Subscriber access provided by UNIV OF ESSEX
Article
Toxic effects and molecular mechanism of different types of silver nanoparticles to the aquatic crustacean Daphnia magna Jing Hou, yue zhou, chunjie wang, Shiguo Li, and Xiangke Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03918 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 3, 2017
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 32
Environmental Science & Technology
1
Toxic effects and molecular mechanism of different types of silver
2
nanoparticles to the aquatic crustacean Daphnia magna
3
Jing Hou a, Yue Zhou a, Chunjie Wang a, Shiguo Li b*, Xiangke Wang a*
4
a
5
Beijing 102206, China
6
b
7
China
8
* Address correspondence to xkwang@ncepu.edu.cn
College of Environmental Science and Engineering, North China Electric Power University,
Research Center for Eco-Environmental Sciences, Chinese Academy of Science, Beijing 100085,
9
ACS Paragon Plus Environment
Environmental Science & Technology
10
ABSTRACT
11
Silver nanoparticles (AgNPs) have been assessed to have a high exposure risk for humans and
12
aquatic organisms. Toxicity varies considerably between different types of AgNPs. This study
13
aimed to investigate the toxic effects of AgNPs with different particle sizes (40 nm and 110 nm)
14
and different surface coatings (sodium citrate and polyvinylpyrrolidone) on Daphnia magna and
15
their mechanisms of action. The results revealed that the citrate-coated AgNPs were more toxic
16
than PVP-coated AgNPs and that the 40-nm AgNPs were more toxic than the 110-nm AgNPs.
17
Transcriptome analysis further revealed that the toxic effects of AgNPs on D. magna were related
18
to the mechanisms of ion binding and several metabolic pathways such as “RNA polymerase”
19
pathway and “protein digestion and absorption” pathway. Moreover, the Principal component
20
analysis (PAC) results found that surface coating was the major factor that determines the
21
toxicities compared with particle size. These results could help us to better understand the possible
22
mechanism of AgNP toxicity in aquatic invertebrates at the transcriptome level and establish an
23
important foundation for revealing the broad impacts of nanoparticles on aquatic environments.
24
KEYWORDS: silver nanoparticle, Daphnia magna, RNA-Seq, aquatic toxicology, mechanism
ACS Paragon Plus Environment
Page 2 of 32
Page 3 of 32
Environmental Science & Technology
25
INTRODUCTION
26
With the development and commercialization of nanotechnology, silver nanoparticles (AgNPs)
27
have broad application prospects in chemical, optical, medical and other industries 1-3 due to their
28
advantages of small size, large specific surface area, high catalytic activity, low melting point,
29
good sintering properties, good electrical conductivity and good antibacterial properties.
30
catalyst, AgNPs can change the absorption and conversion of laser energy to the polymer system,
31
resulting in the change of laser dissociation. AgNPs doped in semiconductors or insulators can
32
obtain a large nonlinear polarizability, and can be used to make the color filters of optoelectronic
33
devices such as optical switches and advanced optical devices. In addition, AgNPs can be made
34
into medical dressings for most of the trauma treatment, to protect against microbial infection.7-9
35
Given these applications, they will eventually enter the freshwater environment such as rivers or
36
lakes and affect aquatic organisms. AgNPs have induced developmental, hepatic, endocrine and
37
reproductive toxicities in various aquatic organisms such as algae, daphnia, and fish.10-12 Once
38
accumulated in aquatic animals, they can enter the human body through the food chain. Therefore,
39
it is necessary to understand the potential impacts of AgNPs on aquatic invertebrates and facilitate
40
a better assessment of their impacts on human health.
