Subscriber access provided by The University of British Columbia Library
Article
TiO2 Nanoparticle Uptake by the Water Flea Daphnia magna via Different Routes is Calcium-Dependent Ling-Yan Tan, Bin Huang, Shen Xu, Zhongbo Wei, Liuyan Yang, and Ai-Jun Miao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01645 • Publication Date (Web): 30 Jun 2016 Downloaded from http://pubs.acs.org on July 5, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 28
Environmental Science & Technology
1
Table of Contents Art Well-dispersed PAA-TiO2-NPs
Passive drinking
PAA-TiO2-NP aggregates
Active ingestion
Low Ca
High Ca
2 3 4
1
ACS Paragon Plus Environment
Environmental Science & Technology
5 6 7 8 9
10
TiO2 Nanoparticle Uptake by the Water Flea Daphnia magna via
11
Different Routes is Calcium-Dependent
12
13
14
Ling-Yan Tan, Bin Huang, Shen Xu, Zhong-Bo Wei, Liu-Yan Yang, Ai-Jun Miao*
15 16 17 18
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,
19
Nanjing University, Nanjing, Jiangsu Province, 210023, China
20 21 22 23 24 25
*Corresponding author:
[email protected] (Email), +86 25 89680255 (Tel.), +86 25
26
89680569 (Fax)
27 2
ACS Paragon Plus Environment
Page 2 of 28
Page 3 of 28
Environmental Science & Technology
28
ABSTRACT: Calcium plays versatile roles in aquatic ecosystems. In this study, we
29
investigated its effects on the uptake of polyacrylate-coated TiO2 nanoparticles
30
(PAA-TiO2-NPs) by the water flea (cladoceran) Daphnia magna. Particle distribution in these
31
daphnids was also visualized using synchrotron radiation-based micro X-ray fluorescence
32
spectroscopy, transmission electron microscopy, and scanning electron microscopy. At low
33
ambient Ca concentrations in the experimental medium ([Ca]dis), PAA-TiO2-NPs were well
34
dispersed and distributed throughout the daphnid; the particle concentration was highest in
35
the abdominal zone and the gut, as a result of endocytosis and passive drinking of the
36
nanoparticles, respectively. Further, Ca induced PAA-TiO2-NP uptake as a result of the
37
increased Ca influx. At a high [Ca]dis, the PAA-TiO2-NPs formed micron-sized aggregates
38
that were ingested by D. magna and concentrated only in its gut, independent of the Ca influx.
39
Our results demonstrated the multiple effects of Ca on nanoparticle bioaccumulation.
40
Specifically, well-dispersed nanoparticles were taken up by D. magna through endocytosis
41
and passive drinking whereas the uptake of micron-sized aggregates relied on active
42
ingestion.
43 44 45
3
ACS Paragon Plus Environment
Environmental Science & Technology
46
INTRODUCTION
47
Engineered nanoparticles are particles < 100 nm in at least two dimensions.1 They are
48
widely used in areas such as medicine, electronics, textiles, and environmental remediation.
49
With rapid advances in nanotechnology and the increased use of nanoparticles, a substantial
50
proportion will inevitably find their way into the aquatic environment. Understanding how
51
nanoparticles accumulate in aquatic organisms is essential to evaluating their ecotoxicity.
52
Although several studies have examined the toxicity of nanoparticles for various aquatic
53
organisms,2-4 relatively little is known about their accumulation kinetics.5,6
54
Water fleas are typical invertebrates universally distributed in fresh waters, and they
55
play important roles in the aquatic food chain. As a representative species of water flea,
56
Daphnia magna has served as a model organism in bioaccumulation studies of conventional
57
and emerging pollutants.7-9 It can accumulate pollutants through dissolved uptake or dietary
58
assimilation. The former applies to molecular pollutants with a sub-nano size; the main route
59
of entry of these agents is the epipodites of the thoracic limbs.10 In this case, uptake is
60
facilitated by a specialized cell membrane located in the gill epithelium of the daphnid that is
61
presumed to participate in the active transport of molecular pollutants.11 By contrast,
62
particulate pollutants are ingested by D. magna and then assimilated in its gut lumen.
63
However, the route of accumulation of nanoparticles, with a size intermediate between
64
molecules and bulk material, is unclear. According to current studies on nanoparticle
65
bioaccumulation, it is well-established that, regardless of their physicochemical
66
characteristics, a substantial proportion of nanoparticles can be accumulated in the gut of D.
67
magna.12-16 Yet, whether the nanoparticles pass through the gut’s epithelial barrier is debated.
68
Nanoparticles have been detected in areas outside the flea gut (e.g., in ovaries and in lipid
69
storage droplets),14-16 but direct evidence that they originated from the gut is lacking. The
70
possibility that nanoparticles may enter the body of Daphnia via routes other than the gut
71
cannot be excluded.
72
Moreover, the aggregation of nanoparticles in the aquatic environment can alter their
73
bioaccumulation.17 Environmental factors such as the pH and ionic strength of the medium as
74
well as the presence of cations and dissolved organic matter can strikingly influence the 4
ACS Paragon Plus Environment
Page 4 of 28
Page 5 of 28
Environmental Science & Technology
75
stability of nanoparticles.18,19 In aquatic ecosystems, for example, Ca concentrations, ranged
76
from < 0.5 mg/L to > 200 mg/L, can strongly influence nanoparticle aggregation.20,21 Calcium
77
is also a vital cation for D. magna, accounting for 2-8% of its dry weight, and low Ca
78
concentrations adversely affect its survival and reproduction.22 At the molecular level, Ca is
79
essential for many cellular processes, including endocytosis,23 which is the major cellular
80
pathway of nanoparticle internalization.
81
Therefore, in the present study, we examined the Ca-dependent accumulation of
82
polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) in D. magna. Specifically, particle
83
distribution in these daphnids at different Ca levels was visualized using synchrotron
84
radiation-based micro X-ray fluorescence spectroscopy (µXRF), scanning electron
85
microscopy (SEM), and transmission electron microscopy (TEM). The results showed that at
86
low Ca concentrations in the experimental medium ([Ca]dis) the PAA-TiO2-NPs were well
87
dispersed but at high [Ca]dis the particles readily aggregated. We then manipulated both the
88
influx and the intrinsic content of Ca in daphnids and examined the impact on PAA-TiO2-NP
89
uptake by D. magna. The overall objective was to elucidate the mechanisms underlying the
90
effects of Ca on the bioaccumulation of PAA-TiO2-NPs.
91
MATERIALS AND METHODS
92
Organisms and PAA-TiO2-NPs. The cladoceran Daphnia magna and its green algal
93
foods (Chlamydomonas reinhardtii and Scenedesmus obliquus) were obtained from the
94
Institute of Hydrobiology, Chinese Academy of Science. The daphnids were raised in aerated
95
tap water at 23.5 oC on a 12:12 h light-dark cycle with an irradiance of 30 µmol photons/m2/s.
96
Their density was kept at one individual per 10 mL water, and the medium was refreshed
97
every other day. Algal mixtures (2.5×104 cells/mL C. reinhardtii and 6×104 cells/mL S.
