lower pH possessed a higher dissolution
than that at a higher pH. In the present study, the dissolution of
AgNWs in the gut of D. magna displayed a similar trend as AgNPs. The
lower pH of the anterior gut possibly accelerated the dissolution of
AgNWs as compared with other part of gut with higher pH (Loza
et al., 2014).
The confocal images suggested that the dissolved Ag+ was
transported to the inner side of D. magna between the gut region and
the gills (Fig. 6). These detected Ag+ fluorescence dots were partly in ac-
cordance with the lipid storage droplets which were scattered along
D. magna's gut (Fig. 6). No evidence existed to suggest the translocation
of AgNWs from gut to lipid droplets in our study. Nevertheless, upon in-
gestion by D. magna, various nanoparticles such as polystyrene nano-
particle and TiO2 nanoparticles were found to be translocated from
gut into lipid droplets in previous studies (Brun et al., 2017; Cui et al.,
2017b; Rosenkranz et al., 2009; Tan et al., 2016). It was likely that the
accumulation of nanoparticles in the lipid droplets was due to their lipo-
philic properties (Moore, 2006). Thus, whether Ag+ accumulation in
lipid droplets was attributed to the translocation of Ag+ from gut to
lipid droplets or the dissolution of AgNWs in the lipid droplets needs
Y. Li and W.-X. Wang
Fig. 6. Transportation of released Ag+ in D. magna at depuration time 0-h (A) and 12-h after exposure to AgNWs-L for 12-h at 50 μg/L. White arrows pointed to Ag+ between the interspace
of gut and gills of D. magna.
to be further studied. These lipid droplets are the sites for the synthesis
of egg-yolk precursor vitellogenin (Brun et al., 2017). This is subse-
quently transported via the hemolymph to the ovaries to form yolk
granules. Indeed, AgNWs was detected in the hemolymph of D. magna
in a previous study (Scanlan et al., 2013), and such pathway may possi-
bly serve as a potential route for maternal transfer of nanoparticles.
4. Conclusion
For the first time, this study examined the kinetics of AgNWs in
D. magna exposed over a wide concentration range (5–500 μg/L) for var-
ious times. The total amount of released Ag+ and their distribution pro-
files in the gut in these exposure cases were also investigated using in
situ bioimaging method. The nanomaterial size showed no significant
influence on the toxic and uptake of AgNWs. Limited depuration in
D. magna was observed for AgNWs. Final body burdens of AgNWs in
D. magna were related to the exposure concentrations, but not affected
by the exposure times during the uptake experiments. Constant value of
released Ag+ was detected irrelevant of the initially accumulated
AgNWs. Around 5.4% of AgNWs was dissolved when equilibrium state
was obtained, which was much less than those of AgNPs. These findings
suggested that Ag+ release played less important roles in AgNWs' toxic-
ity to D. magna than AgNPs did. Despite that no obvious toxic effects
were observed during the experiments, the difficulty in AgNWs
depuration and their dissolved Ag+ may possibly cause chronic toxic ef-
fects to D. magna. In addition, the observed translocation may serve as a
potential route for maternal transfer of AgNWs. Thus, the potential
long-term health risks of AgNWs and the released Ag+ to aquatic organ-
isms and the offspring need to be further studied.
Supporting information to this article can be found online at https://
doi.org/10.1016/j.scitotenv.2021.146933.
CRediT authorship contribution statement
Yiling Li: Conceptualization, Investigation, Writing
Wen-Xiong Wang: Conceptualization, Writing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
8
Science of the Total Environment 782 (2021) 146933
Acknowledgement
We thank the reviewers for their comments and the AIEgen Biotech
Co. This study was supported by grants from the Hong Kong Research
Grants Council (CityU 16102918, 16103120, T21-604/19-R) and the
Natural Science Foundation of China (22076159).
References
Artal, M.C., Holtz, R.D., Kummrow, F., Alves, O.L., Umbuzeiro, G.A., 2013. The role of silver
and nanadium release in the toxicity of silver vanadate nanowires toward Daphnia
similis. Environ. Toxicol. Chem. 32, 908–912.
Baalousha, M., Yang, Y., Vance, M.E., Colman, B.P., McNeal, S., Xu, J., Blaszczak, J., Steele, M.,
Bernhardt, E., Hochella, M.F., 2016. Outdoor urban nanomaterials: the emergence of a
new, integrated, and critical field of study. Sci. Total Environ. 557, 740–753.
