Aggregation Reverses the Carrier Effects of TiO2 Nanoparticles on

Dec 16, 2016 - Our previous study reported that the Ca-dependent aggregation of polyacrylate-coated TiO2 nanoparticles (PAA-TiO2–NPs) determines the...
0 downloads 0 Views 1005KB Size
Subscriber access provided by University of Newcastle, Australia

Article

Aggregation Reverses the Carrier Effects of TiO2 Nanoparticles on Cadmium Accumulation in the Waterflea Daphnia magna 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.6b03951 • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 18, 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 27

Environmental Science & Technology

1

Table of Contents Art

2 3

1

ACS Paragon Plus Environment

Environmental Science & Technology

4 5 6 7

8

Aggregation Reverses the Carrier Effects of TiO2 Nanoparticles

9

on Cadmium Accumulation in the Waterflea Daphnia magna

10 11

12

13

Ling-Yan Tan, Bin Huang, Shen Xu, Zhong-Bo Wei, Liu-Yan Yang, Ai-Jun Miao*

14 15 16 17

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,

18

Nanjing University, Nanjing, Jiangsu Province, 210023, China

19 20 21 22 23

*Corresponding author: [email protected] (Email), +86 25 89680255 (Tel.), +86 25

24

89680569 (Fax)

25

2

ACS Paragon Plus Environment

Page 2 of 27

Page 3 of 27

Environmental Science & Technology

26

ABSTRACT: Our previous study reported that the Ca-dependent aggregation of

27

polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) determines their routes of uptake by

28

the waterflea Daphnia magna. Besides the effects of aggregation on NP bioaccumulation,

29

how this process may influence the bioavailability of NP-adsorbed pollutants remains

30

obscure. In the present study, the aggregation of PAA-TiO2-NPs was also adjusted through Ca.

31

Then the accumulation and toxicity of Cd in D. magna were investigated in the presence and

32

absence of the NPs. Although PAA-TiO2-NPs ameliorated Cd toxicity at both low and high

33

Ca concentrations, the underlying mechanisms differed completely. At low Ca, the metal-NP

34

complexes were accumulated by endocytosis and passive drinking, with both pollutants

35

distributed throughout the daphnid. Nevertheless, Cd accumulation was reduced due to its

36

rapid dissociation from the NPs during the endocytosis of the metal-NP complexes. At high

37

Ca, the metal-NP complexes were actively ingested, Cd accumulation was induced, and both

38

pollutants were concentrated in the daphnid gut. The aggregation-dependent effects of

39

PAA-TiO2-NPs on Cd bioaccumulation were further evidenced by the distinct patterns of

40

metal efflux from D. magna at different Ca concentrations. Overall, Cd adsorption by

41

PAA-TiO2-NPs may either increase or reduce its bioaccumulation, as determined by the

42

aggregation of the NPs.

43 44

3

ACS Paragon Plus Environment

Environmental Science & Technology

45



INTRODUCTION

46

With the rapid development of nanotechnology, an increasing number of engineered

47

nanoparticles (NPs) would be released into aquatic ecosystems. Consequently, the last decade

48

has witnessed the exponential growth of nanotoxicological research in phytoplankton,

49

zooplankton, fish, and other aquatic organisms.1-3 However, most studies have focused on the

50

toxicity of NPs alone, whereas in the environment they interact with trace metals, organic

51

pollutants, and other polluting and non-polluting compounds. The nature of these interactions

52

and their influence on the behavior and effects of the individual components remain largely

53

unknown. Based on limited studies, NPs increase the bioavailability and toxicity of trace

54

metals (or metalloids) in most cases. For instance, CdTe quantum dots facilitate Cu

55

accumulation in zebrafish with synergistic toxicity developing during joint exposure.4 Sun et

56

al.5 found that arsenite oxidation is induced by TiO2-NPs and so is its accumulation in carp

57

(Cyprinus carpio). Moreover, TiO2-NPs pre-accumulated in the gut of the waterflea Daphnia

58

magna were also shown to enhance the subsequent accumulation and toxicity of Cd and Zn in

59

this organism.6 The complexity of NP and metal interactions was further demonstrated by

60

Rosenfeldt et al.7 They reported that TiO2-NPs (P25) increase Ag toxicity while reducing As

61

and Cu toxicity in D. magna. The postulated mechanism underlying these metal-specific

62

toxicity responses to NPs was that As and Cu have a stronger binding affinity with TiO2-NPs

63

as compared to Ag. Therefore, more Ag was liberated from the NPs after the accumulation of

64

the metal-NP complexes in the gastrointestinal tract of D. magna.