98
obliquus) were fed daily to the daphnids. The diet was doubled when D. magna was older
99
than 3 days. Both species of green algae were cultured in WC medium24 under the same
100
environmental conditions as D. magna. In all of the experiments described below, simplified
101
Elendt M7 medium (SM7)25 was the basal exposure medium and 7-day-old D. magna was the
102
objective organism. 5
ACS Paragon Plus Environment
Environmental Science & Technology
103
The PAA-TiO2-NPs (anatase, primary particle size 1-10 nm with an isoelectric point of
104
2.0) were the same as those used by Yang et al.26 and were purchased from Vive Nano
105
(Toronto, Canada). The nanoparticles were coated with sodium polyacrylate (74% of total
106
weight) to improve their stability in aqueous solutions. Their hydrodynamic size in the
107
experimental media was determined using a dynamic light scattering particle sizer (DLS,
108
ZetaPALS, Brookhaven Instruments, NY, USA).
109
Forty-Eight-Hour Uptake Experiment. PAA-TiO2-NP (4.0 mg-Ti/L) uptake by D.
110
magna was investigated in medium containing 0.2 and 2.0 mM Ca (in the form of CaCl2).
111
The concentration of PAA-TiO2-NPs used in the experiment was far below the non-observed
112
effect level (> 400 mg-Ti/L) determined in a preliminary acute toxicity study. Each treatment
113
had three replicates and each replicate comprised 70 individuals. These daphnids were first
114
allowed to evacuate their guts in fresh SM7 alone for 1 h and then transferred to the uptake
115
medium. After 1, 2, 4, 6, 12, 24, and 48 h, 10 individuals per time point were removed and
116
PAA-TiO2-NPs loosely adsorbed onto their carapaces were washed away using 3×100 mL of
117
SM7. The daphnids collected at each time point were digested in 20 mL H2SO4 and 8 g
118
(NH4)2SO4.27 The bioaccumulated Ti ([Ti]daphnia) was quantified using a graphite furnace
119
atomic absorption spectrometer (GFAAS, Thermo Fisher Scientific Inc., Waltham, MA, USA)
120
with a detection limit of 3 µg/L. The sample digestion procedure and the Ti determination
121
method were verified through D. magna samples spiked with known amount of
122
PAA-TiO2-NPs and a recovery of 100 ± 10% was observed.
123
In another 48-h experiment, all procedures were the same except that the uptake media
124
were refreshed every 12 h. The aim of this experiment was to examine whether the potential
125
sedimentation of PAA-TiO2-NPs in the uptake media (especially in the treatment with 2.0 M
126
Ca) influenced their accumulation during the 48-h period. Throughout both experiments, the
127
concentration of PAA-TiO2-NPs suspended in the uptake media was monitored.
128
Six-Hour Uptake Experiment. The procedure for this experiment was similar to that
129
used in the 48-h experiment described above. However, the exposure duration was shortened
130
to 6 h; three time points (1, 3, and 6 h) and five levels of [Ca]dis (0, 0.2, 0.5, 1, and 2 mM)
131
were evaluated. The uptake rate of PAA-TiO2-NPs was calculated as the slope of the linear 6
ACS Paragon Plus Environment
Page 6 of 28
Page 7 of 28
Environmental Science & Technology
132
regression between [Ti]daphnia and the exposure time.26 Additionally, PAA-TiO2-NP uptake in
133
the presence of 2.0 mM EGTA (ethylene glycol tetraacetic acid, a Ca-binding ligand) and at
134
different pH values (6, 7, 8, and 9) was examined at various levels of [Ca]dis (0, 0.5, and 2.0
135
mM in the EGTA experiment; 0, 0.2, and 2.0 mM for different pH values). As EGTA and pH
136
influence Ca speciation and its influx, they may also change the uptake of PAA-TiO2-NPs by
137
D. magna. In this experiment, 0.2 mM (0.5 mM) Ca was applied to maximize the potentially
138
inductive (inhibitory) effect of pH (EGTA). To further explore the mechanisms underlying
139
Ca-dependent PAA-TiO2-NP accumulation, the daphnids were pre-exposed to nifedipine (1
140
mM), amiloride (3.0 mM), or BAY K8644 (1 and 10 µM) for 1 h before the 6-h uptake of the
141
particles was examined. Nifedipine and amiloride are inhibitors of the L-type Ca channel and
142
the Na/Ca (NCX) exchanger, respectively,28 whereas BAY K8644 is an agonist of
143
voltage-gated Ca channels.29
144
Besides the changes in ambient Ca, its speciation, and its influx, as described above, the
145
intrinsic Ca content of the daphnids ([Ca]daphnia) was altered by rearing them in SM7
146
containing 12.5 µM, 0.5 mM, or 5.0 mM Ca for 24 and 48 h. PAA-TiO2-NP uptake was then
147
determined in SM7 at the same level of [Ca]dis (12.5 µM). The Ca content in the daphnids
148
was measured by GFAAS. [Ca]daphnia, or its bioavailable concentration in the cells was further
149
modulated
150
[1,2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester)]. A-23187
151
is a carrier of Ca; thapsigargin induces the liberation of Ca from the endoplasmic reticulum to
152
the cytoplasm;30,31 and BAPTA-AM, as a strong Ca-binding ligand, enters cells by passive
153
diffusion and decreases the cytosolic ionized Ca concentration.32 PAA-TiO2-NP uptake in the
154
presence of 0, 0.01, 0.04, and 0.2 µM A-23187 was performed with a [Ca]dis of 0.2 mM. In
155
addition, the daphnids were pre-exposed to thapsigargin (0, 0.3, 1.0, and 3.0 µM) or
156
BAPTA-AM (0, 0.03, 0.2, and 1.0 mM) for 1 h, after which PAA-TiO2-NP accumulation was
157
quantified in the uptake media alone, without added Ca, thapsigargin, or BAPTA-AM.
using
calcium
ionophore
(A-23187),
thapsigargin,
and
BAPTA-AM
158
PAA-TiO2-NP Elimination Experiment. Seven-day-old daphnids were exposed to 4
159
mg-Ti/L PAA-TiO2-NPs at a [Ca]dis of 0.2 mM for 1 h and then evenly divided into three
160
depuration treatments, containing no PAA-TiO2-NPs. Daphnids in the first two treatments 7
ACS Paragon Plus Environment
Environmental Science & Technology
161
were transferred to fresh SM7 with and without the addition of 0.2 mM EGTA, respectively.
162
Those in the third treatment were heat killed (45 oC for 2 min) and then transferred to fresh
163
SM7. Each treatment consisted of three replicates, and 10 daphnids were collected from each
164
one for the measurement of [Ti]daphnia after 0, 1, 3, 6, 12, 24, and 48 h of depuration.
165
µXRF Studies. D. magna samples for µXRF were prepared following a procedure
166
similar to that described in Laforsch and Tollrian.33 Briefly, the daphnids were exposed to 40
167
mg-Ti/L PAA-TiO2-NPs for 24 h with a [Ca]dis of 0.2 and 2.0 mM. They were then fixed in
168
4% methanol for 10 min before being dehydrated in graded acetone solutions (70, 80, 90,
169
2×98, and 2×100%) for 10 min each. Subsequently, the specimens were immersed in 1.5 mL
170
of HMDS (1,1,1,3,3,3 hexamethyldisilazane); 90% of the HMDS was pipetted out after 30
171
min, and the rest was evaporated in a desiccator. The PAA-TiO2-NP concentrations used in
172
this and the experiments described below were ten times higher than the concentrations used
173
in the uptake and elimination experiments, because of the detection limit of µXRF, TEM, and
174
SEM. Nevertheless, the average hydrodynamic size of PAA-TiO2-NPs remained unchanged.