41
4-6
As a
The physicochemical properties such as size,13 shape,14 surface coating,15 surface charge,16 and 17,18
42
solution chemistry
are important factors to determine AgNPs toxicity. Particle size and
43
surface coating have been considered to be the most determinant factors. For example, 20-nm
44
AgNPs induced more inflammation or cardiac ischemia/reperfusion injury than 110-nm AgNPs in
45
Danio Rerio.19 Locomotor activity experiment demonstrated that Gammarus roeseli exposed to
46
60-nm AgNPs exerted a significantly lower activity compared to those exposed to 30-nm
47
AgNPs.20 The reason why particle size is so determinant of biological toxicity may be dependent
48
on the higher dissolution rate of smaller particles and the pathway by which particles enter the cell
49
membrane. Kim et al. reported that NPs < 50 nm can more easily enter cells through endocytosis
50
than NPs > 50 nm.21 It was also demonstrated that 20-nm AgNPs had significantly greater effects
51
on tail moment, tail DNA intensity and burrowing activity of Nereis diversicolor than 80-nm
52
AgNPs.22 Some contrasting findings demonstrated that AgNPs with larger particle sizes were
53
more toxic.23 It is likely that particle size is not a dominant factor in toxicity. For example,
54
Jimenez-lamana et al. revealed that surface coating rather than size determines the stability of
ACS Paragon Plus Environment
Environmental Science & Technology
Page 4 of 32
55
AgNPs in aquatic ecosystems, with a stability order of PVP-coated AgNPs > citrate-coated
56
AgNPs > lipoic acid-coated AgNPs.24 Similarly, PVP-coated AgNPs were more stable than
57
citrate-coated AgNPs as a result of steric repulsion by the absorbed polymer layer.25 Gum
58
arabic-coated AgNPs has a greater impact on aquatic ecosystems than PVP-coated AgNPs.26 The
59
7-nm PVA-coated AgNPs exhibited higher toxicity to Nitrosomonas europaea than 7-nm
60
Na2ATP-coated AgNPs.27 Thus it can be seen that the influence of AgNPs with different
61
properties on aquatic organisms is complex. To fully understand the toxic effects of AgNPs on
62
aquatic organisms, the physiological and molecular mechanisms behind these complex effects
63
need to be revealed at different levels and perspectives. Although considerable progress has been
64
made towards elucidating the toxicity levels and biological effects, there is less information
65
regarding the molecular mechanisms of toxicity of AgNPs with both different particle sizes and
66
different surface coatings to aquatic organisms, especially aquatic invertebrates.
67
With the rapid development of high-throughput sequencing technologies, the possible
68
physiological functions in specific tissues and organs have explored fully at the transcriptomic
69
level.28-31 RNA sequencing (RNA-Seq) has proven to be a rapid and powerful technique with low
70
noise, high efficiency and massive data output enabling high-throughput sequencing of the whole
71
transcriptome.32 RNA-seq can aid in identifying biomarkers of exposure, differentiating toxicity
72
between AgNPs, detecting casual contaminants in environmental samples, and assessing the
73
mechanisms of response to stressors.33-34 Although RNA-seq has been performed in aquatic
74
species such as Pandalus latirostris,35 Salmo salar,36 Oryzias melastigma,37 Labeo rohita,38
75
Lateolabrax japonicas,39 Litopenaeus vannamei,40 Ostrea edulis,41 Pinctada fucata42 and Patella
76
vulgate,43 the molecular mechanisms underlying the effects of different types of AgNPs on aquatic
77
organisms are still rare.
78
The objectives of the present study were to investigate toxic effects of different types of AgNPs
79
on an aquatic invertebrate D. magna and further explore their mechanisms of action. D. magna is
80
widely used as a model organism to evaluate the invertebrate response to toxicants due to its rapid
81
reproduction, short lifespan, and high sensitivity to environmental pollutants.44-46 To achieve the
82
above objectives, a RNA-seq technology was employed to investigate the toxic effects and
83
molecular
84
110-PVP-AgNP) on D. magna, which will establish an important foundation for revealing the
mechanism
of
AgNPs
(40-cit-AgNP,
110-cit-AgNP,
ACS Paragon Plus Environment
40-PVP-AgNP
and
Page 5 of 32
Environmental Science & Technology
85
toxicity and toxic mechanism of AgNPs in aquatic invertebrates.
86
MATERIALS AND METHODS
87
Daphnia magna Cultivation. D. magna obtained from the Chinese Research Academy of
88
Environmental Sciences were cultured in an illumination incubator with a photoperiod of 16 h/8 h
89
(light/dark) and a temperature of 21±1 °C. D. magna were maintained in filtered and sterilized tap
90
water47 and fed Scenedesmus obliquus at approximately 0.2 mg of carbon per individual per day.
91
The medium was renewed three times a week and aerated overnight before use. S. obliquus
92
obtained from the Institute of Hydrobiology, Chinese Academy of Sciences were grown in Bold's
93
Basal Medium (BBM) with and initial pH of 8.0. Table S1 summarizes the chemical composition
94
of BBM. The algae were cultured at 25±1 °C with a light intensity of 2500 lx and a photoperiod of
95
14 h/10 h (light/dark). The algal cell density was determined using a hemocytometer under a
96
trinocular microscope (B204TR, Chongqing, China) at a magnification of 400×.