65

In the metal-NP interaction studies mentioned above, the aqueous suspensions of the

66

NPs were mostly unstable and the particles tended to form micrometer- or

67

submicrometer-sized aggregates in the experimental medium. Despite the increased

68

application of well-dispersed NPs in a variety of areas,8 their effects on the accumulation and

69

toxicity of trace metals remain unclear. Polyacrylate-coated TiO2-NPs (PAA-TiO2-NPs) were

70

well dispersed in a variety of experimental media (e.g., WC and Dryl’s media) and showed no

71

toxicity to the organisms (Chlamydomonas reinhardtii and Tetrahymena thermophila) used in

72

our previous studies.9, 10 These particles could not be taken up by the green alga C. reinhardtii

73

because of their interception by the cell wall.9 Under this condition, PAA-TiO2-NPs 4

ACS Paragon Plus Environment

Page 4 of 27

Page 5 of 27

Environmental Science & Technology

74

decreased the ambient concentration of free Cd ion by more than 90% in most cases through

75

surface adsorption, thereby reducing metal accumulation and toxicity. Nevertheless, Cd

76

toxicity to C. reinhardtii in the presence of PAA-TiO2-NPs could still be well-described using

77

the free ion activity model (FIAM).11 Unlike C. reinhardtii, the protozoan T. thermophila

78

could endocytose PAA-TiO2-NPs directly,10 such that the NPs serve as carriers of Cd,

79

increase its accumulation and toxicity. For multicellular organisms, their interactions with

80

NPs and trace metals are no doubt much more complex, especially considering the fact that

81

NPs of different aggregation states may have completely different uptake routes. In our

82

previous study,12 the well-dispersed PAA-TiO2-NPs at the low Ca concentration (0.2 mM)

83

were found to be taken up by D. magna through endocytosis and passive drinking. Under this

84

condition, the NPs were distributed throughout the daphnid. By contrast, micrometer-sized

85

aggregates were formed at the high Ca concentration (2.0 mM). In this case, PAA-TiO2-NPs

86

were actively ingested and predominantly concentrated in the daphnid gut. Besides the effects

87

of aggregation on NP uptake route in D. magna, how this process may influence the

88

accumulation and toxicity of trace metals, as adsorbed on the surfaces of the NPs, remains

89

unexplored. Therefore, in the present study, we compared the toxicity of Cd to D. magna in

90

the presence and absence of the well-dispersed PAA-TiO2-NPs and their micrometer-sized

91

aggregates. The aggregation of the NPs were adjusted through the ambient concentration of

92

Ca ([Ca]dis, 0.2 mM vs. 2.0 mM), similar to what was described in Tan et al.11 The uptake and

93

efflux of Cd and PAA-TiO2-NPs by D. magna were also investigated and the distributions of

94

both pollutants in the daphnid were determined, using synchrotron radiation based micro

95

X-ray fluorescence spectrometry (µXRF). Our results provide insights into the underlying

96

mechanisms how Ca-dependent aggregation of NPs may influence their carrier effects on

97

trace metal accumulation and toxicity, and shed new light on the role of NPs in trace-metal

98

bioavailability.

99



MATERIALS AND METHODS

100

Organisms and PAA-TiO2-NPs. The cladoceran Daphnia magna and its algal foods

101

(Chlamydomonas reinhardtii and Scenedesmus obliquus) were obtained from the Institute of

102

Hydrobiology, Chinese Academy of Science. D. magna was raised in aerated tap water at a 5

ACS Paragon Plus Environment

Environmental Science & Technology

103

density of one individual per 10 mL of water. The daphnids were fed daily with an algal

104

mixture of C. reinhardtii (2.5×104 cells/mL) and Scenedesmus obliquus (6×104 cells/mL).