175
µXRF mapping of Ti (KL3, 4.5109 keV) and Ca (KL3, 3.6917 keV) in the daphnids was
176
performed using the BL15U beamline at the Shanghai Radiation Synchrotron Facility
177
(Shanghai, China). The storage ring current was 200-300 mA with an energy level of 3.5
178
GeV. Element maps were obtained by scanning the samples with a 10-keV monochromatic
179
beam, which was focused to 50 × 50 µm2 using K-B optics. The step size and scanning time
180
were 50 µm and 3 s, respectively. X-ray fluorescence was recorded using the 7-element Si (Li)
181
detector combined with a multiple channel analyzer (e2v, UK). The fluorescence data were
182
processed using Pviewer (version 1.0) and 2D Array Image Data Plotter (version 1.0).
183
TEM and SEM Analyses. A procedure similar to that described in a previous study by
184
our group27 was used to process the daphnids. Briefly, after a 24-h exposure to 40 mg-Ti/L
185
PAA-TiO2-NPs, the fleas were fixed in 3% glutaraldehyde prepared in 0.2 M
186
phosphate-buffered saline (PBS). They were then cleaned with PBS, stained in 1% osmium
187
tetroxide, and dehydrated sequentially in acetone solutions (30, 50, 70, 80, 90, and 2×100%).
188
Afterward, they were embedded into epoxy resin, sectioned to 100-nm thickness, and stained
189
with uranyl acetate and lead citrate. The elemental composition of the potentially 8
ACS Paragon Plus Environment
Page 8 of 28
Page 9 of 28
Environmental Science & Technology
190
PAA-TiO2-NP-containing spots in the TEM samples was determined using an
191
energy-dispersive X-ray (EDX) spectrometer (JEM-2100, JEOL, Tokyo, Japan). For SEM
192
analysis, the D. magna samples were fixed and dehydrated following the same procedure as
193
that for µXRF. After the carapace was removed using Teflon forceps, the thoracic limbs were
194
collected and their Ti distribution was mapped by SEM-EDX (S-4800, Hitachi, Tokyo,
195
Japan).
196
Statistical Analysis. Significant differences were defined based on a p value of < 0.05,
197
obtained in a one-way or two-way analysis of variance with posthoc multiple comparisons
198
(Tukey or Tamhane; SPSS 11.0 by SPSS, Chicago, USA). The analysis of variance took into
199
account both the normality (Kolmogorov-Smirnov and Shapiro-Wilk tests) and the
200
homogeneity of variance (Levene’s test) of the data.
201
RESULTS AND DISCUSSION
202
Ca Effects on PAA-TiO2-NP Accumulation. According to the conventional
203
biodynamic model,34 the content of a pollutant in an organism (Cb) first increases with
204
exposure time (t) and then levels off, as described by Eq. (1):
205
Cb =
k u C w (1 − e − ( ke + µ )t ) ke + µ
(1)
206
where Cw represents the pollutant’s concentration in the uptake medium, ku and ke are the
207
uptake and efflux rate constants, respectively, and µ is the organism’s growth rate, which is
208
negligible for D. magna.34 In the present study, a hyperbolic relationship between Cb and t
209
was not observed when the medium in the 48-h uptake experiment was not regularly
210
refreshed (Figure 1a). Thus, [Ti]daphnia increased initially with exposure time, reached its
211
maximum after 12 h, and decreased thereafter at both [Ca]dis (0.2 and 2.0 mM). According to
212
Eq. (1), a hyperbolic relationship between Cb and t requires that ku, ke, and Cw remain
213
constant during the experimental period. When the uptake medium was refreshed every 12 h,
214
[Ti]daphnia in the high-Ca treatment leveled off instead of decreasing after reaching a
215
maximum (Figure 1a). This finding implies that the decrease in Ti accumulation after a 12-h
216
exposure at high Ca levels was primarily due to the reduced concentration of PAA-TiO2-NPs 9
ACS Paragon Plus Environment
Environmental Science & Technology
217
suspended in the uptake medium. This explanation is supported by the formation of
218
PAA-TiO2-NP aggregates with an average size of 1650 nm, which after 12 h settled on the
219
bottom of the container, thus reducing the concentration (4 mg-Ti/L) of suspended
220
PAA-TiO2-NPs by > 70%. Similarly, quantum dot concentration in the gut of D. magna was
221
also found to decrease as a result of the decrease in the water column quantum dot
222
concentration.35 Unlike in the high-Ca (2.0 mM) treatment, the pattern of [Ti]daphnia variation
223
with exposure time remained unchanged, regardless of whether the medium was refreshed, at
224
a [Ca]dis of 0.2 mM. In this case, PAA-TiO2-NPs were well dispersed, with an average size of
225
20 nm, and their suspended concentration remained stable even when the uptake medium was
226
not refreshed. Therefore, the parabolic relationship between [Ti]daphnia and the exposure time
227
in the low-Ca treatment was due to the change in ku, ke, or both. It appears that D. magna has
228
a regulation system to limit excessive accumulation of PAA-TiO2-NPs, similar to what was
229
described in Adam et al.36. These results also suggested that, at the two [Ca]dis, the
230
PAA-TiO2-NPs were taken up by D. magna through disparate routes, which further implied
231
that the nanoparticle uptake pathway might be aggregation/size dependent. This will be
232
discussed below. Nevertheless, it is also likely that the PAA-TiO2-NP uptake route was the
233
same, but their distribution in daphnids or their efflux was Ca-dependent.
234
In addition to the different patterns of [Ti]daphnia variation with exposure time,
235
Ca-related effects on nanoparticle accumulation were evidenced by the significantly (p < 0.05)
236
higher [Ti]daphnia when [Ca]dis was 2.0 mM (Figure 1a). To determine whether this
237
accumulation disparity was simply caused by Ca-induced aggregation, PAA-TiO2-NP uptake
238
rates at five Ca levels were compared (Figure 1b). As [Ca]dis increased from 0 to 0.5 mM, the
239
hydrodynamic size (19.4-20.4 nm) of the PAA-TiO2-NPs remained stable, but their uptake
240
rate was significantly (p < 0.05) increased, by 243%. When [Ca]dis was further enhanced to
241
1.0 and 2.0 mM, massive aggregation occurred, yielding particle sizes of 675.4 and 1561.1
242
nm, respectively. The uptake rates of PAA-TiO2-NPs at these two highest Ca levels were
243
comparable. It seemed that Ca induced the uptake of well-dispersed PAA-TiO2-NPs but had
244
no effect on their micron-sized aggregates. This also suggested different routes of entry for
245
well-dispersed PAA-TiO2-NPs and their micron-sized aggregates in D. magna, a hypothesis 10
ACS Paragon Plus Environment
Page 10 of 28
Page 11 of 28
Environmental Science & Technology
246
later supported by the difference in the distribution of Ti in daphnids at a [Ca]dis of 0.2 vs. 2.0
247
mM (Figure 2). The D. magna in the different treatments was distinguished by the Ca signal
248
(Figure 2a, c, and e), with a negligible amount of Ti in unexposed daphnids (Figure 2b). At a
249
[Ca]dis of 0.2 mM, a substantial amount of Ti accumulated within the daphnids (Figure 2d),
250
mostly in the gut and abdominal zone. Separating these two sites was a region with a
251
relatively low Ti content. Thus, the PAA-TiO2-NPs concentrated in these two tissues may
252
have differed in their origins. Nanoparticle accumulation in the abdominal zone was also
253
evidenced by the pronounced Ti signal in the epipodite and filter setae isolated from the
254
bodies of daphnids (Figure 3a, b). In contrast to the low-Ca treatment (Figure 2d), the
255
PAA-TiO2-NPs were detected only in the daphnid gut when [Ca]dis was 2.0 mM (Figure 2f).