97
Silver Nanoparticle Characterization. Citrate-coated AgNPs (sodium citrate as a stabilizer)
98
with average particle sizes of 40 nm and 110 nm (40-cit-AgNP and 110-cit-AgNP) and
99
PVP-coated AgNPs (0.3% polyvinylpyrrolidone) with average particle sizes of 40 nm and 110 nm
100
(40-PVP-AgNP and 110-PVP-AgNP) were obtained from nanoComposix (San Diego, USA).
101
Transmission electron microscopy (TEM) analysis was used to characterize the size and shape of
102
the AgNPs after 0 h and 24 h of dispersion in the test medium by placing a droplet of the AgNPs
103
on a TEM grid followed by obtaining images using a TEM (Hitachi H-7650B, Japan) at an
104
accelerating voltage of 120 kV. The hydrodynamic size and zeta potential of the AgNPs dispersed
105
in the test medium were measured at 0 h and 24 h using a Zetasizer (Nano ZS, Malvern
106
Instruments, UK). Each time point consisted of three replicates, and five consecutive
107
measurements were made per replicate. Dissolution of the AgNPs was determined by separating
108
dissolved silver from the AgNPs using centrifugal ultrafiltration with a 30-kDa filter (Millipore
109
Amicon Ultra, USA).48 The dispersions were centrifuged at 3000 g for 60 min (Centrifuge 5424,
110
Eppendorf, Germany), and the dissolved silver present in the filtrate was acidified and analyzed in
111
triplicate at 6, 12, 18 and 24 h by ICP-AES (SPECTRO Analytical Instruments GmbH ,
112
Germany).
113
Acute Toxicity Test. D. magna toxicity tests were slightly modified from the U.S. EPA Whole
114
Effluent Toxicity guidelines.49 The 14-day-old D. magna were exposed to six different
ACS Paragon Plus Environment
Environmental Science & Technology
Page 6 of 32
115
concentrations (0, 2, 4, 8, 16, 32, and 64 µg/L) of AgNPs. Each concentration consisted of thirty D.
116
magna with in each of six replicates. D. magna were incubated in incubator without feeding
117
during the 24 h exposure period and examined for lethality after 24 h. The probit analysis method
118
was employed to estimate the median lethal concentration (LC50) values. All subsequent assays
119
were performed using 2 µg/L (close to 1/10 LC50) of 40-cit-AgNP, 110-cit-AgNP, 40-PVP-AgNP
120
and 110-PVP-AgNP, because this level was below that which causes nonspecific gene expression
121
responses.50 At predetermined intervals (6, 12, 18, and 24 h), Thirty D. magna was sampled from
122
each treatment and sacrificed to measure silver concentration in the body. Three biological
123
replicates were conducted for each treatment.
124
RNA Sequencing. Exposures were performed on D. magna using 2 µg/L (close to 1/10 LC50)
125
of 40-cit-AgNP, 110-cit-AgNP, 40-PVP-AgNP and 110-PVP-AgNP for 24 h. Thirty 14-d-old D.
126
magna were pooled together to generate one sample. Three biological replicates were conducted
127
for each treatment. The detailed RNA-Seq method, including total RNA extraction, mRNA
128
enrichment, mRNA fragment, cDNA synthesis, library construction and Illumina sequencing, is
129
shown in the supporting information. RNA-seq library sequencing was carried out on an Illumina
130
HiSeq 4000 instrument with 50 bp based on the cDNA library of D. magna. If the result passed
131
quality control, the NOISeq method was used to screen for differentially expressed genes (DEGs)
132
according to the following criteria: fold change > 1.2 and p-value > 0.5. The website
133
(http://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to generate the Venn diagram.
134
Gene Ontology Enrichment Analysis. GO enrichment analysis provided all GO terms that
135
were significantly enriched in a list of DEGs. All DEGs were mapped to GO terms and gene
136
numbers were calculated for every term in the database (http://www.geneontology.org/).