105

The diet was doubled when D. magna was older than 3 d; the culture medium was refreshed

106

every other day. Daphnids in the stock culture and in all experiments described below were

107

maintained at 24 oC on a 12:12 h light-dark cycle with an irradiance of 30 µmol photons/m2/s.

108

In all of the experiments described below, seven-day-old daphnids were selected as the

109

organism of interest and a simplified Elendt M7 medium (SM7)13 was adopted as the basis of

110

all exposure media for better management of Cd speciation.

111

The PAA-TiO2-NPs used herein were the same as those employed in a previous study

112

by our group10. The particles are coated with hydrophilic sodium polyacrylate (74% of total

113

weight), resulting in a primary particle size of 1-10 nm. Their size distribution in the

114

experimental medium was analyzed using a dynamic light scattering particle sizer (DLS,

115

ZetaPALS, Brookhaven Instruments, NY, USA). A solid state laser (35 mW) with a

116

wavelength of 660 nm served as the light source of DLS and the scattering light was detected

117

at an angle of 90o (recording time = 2 min).

118

Toxicity Testing. Four 24-h toxicity tests were carried out in the experimental medium

119

containing 0.2 or 2.0 mM Ca and 0 or 4 mg-Ti/L PAA-TiO2-NPs but without any addition of

120

algal foods. The nominal total Cd concentration in the medium ([Cd]T) was 0, 10, 20, 50, 100,

121

200, and 300 µg/L, with triplicate samples established for each toxicity test. The daphnids

122

were first allowed to evacuate their guts for 1 h in SM7 in the absence of PAA-TiO2-NPs, Cd,

123

or algal foods. They were subsequently transferred to the toxicity medium with 20 daphnids

124

in each replicate and their immobilization was examined every 3 h over the course of the

125

experiment. At the end of the toxicity tests, all the daphnids of each replicate were taken out,

126

evenly divided into two groups, and were digested in HNO3 (for Cd) or (NH4)2SO4 and

127

H2SO4 (for Ti) following the methods described by Yang et al.10 The Ti and Cd

128

concentrations in the D. magna samples were then quantified by graphite furnace atomic

129

absorption spectrophotometry (GFAAS, Thermo Fisher Scientific Inc., Waltham, MA) and

130

inductively coupled plasma mass spectrometry (ICP-MS, NexION 300, PerkinElmer, MA),

131

respectively. The concentrations of PAA-TiO2-NPs and Cd in the experimental medium were 6

ACS Paragon Plus Environment

Page 6 of 27

Page 7 of 27

Environmental Science & Technology

132

also measured at the beginning and end of each experiment. In the meantime, the adsorption

133

of Cd on PAA-TiO2-NPs was determined using a 10 kilo dalton (kD) ultracentrifuge filter

134

with a pore size of approximately 1 nm (PALL Nanosep series).9

135

Forty-Eight Hour Accumulation Experiment. The 180 daphnids in each of the three

136

replicates were exposed to Cd (10 µg/L) and PAA-TiO2-NPs (4 mg-Ti/L) with [Ca]dis of 0.2

137

and 2.0 mM, respectively. After 0.25, 0.5, 1, 3, 6, 12, 24, 36, and 48 h of exposure, 20

138

daphnids were collected from each replicate and the accumulation of PAA-TiO2-NPs and Cd

139

was measured, similar to the toxicity experiment described above. Considering the relatively

140

low concentration of Cd used in the experiments, its enriched isotope (Cd-111, atomic

141

percent 96.4%, Oak Ridge National Laboratory, TN) was employed as the sole source of

142

added Cd to discriminate its uptake from the background.

143

Six-Hour Uptake Experiment. Similar to the 48-h accumulation experiment, D.

144

magna was exposed to Cd (10 µg/L) and PAA-TiO2-NPs (4 mg-Ti/L) at both [Ca]dis.