Brun, N.R., Beenakker, M.M., Hunting, E.R., Ebert, D., Vijver, M.G., 2017. Brood pouch-
mediated polystyrene nanoparticle uptake during Daphnia magna embryogenesis.
Nanotoxicology 11, 1059–1069.
Chae, Y., An, Y.-J., 2016. Toxicity and transfer of polyvinylpyrrolidone-coated silver nano-
wires in an aquatic food chain consisting of algae, water fleas, and zebrafish. Aquat.
Toxicol. 173, 94–104.
Choi, S., Lee, H., Ghaffari, R., Hyeon, T., Kim, D.H., 2016. Recent advances in flexible and
stretchable bio-electronic devices integrated with nanomaterials. Adv. Mater. 28,
4203–4218.
Cui, R., Chae, Y., Y-J, An, 2017a. Dimension-dependent toxicity of silver nanomaterials on
the cladocerans Daphnia magna and Daphnia galeata. Chemosphere 185, 205–212.
Cui, R., Kim, S.W., Y-J, An, 2017b. Polystyrene nanoplastics inhibit reproduction and in-
duce abnormal embryonic development in the freshwater crustacean Daphnia
galeata. Sci. Rep. 7, 1–10.
Dobias, J., Bernier-Latmani, R., 2013. Silver release from silver nanoparticles in natural wa-
ters. Environ. Sci. Technol. 47, 4140–4146.
Ferry, J.L., Craig, P., Hexel, C., Sisco, P., Frey, R., Pennington, P.L., Fulton, M.H., Decho, Scott
G., Kashiwada S, A.W., Murphy, C.J., Shaw, T.J., 2009. Transfer of gold nanoparticles
from the water column to the estuarine food web. Nature Nanotech. 4, 441–444.
George, S., Lin, S., Ji, Z., Thomas, C.R., Li, L., Mecklenburg, M., Meng, H., Wang, X., Zhang, H.,
Xia, T., Hohman, J.N., Lin, S., Zink, J.I., Weiss, P.S., Nel, A.E., 2012. Surface defects on
plate-shaped silver nanoparticles contribute to its hazard potential in a fish gill cell
line and zebrafish embryos. ACS Nano 6, 3745–3759.
Holbrook, R.D., Murphy, K.E., Morrow, J.B., Cole, K.D., 2008. Trophic transfer of nanoparti-
cles in a simplified invertebrate food web. Nature Nanotech. 3, 352–355.
Langley, D., Giusti, G., Mayousse, C., Celle, C., Bellet, D., Simonato, J.-P., 2012. Flexible trans-
parent conductive materials based on silver nanowire networks: a review. Nanotech-
nology 24, 452001.
Lazareva, A., Keller, A.A., 2014. Estimating potential life cycle releases of engineered
nanomaterials from wastewater treatment plants. ACS Sustain. Chem. Eng. 2,
1656–1665.
Lee, J.H., J-Y, Hwang, Zhu, J., Hwang, H.R., Lee, S.M., Cheng, H., Lee, S., Hwang, S., 2018.
Flexible conductive composite integrated with personal earphone for wireless,
real-time monitoring of electrophysiological signs. ACS Appl. Mater. Inter. 10,
21184–21190.
Li, X., Schirmer, K., Bernard, L., Sigg, L., Pillai, S., Behra, R., 2015. Silver nanoparticle toxicity
and association with the alga Euglena gracilis. Environ. Sci.: Nano 2, 594–602.
Y. Li and W.-X. Wang
Li, H., Wang, C., Hou, T., Li, F., 2017. Amphiphile-mediated ultrasmall aggregation induced
emission dots for ultrasensitive fluorescence biosensing. Anal. Chem. 89, 9100–9107.
Lovern, S.B., Owen, H.A., Klaper, R., 2008. Electron microscopy of gold nanoparticle intake
in the gut of Daphnia magna. Nanotoxicology 2, 43–48.
Loza, K., Diendorf, J., Sengstock, C., Ruiz-Gonzalez, L., Gonzalez-Calbet, J.M., Vallet-Regi, M.,
Köller, M., Epple, M., 2014. The dissolution and biological effects of silver nanoparti-
cles in biological media. J. Mater. Chem. B 2, 1634–1643.