137
Significantly enriched GO terms for the input list of DEGs were calculated using the
138
hypergeometric
139
(http://search.cpan.org/~sherlock/GO-TermFinder/).
test
with
p-value
correction
based
on
'GO::TermFinder'
140
Kyoto Encyclopedia of Genes and Genomes Pathway Analysis. The KEGG was used to
141
identify the biological pathways of DEGs that were affected by AgNPs via comparison to the
142
background genome, which helped to further predict the functions of these DEGs. A KEGG
143
enrichment analysis (hypergeometric test, q-value < 0.05) was performed on all DEGs using the
144
KEGG database (http://www.genome.jp/kegg/).
ACS Paragon Plus Environment
Page 7 of 32
Environmental Science & Technology
145
Hierarchical Clustering Analysis (HCA) and Principal Component Analysis (PCA). HCA
146
is a chemometric tool used to calculate the distance between samples via the Sum of Squares of
147
Deviations algorithm. Genes with similar expression values are clustered at both the row and
148
column level. Only genes that were expressed in all samples of the cluster plan were used to build
149
the heatmap. The heatmap was produced with cluster and javaTreeview software. Expression
150
values for each transcript were log2 transformed before cluster formation. The heatmap was
151
clustered using the complete linkage hierarchical analysis based on Euclidean distance. For the
152
PCA, all data were first unit-variance scaled and mean centered. A two-component model was
153
selected for the distance measurement, which scattered in oval shapes when the datasets were
154
strongly related.
155
Real-time Quantitative PCR. RT-qPCR was performed to validate the reliability of the
156
RNA-seq data. A total of eight DEGs with various expression patterns, including two DEGs
157
involved in 40-cit-AgNP, two DEGs involved in 110-cit-AgNP, two DEGs involved in
158
40-PVP-AgNP, and two DEGs involved in 110-PVP-AgNP, were selected for validation. Total
159
RNA was extracted and purified as described above. The first-strand cDNA was synthesized using
160
the Superscript III First-Strand Synthesis kit (Invitrogen, Carlsbad, CA) following the
161
manufacturer’s instructions. RT-qPCR was performed using SYBR Premix Ex TaqII (Takara,
162
Japan) on a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster, CA). The cycling
163
conditions were as follows: one cycle of 50 °C for 5 min and 95 °C for 2 min for initial
164
denaturation, and 45 cycles of 95 °C for 15 s and 60 °C for 30 s. Each sample was run in triplicate.
165
DmACTIN was used as a reference gene, and primers were designed with Primer-BLAST
166
(www.ncbi.nlm.nih.gov/tools/primer-blast/). The relative quantitative method (2-∆∆CT) was used to
167
calculate the fold change of the selected genes. Data are expressed as the mean ± standard error
168
from samples performed in triplicate.
169
RESULTS
170
Physicochemical Characterization of AgNPs. Figure 1 displays the size, shape, and
171
agglomeration state of the four types of AgNPs after 0 h and 24 h of dispersion in the D. magna
172
test medium. The representative TEM images demonstrated that the primary AgNPs were
173
spherical in shape and uniform in size, confirming the manufacturer’s description of 40 nm or 110
174
nm for all four particles (Figure 1a-d). After 24 h of dispersion, the 40-cit-AgNP aggregated
ACS Paragon Plus Environment
Environmental Science & Technology
175
significantly into irregular shapes (Figure 1e). The 110-cit-AgNPs were discrete without
176
aggregation, although some small spherical particles were obviously dispersed around each
177
nanoparticle (Figure 1f). Both 40-PVP-AgNP and 110-PVP-AgNP showed a monodisperse
178
nanoparticle size distribution and spherical form without clusters or aggregates, indicating greater
179
stabilization of the PVP-coated AgNPs (Figure 1g-h).
180
The TEM results were also confirmed by the hydrodynamic sizes and zeta potentials (Table 1).
181
The hydrodynamic sizes of 40-cit-AgNP, 110-cit-AgNP, 40-PVP-AgNP, and 110-PVP-AgNP in
182
the D. magna test medium were found to change from 43.2 ± 0.3 nm, 94.3 ± 0.6 nm, 45.2 ± 0.2
183
nm, and 89.6 ± 0.9 nm after 0 h of exposure to 209.7 ± 26.5 nm, 89.4 ± 5.6 nm, 38.7 ± 3.1 nm, and
184
86.1 ± 0.7 nm after 24 h of exposure, respectively. Zeta potentials were used to characterize the
185
stability of the dispersed system. The absolute value of the zeta potentials for 40-cit-AgNP and
186
110-cit-AgNP decreased from -31.3 ± 3.9 and -36.7 ± 2.8 after 0 h of exposure to -11.3 ± 3.2 and
187
-18.6 ± 2.2 after 24 h of exposure, respectively, suggesting a significant decrease in the AgNP
188
stability. 40-PVP-AgNP and 110-PVP-AgNP had zeta potentials of -28.3 ± 2.4 and -31.6 ± 2.6
189
after 24 h of exposure, indicating good stability of the PVP-coated AgNPs.