145

However, the duration of this experiment was shortened to 6 h, with samples collected at 0.5,

146

1, 2, 3, and 6 h. At each time point, the bioaccumulation of Cd and PAA-TiO2-NPs was

147

quantified. This experiment was performed at 24 and 4 oC to determine how inhibiting

148

endocytosis (4

149

bioaccumulation. In the latter treatment, the uptake medium was pre-equilibrated at 4 oC

150

overnight before its addition of daphnids and the subsequent uptake was also performed at

151

this temperature.

o

C) may influence the carrier effects of PAA-TiO2-NPs on Cd

152

Efflux Experiment. At each of the two Ca concentrations, 120 daphnids prepared in

153

triplicate samples were exposed to Cd (10 µg/L) with or without the addition of

154

PAA-TiO2-NPs (4 mg-Ti/L) for 16 h. The daphnids were then transferred to another container

155

and fed with algal foods (5×104 cells/mL) for 8 h to ensure their health.14 Neither NPs nor Cd

156

were added during the feeding phase. This process was repeated three times, such that the

157

total exposure duration was 3 d. Afterwards, the daphnids were transferred to fresh SM

158

containing the respective concentrations of Ca and algal foods (5×104 cells/mL) but without

159

PAA-TiO2-NPs and Cd. The concentrations of PAA-TiO2-NPs and Cd retained in the

160

daphnids were determined after 0.5, 1, 2, 3, 4, and 6 d of depuration. At each time point, the 7

ACS Paragon Plus Environment

Environmental Science & Technology

Page 8 of 27

161

depuration medium was also refreshed. After the removal of the molted carapaces and

162

neonates, the old depuration medium was passed through a 0.22 µm membrane. The debris

163

retained on the membrane was defined as feces while the < 0.22 µm filtrate made up the

164

dissolved phase. The distribution of the depurated PAA-TiO2-NPs and Cd in the dissolved

165

phase, molted carapaces, neonates, and feces were analyzed as described in Miao et al.13

166

µXRF Studies. The daphnids were exposed to 100 µg/L Cd at 0.2 and 2.0 mM Ca for

167

24 h with or without the addition of PAA-TiO2-NPs (4 mg-Ti/L). They were then fixed in

168

methanol,

169

hexamethyldisilazane as described in our previous study.12, 15 The distribution of Ti (KL3,

170

4.5109 keV), Cd (L3M5, 3.1338 keV), and Ca (KL3, 3.6917 keV) in daphnids was then

171

mapped by µXRF using a BL15U beamline at the Shanghai Radiation Synchrotron Facility

172

(SSRF, Shanghai, China). The storage ring current was 200-300 mA with an energy of 3.5

173

GeV. The samples were scanned by a 10 keV monochromatic beam, which was focused to 50

174

× 50 µm2 using K-B optics. The step size and scanning time were 50 µm and 3 s, respectively.

175

X-ray fluorescence was recorded by a seven-element Si (Li) detector combined with a

176

multiple channel analyzer (e2v, UK). Fluorescence data were processed using Pviewer

177

(version 1.0) and 2D Array Image Data Plotter (version 1.0).

dehydrated

in

graded

acetone

solutions,

and

dried

in

1,1,1,3,3,3

178

Statistical Analysis. Significant differences (accepted at p < 0.05) were determined in

179

one-way or two-way analyses of variance (ANOVA) with post-hoc multiple comparisons

180

(Tukey or Tamhane) (SPSS 11.0 by SPSS, Chicago, USA). Both the normality (Kolmogorov–

181

Smirnov and Shapiro–Wilk tests) and the homogeneity of variance (Levene’s test) of the data

182

were examined during the ANOVA.

183



RESULTS AND DISCUSSION

184

Toxicity and Bioaccumulation of Cd and PAA-TiO2-NPs. In the absence of

185

PAA-TiO2-NPs, remarkable Cd toxicity was observed at both [Ca]dis (Supporting Information,

186

Figure S1a, c). The EC50 values thus obtained (65.5 and 132.2 µg/L, Figure 1) were higher

187

than those reported by Tan and Wang16 (7.5-24.8 µg/L) because the exposure time used in this

188

work was only half as long (24 h vs. 48 h). Further, the significantly (p < 0.05) higher EC50 8

ACS Paragon Plus Environment

Page 9 of 27

Environmental Science & Technology

189

and thus lower toxicity at the high Ca concentration indicated the amelioration of Cd toxicity

190

by Ca as a result of the uptake competition between these two cations.16, 17 Nevertheless,