256
According to the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory,37,38 nanoparticles
257
aggregate rapidly at high Ca levels due to the screening of their surface charge and the lower
258
aggregation energy barrier. The distance between filtering setae on Daphnia thoracic limbs is
259
approximately 0.5 µm (Figure 3a), and filtration, rather than electrostatic attraction, is the
260
main mechanism for particle capture by daphnids.10,39 This explains the accumulation of the
261
micron-sized PAA-TiO2-NP aggregates in the gut. In contrast to their micron-sized
262
counterparts, well-dispersed PAA-TiO2-NPs with a size of ~20 nm cannot be taken up by
263
daphnids through setae filtration, which suggests a route other than ingestion. Gophen and
264
Geller40 found that particles < 0.5 µm did not accumulate in the daphnid gut. Nevertheless,
265
our detection of the low-level gut accumulation of well-dispersed PAA-TiO2-NPs at low Ca
266
levels suggests passive drinking (also called incidental ingestion) of the experimental
267
medium by the flea.10 A substantial accumulation of well-dispersed gold nanoparticles in the
268
gut tract of D. magna was also reported in Wray and Klaine.8 They further proposed that this
269
phenomenon was caused by incidental ingestion through physicochemical processes such as,
270
gravitational deposition, inertial impaction, motile-particle deposition, and electrostatic
271
interaction.
272
That the Ti signal in the D. magna abdominal zone at a low [Ca]dis reflected true
273
internalization of the well-dispersed PAA-TiO2-NPs rather than simply their attachment to the
274
thoracic limb surface was supported by the following observations. When the 11
ACS Paragon Plus Environment
Environmental Science & Technology
Page 12 of 28
275
nanoparticle-containing daphnids were transferred to fresh SM7, a biphasic decrease in
276
[Ti]daphnia was observed during the 48-h depuration period (Figure 4). After 12 h, > 70% of the
277
PAA-TiO2-NPs were depurated, and only 15% was retained at 48 h. The presence of EGTA in
278
the
279
PAA-TiO2-NP-containing daphnids were killed at the beginning of the experiment, efflux was
280
not significant (p > 0.05). Together, these findings suggested that the Ca-mediated induction
281
of the accumulation of well-dispersed PAA-TiO2-NPs was not simply due to the increased
282
adsorption of the particles onto the daphnids and that depuration was not a passive process
283
occurring by, e.g., surface desorption. Otherwise, EGTA might accelerate the depuration of
284
PAA-TiO2-NPs and a notable elimination of PAA-TiO2-NPs by the heat-killed daphnids
285
might be observed. Therefore, the notable Ti signal in Figure 2d reflected the internalization
286
of the PAA-TiO2-NPs. This conclusion was further supported by the two-orders-of-magnitude
287
lower accumulation of Ti in heat-killed (0.02-0.06 mg-Ti/g-dw) than in living daphnids.
288
Moreover, TEM images of tissue slices from D. magna abdominal zone showed a substantial
289
internalization of PAA-TiO2-NPs into epipodite cells (Figure 3c, d), where they were
290
primarily concentrated in micron-sized endosomes. Research in daphnids, although limited,
291
has shown that accumulated nanoparticles are either confined to the gut lumen or pass
292
through the gut’s epithelial barrier to specific anatomic sites, such as the ovaries and
293
lipid-storage droplets. Heinlaan et al.12 stated that the implicit internalization of CuO
294
nanoparticles via D. magna midgut epithelial cells was not obvious. Khan et al.13 found no
295
evidence for trans-epithelial alimentary uptake of ingested Au nanoparticles by D. magna
296
either. By contrast, fluorescent polystyrene nanoparticles were observed to accumulate in D.
297
magna lipid-storage droplets, suggesting their penetration of gut epithelial cells.14 Similarly,
298
quantum dots of various surface coatings concentrated in the D. magna gastrointestinal tract,
299
brood chamber, and exoskeleton.15 Nevertheless, direct evidence for the translocation of these
300
nanoparticles from the gut to the objective location is lacking. Other entry routes may thus
301
exist, as supported not only by the results of the present study but also by those of other
302
researchers. For example, although Ag nanoparticles accumulated in D. magna ovaries,16
303
there was no evidence that they had penetrated the gastrointestinal tract barrier and their
304
concentration in the gut lumen was low.
efflux
medium
had
no
effect
on
depuration.
12
ACS Paragon Plus Environment
By
contrast,
when
the
Page 13 of 28
Environmental Science & Technology
305
Manipulation of Ambient Ca Speciation and Its Influx. To further elucidate the
306
mechanisms underlying the Ca-dependent accumulation of well-dispersed PAA-TiO2-NPs
307
and to confirm different uptake routes of well-dispersed PAA-TiO2-NPs and their
308
micron-sized aggregates, the effects of EGTA, pH, and Ca channel inhibitors/agonist on
309
PAA-TiO2-NP uptake were investigated. A pre-requisite of these experiments is that the
310
nanoparticle aggregation status was unaffected by EGTA, pH, and Ca channel
311
inhibitors/agonist. In the presence of 2.0 mM EGTA, free Ca ions in the medium were
312
reduced dramatically, such that they contributed < 1% to the total Ca. Under this condition,
313
PAA-TiO2-NP uptake was suppressed by 65.7% when the [Ca]dis was 0.5 mM, whereas
314
inhibition was not significant (p > 0.05) at a [Ca]dis of 2.0 mM (Figure 5a). PAA-TiO2-NP
315
uptake suppression in the first case was not caused by EGTA itself or by cations other than Ca
316
(e.g., K, Na, and Mg), considering the fact that neither EGTA nor EDTA, as common
317
metal-binding ligands, influenced the uptake of well-dispersed PAA-TiO2-NPs in the absence
318
of Ca. Moreover, PAA-TiO2-NPs were well-dispersed with an average particle size of 20 nm
319
and their uptake remained constant in medium containing 0-1.0 mM Mg but without Ca
320
(Supporting Information, Figure S1). All these phenomena imply that the uptake of
321
well-dispersed PAA-TiO2-NPs is Ca-dependent rather than a general effect of cations and this
322
process is determined by the concentration of free Ca ions rather than the total Ca
323
concentration.