190
Acute Toxicity. The 24-h LC50 values for 40-cit-AgNP, 110-cit-AgNP, 40-PVP-AgNP, and
191
110-PVP-AgNP were 8.90 µg/L, 17.43 µg/L, 24.97 µg/L, and 38.35 µg/L, respectively (Figure S1).
192
The rank order of toxicity to D. magna based on the LC50 values was 40-cit-AgNP >
193
110-cit-AgNP > 40-PVP-AgNP > 110-PVP-AgNP. Two trends related to toxicity were
194
summarized: citrate-coated AgNPs were more toxic than PVP-coated AgNPs, and 40-nm AgNPs
195
were more toxic than 110-nm AgNPs.
196
Dissolution of Silver Nanoparticles. The particle dissolution rates were determined because
197
particle dissolution plays a key role in the toxicity of AgNPs to aquatic organisms. All four
198
particles demonstrated a significant increase in Ag shedding after 6, 12, 18, and 24 h of incubation
199
in the D. magna test medium (Figure 2). The dissolution rates within 24 h followed the sequence
200
40-cit-AgNP > 110-cit-AgNP > 40-PVP-AgNP > 110-PVP-AgNP. The citrate-coated AgNPs
201
exhibited a higher level of dissolution than the PVP-coated AgNPs, and the 40-nm AgNPs were
202
more rapid than the 110-nm AgNPs for the same type of surface coating with the exception of
203
40-PVP-AgNP at 6 h and 40-cit-AgNP at 24 h.
204
Uptake of Silver Nanoparticles. As shown in Figure 3, the uptake of the four types of AgNPs
ACS Paragon Plus Environment
Page 8 of 32
Page 9 of 32
Environmental Science & Technology
205
showed a general increase during the 24 h. The uptake of 40-nm AgNPs by D. magna is greater
206
than that of 110-nm AgNPs at the same time point, while there was no significant difference in the
207
uptake between citrate-coated AgNPs and PVP-coated AgNPs. The difference in body burden
208
during this period could be explained by particle size. The body burden values of 40-cit-AgNP at
209
6, 12, 18, and 24 h increased by 116%, 138%, 108% and 122% compared with 110-cit-AgNP. The
210
body burden values of 40-PVP-AgNP at 6, 12, 18, and 24 h increased by 172%, 224%, 66% and
211
141% compared with 110-PVP-AgNP.
212
Analysis of Differentially Expressed Genes. To discriminate the toxic effects induced by the
213
different types of AgNPs, a total of 1262, 974, 664, and 594 DEGs for 40-cit-AgNP,
214
110-cit-AgNP, 40-PVP-AgNP, and 110-PVP-AgNP were identified using a cutoff of p-value > 0.5
215
and fold change > 1.2 (Table 2). The lists of DEGs for each exposure are presented in File S1. The
216
gene expression data were correlated with the acute toxicity data. The 40-cit-AgNP caused the
217
most abundant candidate genes, followed by 110-cit-AgNP and then 40-PVP-AgNP;
218
110-PVP-AgNP resulted in the weakest response. The number of down-regulated genes exceeded
219
the number of up-regulated genes for all four exposures. The distributions and possible relations
220
of DEGs between all treatments are demonstrated in a Venn diagram (Figure 4). Venn diagram
221
analysis revealed that 120 (5.2%) DEGs were commonly altered by all four exposures. There were
222
729 (31.6%), 444 (19.2%), 147 (6.4%), and 214 (9.3%) DEGs for 40-cit-AgNP, 110-cit-AgNP,
223
40-PVP-AgNP, and 110-PVP-AgNP, respectively, that were altered by only one of the treatments
224
and not by any other treatments. The number of DEGs altered by citrate-coated AgNPs (319 genes,
225
13.8%) was greater than the number of altered by PVP-coated AgNPs (223 genes, 9.6%).