191

other mechanisms were also involved because the disparate levels of Cd accumulation do not

192

fully explain the difference in toxicity at these two Ca concentrations (Supporting

193

Information, Figure S2). For instance, Ca may affect the membrane permeability as well as

194

other physiological parameters of D. magna.18

195

When PAA-TiO2-NPs (4 mg-Ti/L) were applied in the toxicity medium, there was no

196

daphnid mortality at any of the Cd concentrations tested during the 24-h exposure period

197

(Supporting Information, Figure S1b, d). In this experiment, the NPs were well dispersed (20

198

nm) at the low Ca concentration, but formed micrometer-sized aggregates (1650 nm) when

199

[Ca]dis was high. Moreover, > 90% of the Cd in the toxicity medium was adsorbed by

200

PAA-TiO2-NPs with no significant (p > 0.05) difference observed between the two Ca levels

201

(Supporting Information, Figure S3). Yang et al.9 also found that incubation of the green alga

202

C. reinhardtii with PAA-TiO2-NPs relieve Cd toxicity. In their study, such toxicity alleviation

203

was explained by the adsorption of Cd to the particle surfaces and Cd uptake was thus

204

inhibited as the NPs cannot enter the algal cells. By contrast, the protozoan T. thermophila

205

can endocytose PAA-TiO2-NPs directly.10 In this case, particle addition induces Cd

206

accumulation, changes the subcellular distribution of the metal, and exacerbates its toxicity.

207

These distinct responses suggest that NP effects on metal toxicity are organism-specific and

208

would further be influenced by the nature of the NPs and the type of metal. For instance,

209

Rosenfeldt et al.7 reported that TiO2-NPs (P25) could either induce or reduce the toxicity of

210

Ag or Cu to D. magna depending on the alteration of metal accumulation by the NPs. In

211

addition, the same authors found that, in the presence of P25, the toxicity of As was alleviated

212

while its uptake was increased.

213

The sophisticated effects of NPs on metal toxicity were also evidenced by the Cd

214

accumulation results of the present study (Figure 2a, b). At a [Ca]dis of 2.0 mM,

215

PAA-TiO2-NPs (4 mg-Ti/L) induced Cd uptake, and thus [Cd]daphnia increased linearly from

216

1.2 µg/g-dw (1.8 µg/g-dw) at the lowest Cd concentration to 941.4 µg/g-dw (101.4 µg/g-dw)

217

at a [Cd]T of 300 µg/L in the presence (absence) of PAA-TiO2-NPs. By contrast, in the 9

ACS Paragon Plus Environment

Environmental Science & Technology

218

toxicity medium with a [Ca]dis of 0.2 mM, PAA-TiO2-NPs (4 mg-Ti/L) reduced [Cd]daphnia by

219

more than one order of magnitude in the different Cd concentration treatments, from

220

42.8-593.2 µg/g-dw in the absence of the NPs to 1.6-19.9 µg/g-dw in the presence of the NPs.

221

These results clearly indicated that the well-dispersed PAA-TiO2-NPs and their

222

micrometer-sized aggregates had opposing effects on Cd accumulation despite their

223

alleviation of Cd toxicity at both aggregation states.

224

To elucidate the underlying mechanism for the aggregation-dependent impacts of NPs

225

on Cd accumulation, the PAA-TiO2-NP content of the daphnids in each treatment was

226

quantified at the end of the toxicity tests (Figure 2c). Although the adsorption of Cd to the

227

particle surfaces correlated positively with [Cd]T, as shown in our previous study,9

228

PAA-TiO2-NP accumulation was independent of the Cd concentration, but was significantly

229

(p < 0.05) induced when the micrometer-sized aggregates were formed at the high Ca

230

concentration. This was evidenced by the increase in [Ti]daphnia from 4.8-5.9 mg-Ti/g-dw to

231

11.8-13.4 mg-Ti/g-dw at a [Ca]dis of 0.2 and 2.0 mM, respectively (Figure 2c). According to

232

our earlier findings,12 the increased accumulation of PAA-TiO2-NPs could be mainly

233

attributed to the Ca-related aggregation of the NPs. Namely, the well-dispersed NPs at the

234

low Ca concentration were taken up by the daphnids mainly through endocytosis and passive

235

drinking, whereas active ingestion was the major route by which they accumulated the

236

micrometer-sized aggregates at the high Ca concentration.