324
The results of the EGTA experiment suggested two possible mechanisms for the
325
observed effects of free Ca ions. (1) The free Ca ion concentration determines the amount of
326
Ca adsorbed on the surfaces of PAA-TiO2-NPs and D. magna, which in turn influences
327
attachment of the nanoparticles to the target sites and their subsequent internalization.41 A
328
positive correlation between cations such as Ca, Mg, and Na and the attachment efficiency of
329
nanoparticles (e.g., fullerene and α-Fe2O3) was previously reported.21,41 (2) The influx of Ca
330
and its bioavailability in the cytosol or certain micro-domains of D. magna cells depend on its
331
ambient free ion concentration. Endocytosis, as the main form of cellular nanoparticle
332
internalization, has been related to the influx or intracellular content of Ca. Lew et al.42
333
showed that Fc-receptor-triggered phagocytosis in human neutrophils depends on 13
ACS Paragon Plus Environment
Environmental Science & Technology
334
intracellular Ca transients. Our preliminary experiment in the protozoan Tetrahymena
335
thermophila also demonstrated that Ca induced the endocytosis of well-dispersed
336
PAA-TiO2-NPs (Supporting Information, Figure S2). In a study in rodents, Ca influx initiated
337
all forms of endocytosis at a single nerve terminal in response to the speeding up of
338
membrane invagination and fission.43 The Ca sensor mediating these forms of endocytosis is
339
calmodulin, which activates the phosphatase calcineurin and thus targets numerous endocytic
340
proteins.23
341
To determine which of the two mechanisms proposed above is the more likely one in D.
342
magna, PAA-TiO2-NP uptake at different pH values but with the same [Ca]dis was compared.
343
At the four pH values tested (6-9), the free Ca ion concentration in the medium remained
344
constant for each [Ca]dis, particularly at pH 6, 7, and 8. Nevertheless, when [Ca]dis was 0.2
345
mM, the PAA-TiO2-NP uptake rate increased gradually, from 0.28 mg-Ti/g-dw/h at pH 6 to
346
0.86 mg-Ti/g-dw/h at pH 8 and leveled off thereafter (Figure 5b). By contrast, when [Ca]dis
347
was 0 or 2.0 mM, PAA-TiO2-NP uptake was independent of ambient pH. This discrepancy
348
implied that Ca plays an important role in the effects of pH on the uptake of well-dispersed
349
PAA-TiO2-NPs. According to the free ion activity model (FIAM) as proposed for aquatic
350
organisms,44 Ca adsorption and its uptake should increase with increasing pH, as the
351
competition from H+ ions decreases. It seems that the uptake of well-dispersed
352
PAA-TiO2-NPs was related to Ca influx. Nevertheless, Ca adsorption on PAA-TiO2-NPs, as
353
measured by GFAAS, also rose from 520 to 628 mg g-Ti-1 as the pH was increased from 6 to
354
9, although it was less influenced by pH than by EGTA (248 and 570 mg g-Ti-1 of Ca on
355
PAA-TiO2-NPs in the presence and absence of 2.0 mM EGTA). Therefore, the first possibility
356
of the above-proposed mechanisms cannot be excluded.
357
As inhibitors of the L-type Ca channel and the NCX exchanger, nifedipine and
358
amiloride inhibit Ca uptake.20 Although neither inhibitor altered Ca speciation in the medium
359
or its adsorption onto PAA-TiO2-NPs and the D. magna surface, at a [Ca]dis of 0.5 mM
360
PAA-TiO2-NP uptake decreased by 33.9 and 79.8% in the presence of nifedipine and
361
amiloride, respectively (Figure 5c). By contrast, at a [Ca]dis of 2.0 mM the effects were not
362
significant (p > 0.05), with PAA-TiO2-NP uptake remaining in the range of 0.96-1.38 14
ACS Paragon Plus Environment
Page 14 of 28
Page 15 of 28
Environmental Science & Technology
363
mg-Ti/g-dw/h. Similar to the high-Ca treatment, nifedipine did not suppress PAA-TiO2-NP
364
uptake in medium without added Ca; amiloride, however, reduced PAA-TiO2-NP uptake by
365
74.5%, suggesting that its effects were Ca-independent. In fact, amiloride also inhibits
366
Na+/H+ exchangers and may therefore limit endocytosis by lowering the submembranous pH
367
and preventing Rac1 and Cdc42 signaling.45 Nevertheless, the possibility that amiloride
368
altered Ca translocation between the different compartments of D. magna cells and thereby
369
reduced the intracellularly bioavailable Ca cannot be excluded. As for the Ca channel agonist
370
BAY K8644, its effect on PAA-TiO2-NP uptake was dependent on [Ca]dis, with induction
371
observed at 0.2 mM but not at 0 or 2.0 mM (Figure 5d). Collectively, the results from the
372
inhibitor and agonist experiments clearly demonstrated that Ca influx or its bioavailable
373
concentration in D. magna cells plays a critical role in the uptake of well-dispersed
374
PAA-TiO2-NPs. Further, the completely different responses of the uptake of well-dispersed
375
PAA-TiO2-NPs and their micron-sized aggregates to EGTA, pH, nifedipine, amiloride, and
376
BAY K8644 also suggested their different routes of entry in daphnids.
377
Effects of Intracellular Ca Bioavailability. We then asked whether the Ca-dependent
378
uptake of well-dispersed PAA-TiO2-NPs was attributable to the change in Ca influx itself or
379
to fluctuations of its bioavailability in the cytosol or certain micro-domains of D. magna cells.
380
To answer this question, [Ca]daphnia was manipulated as follows. When the daphnids were
381
pre-cultured in media with a [Ca]dis of 0.0125, 0.5, and 5 mM, [Ca]daphnia was, respectively,
382
36.3, 37.2, and 49.3 mg/g after 24 h and 18.3, 37.5, and 42.8 mg/g after 48 h (Figure 6a) as
383
measured by GFAAS. The daphnids look healthy at all three Ca levels based on their
384
swimming behavior. At both time points, the absence of a significant (p > 0.05) alteration of
385
PAA-TiO2-NP uptake could be explained by the fact that [Ca]dis (0.0125 mM) was the same
386
in the uptake media of the different treatments such that Ca influx was the same for the
387
different Ca-containing daphnids. Alternatively, although [Ca]daphnia changed, how the
388
intracellular Ca speciation may vary was unclear and thus the free Ca concentration in the
389
cytosol or other micro-domains of the daphnids may have remained unchanged. In addition to
390
allowing the daphnids to acclimate to media with different [Ca]dis, the lipophilic Ca-binding
391
reagent BAPTA-AM was applied to reduce the intracellular concentration of free Ca ions. No 15
ACS Paragon Plus Environment
Environmental Science & Technology
392
significant (p > 0.05) effects were found in either case (Figure 6b). When the Ca
393
concentration in the cytosol of daphnids was increased by the addition of a calcium ionophore
394
or thapsigargin, particle uptake was unaffected (Figure 6b). Taken together, these results
395
suggest that the influx of Ca through the plasma membrane, but not its concentration in
396
daphnids, determines the uptake of well-dispersed PAA-TiO2-NPs. Moreover, the uptake of
397
dissolved Ca occurred primarily in D. magna epipodites, where well-dispersed
398
PAA-TiO2-NPs concentrate,10 suggesting that the nanoparticles are taken up by the thoracic
399
limbs. During the Ca transients that occur in all organisms, local Ca concentrations may
400
increase to tens of µM upon the opening of Ca channels and then fall dramatically during
401
their closure.46 Therefore, it is also likely that in a multicellular organism such as D. magna
402
the addition of Ca ionophore, thapsigargin, or BAPTA-AM did not change the Ca
403
concentration in the specific micro-domains responsible for PAA-TiO2-NP uptake.