226
Similarly, the number of DEGs altered by 40-nm AgNPs (312 genes, 13.5%) was greater than the
227
number altered by 110-nm AgNPs (213 genes, 9.2%). Sequencing data have been submitted to the
228
Sequence Read Archive (SRA, https://submit.ncbi.nlm.nih.gov/subs/sra/) under the accession
229
number PRJNA389206.
230
GO Enrichment Analysis of DEGs. To further study the functions of the DEGs, they are
231
subjected to gene ontology (GO) functional enrichment analysis, which is a gene-function
232
classification system used to describe the properties of genes and their products according to three
233
characteristics: cellular components, molecular function, and biological processes. The GO
234
enrichment analysis results indicated that 29, 15, 8, and 24 GO terms were significantly enriched
ACS Paragon Plus Environment
Environmental Science & Technology
235
in the D. magna exposed to 40-cit-AgNP, 110-cit-AgNP, 40-PVP-AgNP, and 110-PVP-AgNP,
236
respectively (File S2). The GO terms that existed in at least two exposure conditions are
237
summarized in Figure 5. Most of the GO terms are related to ion binding. Transition metal ion
238
binding (GO:0046914), ion binding (GO:0043167), and iron ion binding (GO:0005506) are the
239
common GO terms for three of the exposure conditions. Cation binding (GO:0043169) is the only
240
common GO term for the 40-nm AgNPs (40-cit-AgNP and 40-PVP-AgNP). Protein complex
241
(GO:0043234), transporter activity (GO:0005215), substrate-specific transporter activity
242
(GO:0022892), gas transport (GO:0015669), single-organism transport (GO:0044765), and
243
single-organism localization (GO:1902578) are common GO terms for the 110-nm AgNPs
244
(110-cit-AgNP and 110-PVP-AgNP). Notably, gas transport (GO:0015669) is the most
245
statistically significant term for both 110-cit-AgNP (p = 7.61×10-9) and 110-PVP-AgNP (p =
246
3.05×10-8).
247
KEGG Pathway Analysis of DEGs. KEGG analysis was further performed to identify the
248
enrichment pathways in D. magna for each treatment. DEGs for 40-cit-AgNP, 110-cit-AgNP,
249
40-PVP-AgNP, and 110-PVP-AgNP were found to be enriched in 252, 225, 184, and, 192 KEGG
250
pathways, respectively (File S3). Taking a q-value < 0.05 as a threshold, the most represented
251
pathways are summarized in Table 3. Hippo signaling (q = 0.0211) is the most statistically
252
significant pathway for 40-cit-AgNP. RNA polymerase (q = 8.55×10-30) is the most statistically
253
significant pathway for 110-cit-AgNP. Pyrimidine metabolism (q = 1.60×10-5) and lysosom (q =
254
8.62×10-5) are the most statistically significant pathways for 40-PVP-AgNP. Protein digestion and
255
absorption (q = 7.64×10-5) is the most statistically significant pathway for 110-PVP-AgNP. RNA
256
polymerase is the only in-common pathway for the citrate-coated AgNPs (40-cit-AgNP and
257
110-cit-AgNP). Protein digestion and absorption is the only in-common pathway for the
258
PVP-coated AgNPs (40-PVP-AgNP and 110-PVP-AgNP).
259
Hierarchical Clustering Analysis. HCA was performed to obtain more information about the
260
similarities or differences between groups and between samples by calculating the distance
261
between them. As shown in Figure S2, the dendrogram presented in the top axis was generated by
262
identifying relatively homogenous clusters of samples based on their similarities. Each row
263
represents a single gene and each column represents an experimental sample. Different colors
264
indicate different gene expression levels. As seen in the dendrogram, the expression profiles
ACS Paragon Plus Environment
Page 10 of 32
Page 11 of 32
Environmental Science & Technology
265
induced by the fifteen samples were clustered into two distinct clusters. The left cluster can be
266
further divided into two subclusters. One is comprised of two 110-PVP-AgNP samples. The other
267
is comprised of one 110-PVP-AgNP sample and three control samples. The right cluster is formed
268
by the 40-cit-AgNP group, 40-PVP-AgNP group, and 110-cit-AgNP group. Three replicate
269
samples are clustered in one group. Each group is independent of the others and has no overlap.
270
The 40-cit-AgNP group is clearly discernible from the rest of groups and forms a subcluster. The
271
110-cit-AgNP and 40-PVP-AgNP groups are mixed to form another subcluster. These results
272
indicate that most of the treatments exhibited a good reproducibility, and the different types of
273
AgNPs can be discriminated from each other at relatively low concentrations.