237

Based on the assumption that the metal-NP complexes remain stable during the

238

accumulation process and that the non-NP-associated metal had a negligible contribution to

239

its accumulation, we calculated the amount of Cd entering the daphnids together with

240

PAA-TiO2-NPs, using [Ti]daphnia (Figure 2c) and the adsorption of Cd by the particles. The

241

value was close to that measured at the high Ca concentration (Figure 2b), suggesting that the

242

carrier effects of PAA-TiO2-NPs (in the form of aggregates) played a predominant role in Cd

243

accumulation under this condition. In this case, the contribution of the non-adsorbed metal to

244

its accumulation was also negligible. By contrast, the measured Cd accumulation was only

245

5.1-17.0% of that predicted at the low Ca concentration (Figure 2a). Considering the

246

contribution of non-NP-associated Cd to [Cd]daphnia, PAA-TiO2-NPs (in the well-dispersed 10

ACS Paragon Plus Environment

Page 10 of 27

Page 11 of 27

Environmental Science & Technology

247

form) could hardly have served as effective Cd carriers despite their substantial accumulation

248

in D. magna. This result further implies that Cd dissociated from the well-dispersed

249

PAA-TiO2-NPs either before or after the uptake of the metal-NP complexes, as further

250

discussed below.

251

Besides the investigation of [Ti]daphnia and [Cd]daphnia in the different Cd concentration

252

treatments at the end of each toxicity test, the variation of these two parameters with exposure

253

time was examined in a 48-h accumulation experiment. Similar to our previous study,12 a

254

parabolic correlation between [Ti]daphnia and exposure time was observed (Figure 3a). This

255

relationship reflected settlement of the PAA-TiO2-NP aggregates at the high Ca level and the

256

variation in the uptake and efflux rate constants with exposure time for the well-dispersed

257

NPs at the low Ca level. Similar to [Ti]daphnia, [Cd]daphnia also exhibited a parabolic correlation

258

with exposure time at the high Ca concentration (Figure 3b). Under this condition, the actual

259

value of [Cd]daphnia was close to that predicted from [Ti]daphnia at all time points. By contrast,

260

[Cd]daphnia rose steadily with increasing exposure time at a [Ca]dis of 0.2 mM and was

261

approximately 3.2-16.8% of the predicted value, consistent with the results of the toxicity

262

experiment described above. This discrepancy was already apparent after only 15 min of

263

exposure, indicative of the rapid dissociation of Cd from the well-dispersed PAA-TiO2-NPs.

264

The aggregation-dependent uptake of PAA-TiO2-NPs and the opposite effects of the

265

particles at different aggregation states on Cd accumulation were also evidenced by the

266

distinct Ti and Cd distributions in D. magna at the two Ca levels. The daphnids were

267

visualized through their µXRF signal of Ca (Figure 4a-d). At a [Ca]dis of 2.0 mM,

268

PAA-TiO2-NPs and Cd were mainly concentrated in the gut (Figure 4e, i), but a considerable

269

amount of Cd was also detected in other areas of D. magna, at sites similar to those

270

determined in the absence of the NPs (Figure 4g, h). In the latter case, Cd was mainly taken

271

up from the experimental medium as free ions. This uptake route may thus have played a

272

critical role in non-gut Cd accumulation in the presence of PAA-TiO2-NP aggregates. At a

273

[Ca]dis of 0.2 mM, the gut PAA-TiO2-NP signal was at least one order of magnitude weaker

274

than at a [Ca]dis of 2.0 mM, although there was only a 50% reduction in [Ti]daphnia at the low

275

concentration of Ca. Therefore, PAA-TiO2-NP distribution in non-gut areas of D. magna 11

ACS Paragon Plus Environment

Environmental Science & Technology

Page 12 of 27

276

would be expected. In fact, our previous study demonstrated that the well-dispersed

277

PAA-TiO2-NPs, at a concentration 10 times higher than used herein, were distributed