404
Overall, our study demonstrated the multiple effects of Ca on PAA-TiO2-NP
405
accumulation. When the Ca level in the medium was low (0-0.5 mM), the PAA-TiO2-NPs
406
were well dispersed and their hydrodynamic size was approximately 20 nm. In this case,
407
passive drinking and endocytosis were the dominant routes for PAA-TiO2-NP accumulation.
408
The nanoparticles taken up by these two routes were distributed throughout the daphnids,
409
with the highest concentrations in the abdominal zone and gut as a result of endocytosis and
410
passive drinking, respectively. When the Ca level in the medium was high (≥ 1 mM), the
411
PAA-TiO2-NPs formed micron-sized aggregates that were actively ingested by D. magna,
412
such that they were exclusively located in the gut. In addition to the effects of [Ca]dis on
413
nanoparticle aggregation and uptake route, the influx of Ca and possibly its concentration in
414
specific micro-domains of the organism could facilitate the uptake of well-dispersed
415
PAA-TiO2-NPs. The Ca-dependent uptake and distribution of PAA-TiO2-NPs we observed
416
herein are likely able to be extended to other nanoparticles and other aquatic organisms.
417
Therefore, the uptake pathways identified in the present study should be considered in risk
418
evaluations of nanoparticles.
419
420
ACKNOWLEDGEMENTS We thank three anonymous reviewers and Dr. Qiaoguo Tan for their instructive 16
ACS Paragon Plus Environment
Page 16 of 28
Page 17 of 28
Environmental Science & Technology
421
comments on this paper. The financial support offered by Chinese public science and
422
technology research funds projects of ocean (grant no. 201505034) and by the National
423
Natural Science Foundation of China (grant nos. 41271486, 41001338, and 21237001) to A. J.
424
Miao made this work possible.
425
SUPPORTING INFORMATION
426
Additional figures showing PAA-TiO2-NP uptake at different levels of Mg as well as the
427
effects of EGTA on PAA-TiO2-NP uptake by Tetrahymena thermophila are included. This
428
material is available free of charge on the ACS Publications Website.
429 430
17
ACS Paragon Plus Environment
Environmental Science & Technology
431
432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473
(1) Klaine, S. J.; Alvarez, P. J. J.; Batley, G. E.; Fernandes, T. F.; Handy, R. D.; Lyon, D. Y.; Mahendra, S.; McLaughlin, M. J.; Lead, J. R. Nanomaterials in the environment: Behavior, fate, bioavailability, and effects. Environ. Toxicol. Chem. 2008, 27, (9), 1825-1851. (2) Bondarenko, O.; Juganson, K.; Ivask, A.; Kasemets, K.; Mortimer, M.; Kahru, A. Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: A critical review. Arch. Toxicol. 2013, 87, (7), 1181-1200. (3) Soni, D.; Naoghare, P. K.; Saravanadevi, S.; Pandey, R. A. Release, transport and toxicity of engineered nanoparticles. In Reviews of Environmental Contamination and Toxicology; Whitacre, D. M., Ed.; Springer International Publishing: New York City 2015; pp 1-47. (4) Nam, D. H.; Lee, B. C.; Eom, I. C.; Kim, P.; Yeo, M. K. Uptake and bioaccumulation of titanium- and silver-nanoparticles in aquatic ecosystems. Mol. Cell. Toxicol. 2014, 10, (1), 9-17. (5) Wang, Y.; Miao, A. J.; Luo, J.; Wei, Z. B.; Zhu, J. J.; Yang, L. Y. Bioaccumulation of CdTe quantum dots in a freshwater alga Ochromonas danica: A kinetics study. Environ. Sci. Technol. 2013, 47, (18), 10601-10610. (6) Croteau, M. N.; Misra, S. K.; Luoma, S. N.; Valsami-Jones, E. Bioaccumulation and toxicity of CuO nanoparticles by a freshwater invertebrate after waterborne and dietborne exposures. Environ. Sci. Technol. 2014, 48, (18), 10929-10937. (7) Zhao, C. M.; Wang, W. X. Biokinetic uptake and efflux of silver nanoparticles in Daphnia magna. Environ. Sci. Technol. 2010, 44, (19), 7699-7704. (8) Wray, A. T.; Klaine, S. J. Modeling the influence of physicochemical properties on gold nanoparticle uptake and elimination by Daphnia magna. Environ. Toxicol. Chem. 2015, 34, (4), 860-872. (9) Li, W. M.; Wang, W. X. Distinct biokinetic behavior of ZnO nanoparticles in Daphnia magna quantified by synthesizing Zn-65 tracer. Water Res. 2013, 47, (2), 895-902. (10) Smirnov, N. Physiology of the Cladocera. Academic Press: Cambridge, Massachusetts, USA, 2013. (11) Kikuchi, S. A unique cell-membrane with a lining of repeating subunits on the cytoplasmic side of presumably ion-transporting cells in the gill epithelium of Daphnia magna (Crustacea, Cladocera). J. Submicr. Cytol. Path. 1982, 14, (4), 711-715. (12) Heinlaan, M.; Kahru, A.; Kasemets, K.; Arbeille, B.; Prensier, G.; Dubourguier, H. C. Changes in the Daphnia magna midgut upon ingestion of copper oxide nanoparticles: A transmission electron microscopy study. Water Res. 2011, 45, (1), 179-190. (13) Khan, F. R.; Kennaway, G. M.; Croteau, M. N.; Dybowska, A.; Smith, B. D.; Nogueira, A. J. A.; Rainbow, P. S.; Luoma, S. N.; Valsami-Jones, E. In vivo retention of ingested Au NPs by Daphnia magna: No evidence for trans-epithelial alimentary uptake. Chemosphere 2014, 100, 97-104. (14) Rosenkranz, P.; Chaudhry, Q.; Stone, V.; Fernandes, T. F. A comparison of nanoparticle and fine particle uptake by Daphnia magna. Environ. Toxicol. Chem. 2009, 28, (10), 2142-2149. (15) Feswick, A.; Griffitt, R. J.; Siebein, K.; Barber, D. S. Uptake, retention and
REFERENCES
18
ACS Paragon Plus Environment
Page 18 of 28
Page 19 of 28
474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517
Environmental Science & Technology