Mandal, B., Rameshbabu, A.P., Soni, S.R., Ghosh, A., Dhara, S., Pal, S., 2017. In situ silver
nanowire deposited cross-linked carboxymethyl cellulose: a potential transdermal
anticancer drug carrier. ACS Appl. Mater. Inter. 9, 36583–36595.
Maynard, A.D., Aitken, R.J., Butz, T., Colvin, V., Donaldson, K., Oberdörster, G., Philbert,
M.A., Ryan, J., Seaton, A., Stone, V., Tinkle, S.S., Tran, L., Walker, N.J., Warheit, D.B.,
2006. Safe handling of nanotechnology. Nature 444, 267–269.
Moore, M., 2006. Do nanoparticles present ecotoxicological risks for the health of the
aquatic environment? Environ. Int. 32, 967–976.
Petersen, E.J., Akkanen, J., Kukkonen, J.V.D., Weber Jr., W.J., 2009. Biological uptake and
depuration of carbon nanotubes by Daphnia magna. Environ. Sci. Technol. 43,
2969–2975.
Poynton, H.C., Lazorchak, J.M., Impellitteri, C.A., Blalock, B.J., Rogers, K., Allen, H.J.,
Loguinov, A., Heckman, J.L., Govindasmawy, S., 2012. Toxicogenomic responses of
nanotoxicity in Daphnia magna exposed to silver nitrate and coated silver nanoparti-
cles. Environ. Sci. Technol. 46, 6288–6296.
Ribeiro, F., Van Gestel, C.A., Pavlaki, M.D., Azevedo, S., Soares, A.M., Loureiro, S., 2017. Bio-
accumulation of silver in Daphnia magna: waterborne and dietary exposure to nano-
particles and dissolved silver. Sci. Total Environ. 574, 1633–1639.
Rosenkranz, P., Chaudhry, Q., Stone, V., Fernandes, T.F., 2009. A comparison of nanoparticle
and fine particle uptake by Daphnia magna. Environ. Toxicol. Chem. 28, 2142–2149.
Sannicolo, T., Lagrange, M., Cabos, A., Celle, C., Simonato, J.P., Bellet, D., 2016. Metallic
nanowire-based transparent electrodes for next generation flexible devices: a review.
Small 12, 6052–6075.
Scanlan, L.D., Reed, R.B., Loguinov, A.V., Antczak, P., Tagmount, A., Aloni, S., Nowinski, D.T.,
Luong, P., Tran, C., Karunaratne, N., Pham, D., Lin, X.X., Falciani, F., Higgins, C.P.,
Ranville, J.F., Vulpe, C.D., Gilbert, B., 2013. Silver nanowire exposure results in inter-
nalization and toxicity to Daphnia magna. ACS Nano 7, 10681–10694.
Sekine, R., Moore, K.L., Matzke, M., Vallotton, P., Jiang, H., Hughes, G.M., Kirby, J.K., Donner,
E., Grovenor, C.R.M., Svendsen, C., Lombi, E., 2017. Complementary imaging of silver
nanoparticle interactions with green algae: dark-field microscopy, electron micros-
copy, and nanoscale secondary ion mass spectrometry. ACS Nano 11, 10894–10902.
Sohn, E.K., Johari, S.A., Kim, T.G., Kim, J.K., Kim, E., Lee, J.H., Chung, Y.S., Yu, I.J., 2015.
Aquatic toxicity comparison of silver nanoparticles and silver nanowires. Biomed.
Res. Int. 2015.
Stegemeier, J.P., Schwab, F., Colman, B.P., Webb, S.M., Newville, M., Lanzirotti, A., Winkler,
C., Wiesner, M.R., Lowry, G.V., 2015. Speciation matters: bioavailability of silver and
silver sulfide nanoparticles to alfalfa (Medicago sativa). Environ. Sci. Technol. 49,
8451–8460.
Tan, C., W-H, Fan, W-X, Wang, 2012. Role of titanium dioxide nanoparticles in the ele-
vated uptake and retention of cadmium and zinc in Daphnia magna. Environ. Sci.
Technol. 46, 469–476.