274
Principal Component Analysis. PCA is commonly used to obtain more information about the
275
main variables and classify different samples by reducing redundant information in the data. As
276
illustrated in Figure 6, dots with the same color represent the same treatment. Each treatment
277
consists of three replicates. The PCA result is in accord with the HCA result. Dots with the same
278
color are relatively close to each other and far from the other dots, indicating a closer relationship
279
among the replicate samples, thus further verifying the reproducibility and reliability of the
280
RNA-seq data. The fifteen sample dots are classified into five groups. The principal component 1
281
values for all of the samples are negative. Note that principal component 2 separates 40-cit-AgNP
282
and 110-cit-AgNP (positive scores) from 40-PVP-AgNP and 110-PVP-AgNP (negative scores),
283
the former being citrate-coated AgNPs and the latter being PVP-coated AgNPs. Furthermore,
284
40-PVP-AgNP is split between positive and negative scores (close to zero) for principal
285
component 2, indicating that the difference is mainly between the group formed by 40-cit-AgNP
286
and 110-cit-AgNP and the group formed by 110-PVP-AgNP and the control. Therefore, surface
287
coating may be the major factor that determines the toxicity of AgNPs to D. magna compared
288
with particle size.
289
Validation of RNA-Seq. To validate the reliability of the data obtained from RNA-Seq, a total
290
of eight DEGs, including four up-regulated DEGs and four down-regulated DEGs from the
291
40-cit-AgNP, 110-cit-AgNP, 40-PVP-AgNP and 110-PVP-AgNP treatments, were selected and
292
validated by RT-qPCR. Detailed information about the primers including gene ID, gene symbol,
293
gene description, product size and primer sequences is provided in Table S2. As shown in Figure
294
S3, the RT-qPCR results for the eight DEGs show a high degree of correlation with the trend
ACS Paragon Plus Environment
Environmental Science & Technology
295
revealed by RNA-Seq. A positive correlation exists (R2=0.933) between the expression level of
296
DEGs from RNA-Seq and RT-qPCR, confirming the high reproducibility and reliability of the
297
RNA-Seq data.
298
DISCUSSION
299
Coating-specific and size-dependent toxicities of AgNPs were demonstrated in the present study.
300
The LC50 data showed that citrate-coated AgNPs were more toxic than PVP-coated AgNPs and
301
that 40-nm AgNPs were more toxic than 110-nm AgNPs. Similar results were obtained from
302
previous studies. Poynton et al. found that the LC50 values of AgNPs in 10-day-old D. magna
303
were 1.8 µg/L for citrate-coated AgNPs and 10.6 µg/L for PVP-coated AgNPs.51 The acute
304
toxicity of AgNPs in D. magna demonstrated that citrate-coated AgNPs were less toxic than
305
uncoated AgNPs, and the larger citrate-coated AgNPs (60 nm and 100 nm) exhibited
306
approximately 7.5-fold lower toxicity relative to the smaller citrate-coated AgNPs (20 nm).52 Our
307
results were also in agreement with a study on Caenorhabditis elegans, where it was demonstrated
308
that the PVP coating reduced the toxicity of the AgNPs significantly and that 8 nm PVP-coated
309
AgNPs were more toxic than 38-nm PVP-coated AgNPs.53
310
Moreover, the toxicity of the AgNPs is directly related to the aggregation of their particles.
311
Aggregation can reduce toxicity.54,55 The aggregated AgNPs formed larger clusters that reduced
312
the uptake by cells, whereas the dispersed AgNPs more easily crossed cell membranes and
313
inhibited cell growth.23 However, in our study, 40-cit-AgNPs were more toxic than 40-PVP-
314
AgNPs even though 40-cit-AgNPs tended to aggregate more. Because the toxicity of AgNPs is not
315
determined by a single factor, a number of factors act in combination to mediate the toxicity of
316
AgNPs. This is in accordance with previous investigation which has confirmed that SO2-coated
317
silver nanowires (AgNWs) were more toxic to D. magna than PVP-coated AgNWs, but the
318
SO2-coated AgNWs aggregated and settled more.