internalization of quantum dots in Daphnia is influenced by particle surface functionalization. Aquat. Toxicol. 2013, 130, 210-218. (16) Georgantzopoulou, A.; Balachandran, Y. L.; Rosenkranz, P.; Dusinska, M.; Lankoff, A.; Wojewodzka, M.; Kruszewski, M.; Guignard, C.; Audinot, J. N.; Girija, S.; Hoffmann, L.; Gutleb, A. C. Ag nanoparticles: size- and surface-dependent effects on model aquatic organisms and uptake evaluation with NanoSIMS. Nanotoxicology 2013, 7, (7), 1168-1178. (17) Kwon, D.; Jeon, S. K.; Yoon, T. H. Impact of agglomeration on the bioaccumulation of sub-100 nm sized TiO2. Colloid. Surface. B 2014, 116, 277-283. (18) Navarro, E.; Baun, A.; Behra, R.; Hartmann, N. B.; Filser, J.; Miao, A. J.; Quigg, A.; Santschi, P. H.; Sigg, L. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 2008, 17, (5), 372-386. (19) Quigg, A.; Chin, W. C.; Chen, C. S.; Zhang, S.; Jiang, Y.; Miao, A. J.; Schwehr, K. A.; Xu, C.; Santschi, P. H. Direct and indirect toxic effects of engineered nanoparticles on algae: Role of natural organic matter. ACS Sustainable Chem. Eng. 2013, 1, (7), 686-702. (20) Tan, Q. G.; Wang, W. X. Interspecies differences in calcium content and requirement in four freshwater cladocerans explained by biokinetic parameters. Limnol. Oceanogr. 2010, 55, (3), 1426-1434. (21) Chen, K. L.; Mylon, S. E.; Elimelech, M. Aggregation kinetics of alginate-coated hematite nanoparticles in monovalent and divalent electrolytes. Environ. Sci. Technol. 2006, 40, (5), 1516-1523. (22) Tan, Q. G.; Wang, W. X. The regulation of calcium in Daphnia magna reared in different calcium environments. Limnol. Oceanogr. 2009, 54, (3), 746-756. (23) Wu, L. G.; Hamid, E.; Shin, W.; Chiang, H. C. Exocytosis and endocytosis: Modes, functions, and coupling mechanisms. Ann. Rev. Physiol. 2014, 76, 301-331. (24) Guillard, R. R.; Lorenzen, C. J. Yellow-green algae with chlorophyllide C. J. Phycol. 1972, 8, (1), 10-14. (25) Miao, A. J.; Wang, N. X.; Yang, L. Y.; Wang, W. X. Accumulation kinetics of arsenic in Daphnia magna under different phosphorus and food density regimes. Environ. Toxicol. Chem. 2012, 31, (6), 1283-1291. (26) Yang, W. W.; Wang, Y.; Huang, B.; Wang, N. X.; Wei, Z. B.; Luo, J.; Miao, A. J.; Yang, L. Y. TiO2 nanoparticles act as a carrier of Cd bioaccumulation in the ciliate Tetrahymena thermophila. Environ. Sci. Technol. 2014, 48, (13), 7568-7575. (27) Yang, W. W.; Miao, A. J.; Yang, L. Y. Cd2+ toxicity to a green alga Chlamydomonas reinhardtii as influenced by its adsorption on TiO2 engineered nanoparticles. Plos One 2012, 7, (3) e32300. (28) Granado e Sa, M.; Baptista, B. B.; Farah, L. S.; Leite, V. P.; Zanotto, F. P. Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei. J. Comp. Physiol. B 2010, 180, (3), 313-321. (29) Benaim, G.; Garcia-Marchan, Y.; Reyes, C.; Uzcanga, G.; Figarella, K. Identification of a sphingosine-sensitive Ca2+ channel in the plasma membrane of Leishmania mexicana. Biochem. Bioph. Res. Co. 2013, 430, (3), 1091-1096. (30) Verma, A.; Bhatt, A. N.; Farooque, A.; Khanna, S.; Singh, S.; Dwarakanath, B. S. Calcium ionophore A23187 reveals calcium related cellular stress as "I-Bodies": An old actor in a new role. Cell Calcium 2011, 50, (6), 510-522. 19
ACS Paragon Plus Environment
Environmental Science & Technology
518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
(31) Lytton, J.; Westlin, M.; Hanley, M. R. Thapsigargin inhibits the sarcoplasmic or endoplasmic-reticulum Ca-ATPase family of calcium pumps. J. Biol. Chem. 1991, 266, (26), 17067-17071. (32) Cousin, M. A.; Robinson, P. J. Ca2+ influx inhibits dynamin and arrests synaptic vesicle endocytosis at the active zone. J. Neurosci. 2000, 20, (3), 949-957. (33) Laforsch, C.; Tollrian, R. A new preparation technique of daphnids for Scanning Electron Microscopy using hexamethyldisilazane. Arch. Hydrobiol. 2000, 149, (4), 587-596. (34) Tsui, M. T. K.; Wang, W. X. Biokinetics and tolerance development of toxic metals in Daphnia magna. Environ. Toxicol. Chem. 2007, 26, (5), 1023-1032. (35) Jackson, B. P.; Pace, H. E.; Lanzirotti, A.; Smith, R.; Ranville, J. F. Synchrotron X-ray 2D and 3D elemental imaging of CdSe/ZnS quantum dot nanoparticles in Daphnia magna. Anal. Bioanal. Chem. 2009, 394, (3), 911-917. (36) Adam, N.; Leroux, F.; Knapen, D.; Bals, S.; Blust, R. The uptake and elimination of ZnO and CuO nanoparticles in Daphnia magna under chronic exposure scenarios. Water Res. 2015, 68, 249-261. (37) Derjaguin, B.; Landau, L. Theory of stability of highly charged liophobic sols and adhesion of highly charged particles in solutions of electrolytes. Zh. Eksp. Teor. Fiz. 1945, 15, (11), 663-682. (38) Verwey, E. J. W.; Overbeek, J. T. G. Theory of the Stability of Lyophobic Colloids; Elsevier: Amsterdam, 1948. (39) Fryer, G. Functional morphology and the adaptive radiation of the Daphniidae (Branchipoda, Anomopoda). Philos. T. Roy. Soc. B 1991, 331, (1259), 1-99. (40) Gophen, M.; Geller, W. Filter mesh size and food particle uptake by Daphnia. Oecologia 1984, 64, (3), 408-412. (41) Chen, K. L.; Elimelech, M. Aggregation and deposition kinetics of fullerene (C-60) nanoparticles. Langmuir 2006, 22, (26), 10994-11001. (42) Lew, D. P.; Andersson, T.; Hed, J.; Divirgilio, F.; Pozzan, T.; Stendahl, O. Ca2+-dependent and Ca2+-independent phagocytosis in human-neutrophils. Nature 1985, 315, (6019), 509-511. (43) Wu, X. S.; McNeil, B. D.; Xu, J.; Fan, J.; Xue, L.; Melicoff, E.; Adachi, R.; Bai, L.; Wu, L. G. Ca2+ and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal. Nat. Neurosci. 2009, 12, (8), 1003-1010. (44) Campbell, P. G. C. Interactions between trace metals and aquatic organisms: A critique of the free-ion activity model. In Metal Speciation and Bioavailability in Aquatic Systems; Tessier, A., Turner, D. R., Eds.; John Wiley & Sons Ltd.: Chichester, UK, 1995; pp 45-102. (45) Koivusalo, M.; Welch, C.; Hayashi, H.; Scott, C. C.; Kim, M.; Alexander, T.; Touret, N.; Hahn, K. M.; Grinstein, S. Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J. Cell Biol. 2010, 188, (4), 547-563. (46) Hosoi, N.; Holt, M.; Sakaba, T. Calcium dependence of exo- and endocytotic coupling at a glutamatergic synapse. Neuron 2009, 63, (2), 216-229
559 560 20
ACS Paragon Plus Environment
Page 20 of 28
Page 21 of 28
Environmental Science & Technology
561
Figure Legends
562
Figure 1. (a) Variations in the Ti content in Daphnia magna ([Ti]daphnia) with exposure time at
563
ambient Ca concentrations ([Ca]dis) of 0.2 and 2.0 mM, as determined in a 48-h uptake
564
experiment. The medium was either refreshed (0.2-re and 2-re) or not (0.2 and 2). (b)
565
PAA-TiO2-NP uptake rates at different [Ca]dis during a 6-h uptake experiment. The values of
566
the bars with different letters on the top are statistically significant (p < 0.05) from each other.