Science of the Total Environment 782 (2021) 146933
Tan, L.-Y., Huang, B., Xu, S., Wei, Z.-B., Yang, L.-Y., Miao, A.-J., 2016. TiO2 nanoparticle up-
take by the water flea Daphnia magna via different routes is calcium-dependent.
Environ. Sci. Technol. 50, 7799–7807.
Tervonen, K., Waissi, G., Petersen, E.J., Akkanen, J., Kukkonen, J.V., 2010. Analysis of
fullerene-C60 and kinetic measurements for its accumulation and depuration in
Daphnia magna. Environ. Toxicol. Chem. 29, 1072–1078.
Toybou D., Celle C., Aude-Garcia C., Rabilloud T., Simonato J-P., 2019. A toxicology-
informed, safer by design approach for the fabrication of transparent electrodes
based on silver nanowires. Environ. Sci. Nano 6, 684–694.
Visnapuu M., Joost U., Juganson K., Künnis-Beres K., Kahru A., Kisand V., Ivask A., 2013.
Dissolution of silver nanowires and nanospheres dictates their toxicity to Escherichia
coli. Biomed. Res. Int. 2013, 819252.
Wang, C., X-L, Chang, Shi, Q., Zhang, X., 2018. Uptake and transfer of 13C-fullerenols from
Scenedesmus obliquus to Daphnia magna in an aquatic environment. Environ. Sci.
Technol. 52, 12133–12141.
Wegner, S.V., Arslan, H., Sunbul, M., Yin, J., He, C., 2010. Dynamic copper (I) imaging in
mammalian cells with a genetically encoded fluorescent copper (I) sensor. J. Am.
Chem. Soc. 132, 2567–2569.
Xie, S., Wong, A.Y., Kwok, R.T., Li, Y., Su, H., Lam, J.W.Y., Chen, S., Tang, B.Z., 2018.
Fluorogenic Ag+–tetrazolate aggregation enables efficient fluorescent biological sil-
ver staining. Angew. Chem. Int. Ed. 57, 5750–5753.
Yan, N., Tang, B.Z., W-X, Wang, 2018a. In vivo bioimaging of silver nanoparticle dissolu-
tion in the gut environment of zooplankton. ACS Nano 12, 12212–12223.
Yan, N., Xie, S., Tang, B.Z., W-X, Wang, 2018b. Real-time monitoring of the dissolution ki-
netics of silver nanoparticles and nanowires in aquatic environments using an
aggregation-induced emission fluorogen. Chem. Comm. 54, 4585–4588.
Yang, Y., Xu, S., Xu, G., Liu, R., Xu, A., Chen, S., Wu, L., 2019. Effects of ionic strength on
physicochemical properties and toxicity of silver nanoparticles. Sci. Total Environ.
647, 1088–1096.
Zhang, L., Wang, W.-X., 2018. Dominant role of silver ions in silver nanoparticle toxicity to
a unicellular alga: evidence from luminogen imaging. Environ. Sci. Technol. 53,
494–502.
Zhang, W., Yao, Y., Sullivan, N., Chen, Y., 2011. Modeling the primary size effects of citrate-
coated silver nanoparticles on their ion release kinetics. Environ. Sci. Technol. 45,
4422–4428.
Zhang, Y., Xia, J., Xu, J., Sun, B., Wu, W., Zhu, L., 2018. Impacts of surfactants on dissolution
and sulfidation of silver nanowires in aquatic environments. Environ. Sci.: Nano 5,
2452–2460.
Zhang, Y., Xu, J., Yang, Y., Sun, B., Wang, K., Zhu, L., 2020. Impacts of proteins on dissolu-
tion and sulfidation of silver nanowires in an aquatic environment: importance of
surface charges. Environ. Sci. Technol. 54, 5560–5568.
Zhao, C.-M., Wang, W.-X., 2010. Biokinetic uptake and efflux of silver nanoparticles in
Daphnia magna. Environ. Sci. Technol. 44, 7699–7704.
Zhao, C.-M., Wang, W.-X., 2012. Size-dependent uptake of silver nanoparticles in Daphnia
magna. Environ. Sci. Technol. 46, 11345–11351.
Zhu, X., Wang, J., Zhang, X., Chang, Y., Chen, Y., 2010. Trophic transfer of TiO2 nanoparti-
cles from daphnia to zebrafish in a simplified freshwater food chain. Chemosphere
79, 928–933.