319
Most of the previous studies showed that AgNP toxicities were caused by the release of silver
320
ions, whether they are different coatings or particle sizes. The dissolution rates were
321
time-dependent and varied considerably between the different types of AgNPs. Citrate-coated
322
AgNPs were more reactive than PVP-coated AgNPs. It might be that the stability of the PVP
323
coating enabled the silver to be fully encapsulated.56 The stability of the PVP-coated AgNPs was
324
also confirmed by the zeta potentials. The absolute values of the zeta potentials for the
ACS Paragon Plus Environment
Page 12 of 32
Page 13 of 32
Environmental Science & Technology
325
citrate-coated AgNPs decreased significantly after 24 h of exposure, suggesting a significant
326
decrease in the AgNP stability and explaining the observed aggregation; in contrast, no obvious
327
changes were observed for the PVP-coated AgNPs, indicating good stability. This result is
328
consistent with previous studies in which citrate-coated AgNPs had a higher dissolution rate than
329
PVP-coated AgNPs.57 The PVP-coated AgNPs were more stable than the citrate-coated AgNPs as
330
a result of steric repulsion by the absorbed polymer layer.25 These findings indicated that the
331
surface coating was not an impervious barrier and that Silver ion can be released from AgNPs and
332
induce toxicity. Meanwhile, there was a significant difference in the toxicity between 40-nm
333
AgNPs and 110-nm AgNPs. The 40-nm AgNPs had higher dissolution rates than 110-nm AgNPs
334
for the same types of surface coating. This may be caused by differences in the release of silver
335
ions.
336
110-nm citrate-coated AgNPs,58 and in which 7-nm AgNPs showed higher dissolution rates than
337
40-nm AgNPs due to the higher specific surface areas and higher enthalpies of formation of the
338
smaller particles.59 Silver ion can be also released from AgNPs in gut or fluids, which is the main
339
mechanism of the toxic effects of AgNPs and AgNPs sterilization. AgNP can be ionized in body
340
fluids or gut, and their interaction with H2O2 is considered to be one of the factors of the release of
341
Ag+ in gut, From the mechanism, the surface of AgNPs can quickly release free Ag+ after
342
oxidation. Therefore,the toxic effects could be also explained by the body burden values. The
343
uptake of 40-nm AgNPs by D. magna is greater than that of 110-nm AgNPs at the same time point.
344
Body burden of the four types of AgNPs showed a general increase during the first 24 h. The
345
similar results were also concluded from the uptake behaviors of carbon nanotubes by Eisenia
346
foetida.60 The uptake of graphene by D. magna across the range of concentrations (25, 50, 100 and
347
250 µg/L) increased during the first 24 h.61
For
example,
20-nm citrate-coated
AgNPs
released
silver
ions
faster
than
348
The toxicity effects of different AgNPs to D. magna were further confirmed by comparative
349
transcriptome analysis. The number of DEGs in D. magna that were altered by citrate-coated
350
AgNPs was significantly greater than that by PVP-coated AgNPs, and the number of DEGs in D.
351
magna that were altered by 40-nm AgNPs was significantly greater than the number altered by
352
110-nm AgNPs, supporting the coating-specific and size-dependent toxicity of AgNPs to D.
353
magna. GO enrichment analysis of the DEGs indicated that the number of GO terms in the
354
citrate-coated AgNPs was greater than the number in the PVP-coated AgNPs, which was
ACS Paragon Plus Environment
Environmental Science & Technology
355
consistent with the results of silver ion release. However, 40-nm was not greater than 110-nm for
356
the PVP-coated AgNPs, which may be caused by the large number of unannotated genes in the D.
357
magna genome database. Note that most of the DEGs in D. magna that were induced by AgNPs
358
were enriched in ion binding-related terms, including “transition metal ion binding”, “ion binding”,
359
“iron ion binding” and “cation binding”, which is consistent with the release trend of silver ion.
360
Ion binding is crucial for numerous biological processes, particularly protein activities involved in
361
these processes. The binding processes between metal ions and active sites of key enzymes in
362
vertebrates are regarded as the basis for enzyme activation. Approximately 1/3 of proteins in
363
vertebrates bind at least one metal ion, including potassium, calcium, sodium, magnesium, copper,
364
iron, zinc and manganese ions.62,
365
proteins upon binding, which stabilize the protein structure and participate in enzyme catalysis.64
366
In this study, the GO results were consistent with earlier studies on aquatic animals. For example,
367
the metal ion binding related genes of zebrafish embryos were significantly influenced by
368
AgNPs.65 D. magna that were exposed to CuO NPs for 96 h (