567
Data are the mean ± standard deviation (n = 3).
568
Figure 2. Distribution of (a, c, e) Ca and (b, d, f) Ti in Daphnia magna (a, b) during a control
569
treatment in the absence of PAA-TiO2-NPs or (c-f) when pre-exposed to 40 mg-Ti/L
570
PAA-TiO2-NPs for 3 h with a [Ca]dis of (c, d) 0.2 and (e, f) 2.0 mM, as determined by
571
synchrotron radiation-based micro X-ray fluorescence spectroscopy (µXRF). White arrows
572
indicate the accumulation of PAA-TiO2-NPs in the gut (GT) and abdominal area (AD) of D.
573
magna.
574
Figure 3. (a) Scanning electron microcopy (SEM) image of the thoracic limb of Daphnia
575
magna pre-exposed to 40 mg-Ti/L PAA-TiO2-NPs for 24 h. (b) Ti mapping (red dots) of the
576
thoracic limb using SEM-EDX. Arrows indicate the areas with a high Ti signal. (c)
577
Distribution of PAA-TiO2-NPs in cells captured in a tissue slice from the abdominal zone of
578
D. magna pre-exposed to 40 mg-Ti/L PAA-TiO2-NPs for 24 h. Arrows indicate the location
579
of PAA-TiO2-NPs. (d) Representative energy dispersive X-ray spectrum of the PAA-TiO2-NP
580
-containing spots in (c).
581
Figure 4. The proportion of PAA-TiO2-NPs retained in Daphnia magna pre-exposed to 4
582
mg-Ti/L PAA-TiO2-NPs at a [Ca]dis of 0.2 mM for 1 h during a 48-h depuration period (Ctrl).
583
In the other two treatments, either the fleas were heat killed before the depuration (Heat) or
584
2.0 mM EGTA was added to the efflux medium (EGTA). Data are the mean ± standard
585
deviation (n = 3).
586
Figure 5. (a) The rate of PAA-TiO2-NP uptake by Daphnia magna in medium containing 0,
587
0.5, or 2.0 mM Ca with (EGTA) or without (None) 2.0 mM EGTA. EDTA (2.0 mM) was also
588
added to the treatment without Ca. (b) The rate of PAA-TiO2-NP uptake by D. magna in 21
ACS Paragon Plus Environment
Environmental Science & Technology
589
medium containing 0, 0.2, or 2.0 mM Ca and with an ambient pH value of 6, 7, 8, or 9. (c)
590
The rate of PAA-TiO2-NP uptake by D. magna pre-exposed to no inhibitors (Ctrl), 1.0 mM
591
nifedipine (Nif), or 3.0 mM amiloride (Ami) in uptake medium containing 0, 0.5, or 2.0 mM
592
Ca. (d) The rate of PAA-TiO2-NP uptake by D. magna pre-exposed to 0, 1, and 10 µM BAY
593
K8644 in uptake medium containing 0, 0.2, or 2.0 mM Ca. The values of the bars with
594
different letters on the top are statistically significant (p < 0.05) from each other. Data are the
595
mean ± standard deviation (n = 3).
596
Figure 6. (a) The rate of PAA-TiO2-NP uptake by Daphnia magna pre-cultured in medium
597
containing 12.5 µM, 0.5 mM, or 5.0 mM Ca for 24 and 48 h. (b) The rate of PAA-TiO2-NP
598
uptake by D. magna in the presence of 0, 0.01, 0.04, and 0.2 µM (A–D) Ca ionophore (Ionop)
599
with a [Ca]dis of 0.2 mM. Alternatively, D. magna was pre-exposed to different concentrations
600
of thapsigargin (Thap, 0, 0.3, 1.0, and 3.0 µM for A–D) or BAPTA-AM (BAPTA, 0, 0.03,
601
0.2, and 1.0 mM for A–D) for 1 h before the accumulation of PAA-TiO2-NPs in the uptake
602
media alone, without added Ca, thapsigargin, or BAPTA-AM. Data are the mean ± standard
603
deviation (n = 3).
604
22
ACS Paragon Plus Environment
Page 22 of 28
Page 23 of 28
Figure 1 [Ti]daphnia (mg-Ti/g-dw)
605
Environmental Science & Technology
10
0
PAA-TiO2-NP uptake rate (mg-Ti/g-dw/h)
0
606 607
a
2 2-re
0.2 0.2-re
20
10
20 30 40 Time (h)
50
2 b 1
ab
ab ab
a 0
0
0.2 0.5
1
2
[Ca]dis (mM)
23
ACS Paragon Plus Environment
b
Environmental Science & Technology
608
Page 24 of 28
Figure 2
Ca
Ti
GT
a
0
b Ca
Ti
GT
c
0
d
AD
0
Ca
Ti
GT
e
f
609 610
24
ACS Paragon Plus Environment
0
Page 25 of 28
611
Environmental Science & Technology
Figure 3
a
b
5 µm
c
d
C 400 Intensity
O Na 200 Ti
Cl 0 0
612 613 614
1
2 3 Energy (keV)
25
ACS Paragon Plus Environment
4
5
Environmental Science & Technology
Figure 4 PAA-TiO2-NPs retained (%)
615
616
100
10 Heat EGTA Ctrl
1 0
10
20
30
40
Depuration time (h)
617
26
ACS Paragon Plus Environment
50
Page 26 of 28
Page 27 of 28
618
Environmental Science & Technology
Figure 5
PAA-TiO2-NP uptake rate (mg-Ti/g-dw/h)
4
None EGTA EDTA
a
1.0 a
2
1.5
a a
0.5
0
3
0.5 Ctrl Nif Ami
2
0.0
2.0
c a a
ab b
0
0.2
ab
c
0.5 [Ca]dis (mM)
a a
0
2.0
a
2.0
d
a
1 a
a
0 µM 1 µM 10 µM
2
1 0
0
3
a
b
b
a
a
a
b
a a a
0
pH=6 pH=7 c bc pH=8 pH=9 aaa b a
b
a a a
a
a
0
0.2 [Ca]dis (mM)
619 620 621 622
27
ACS Paragon Plus Environment
2.0
Environmental Science & Technology
623
Figure 6
PAA-TiO2-NP uptake rate (mg-Ti/g-dw/h)
1.0
a
12.5 µM 0.5 mM 5 mM
.5
0.0 1.5
24h
48h
A B
C D
b
1.0 .5 0.0
624
Page 28 of 28
Ionop
Thap BAPTA Treatments
625 626
28
ACS Paragon Plus Environment