278

throughout the body of D. magna, especially in the gut and abdominal zone.12 In the present

279

study, a detectable amount of Ti was also found in the abdominal zone of D. magna (Figure

280

4j), but its signal was relatively weak, suggesting that PAA-TiO2-NP accumulation in this

281

area was close to the detection limit (at the level of µg/g) of µXRF. Nevertheless, at the low

282

Ca concentration, PAA-TiO2-NPs had negligible effects on Cd distribution, which was

283

instead comparable to the pattern obtained in the absence of the NPs (Figure 4f-h). This

284

phenomenon supports our previous hypothesis that PAA-TiO2-NPs could hardly have served

285

as effective Cd carriers when they were well dispersed at a [Ca]dis of 0.2 mM.

286

Most studies about the effects of NPs on metal accumulation by multicellular animals

287

have reported a remarkable induction. For instance, TiO2-NPs increased the Cd concentration

288

in carp (Cyprinus carpio) by 146% during a 25-d exposure period.19 Similarly, CdTe quantum

289

dots facilitated Cu accumulation in zebrafish.4 Only one study, by Rosenfeldt et al.7, reported

290

a reduction (by 14-fold) of the Cu body burden in D. magna in the presence of TiO2-NPs.

291

According to the underlying mechanisms proposed by those authors, the decrease in Cu

292

accumulation was caused by the rapid agglomeration and sedimentation of the metal-NP

293

complexes. Nevertheless, the aqueous suspensions of the NPs used in these studies were

294

unstable and micrometer- or submicrometer-sized aggregates were formed during the

295

experimental period.

296

In

the

present

study,

PAA-TiO2-NPs

either

stimulated

or

reduced

metal

297

bioaccumulation, depending on the aggregation and uptake routes of the particles. Thus, the

298

micrometer-sized aggregates that formed at a [Ca]dis of 2.0 mM were actively ingested by D.

299

magna and were concentrated in the gut, together with Cd (Figure 4e, i). This suggested that

300

most of the Cd was still associated with the NPs—or at least did not penetrate the epithelial

301

barrier even though the daphnid gut is weakly acidic (pH ~ 6.0)20— and was consistent with

302

our observation that at a pH of 6-9 Cd adsorption on PAA-TiO2-NPs varied by < 35%

303

(Supporting Information, Figure S4a). However, at a [Ca]dis of 0.2 mM, the well-dispersed

304

PAA-TiO2-NPs were taken up by passive drinking and endocytosis. Passive drinking resulted 12

ACS Paragon Plus Environment

Page 13 of 27

Environmental Science & Technology

305

in the gut accumulation of the NPs and their associated Cd, similar to the fates of both

306

pollutants at the high Ca concentration. This implies an increase, rather than a reduction in

307

Cd uptake via this route. By contrast, the endocytosis of PAA-TiO2-NPs led to the

308

distribution of the particles throughout the daphnids, with the highest concentration detected

309

in the abdominal area. A decrease in the accumulation of Cd via its endocytosis with NPs was

310

therefore expected, considering the lower overall uptake of Cd at the low Ca concentration.

311

As reported above, [Cd]daphnia was much lower than the concentration predicted from the

312

accumulation of the well-dispersed PAA-TiO2-NPs and their adsorption of Cd in the toxicity

313

and 48-h uptake experiments. Therefore, a prerequisite for the decreased accumulation of Cd

314

was its dissociation from the PAA-TiO2-NP surfaces during endocytosis of the metal-NP

315

complexes at the low Ca concentration. Endocytosis is temperature-dependent and it ceases at an ambient temperature of 4 oC.21,

316 317

22

We therefore compared PAA-TiO2-NP and Cd accumulation in D. magna at 4 oC and 24 oC

318

and at the two Ca concentrations. At the high Ca concentration, as the ambient temperature

319

dropped from 24 to 4 oC, the accumulation of PAA-TiO2-NPs and Cd was similarly

320

suppressed, declining by 74.0-84.9% and 55.1-72.4%, respectively. At the low Ca

321

concentration, the accumulation of PAA-TiO2-NPs was more severely inhibited than that of

322

Cd. Thus, [Ti]daphnia was 8.0-13.4% and [Cd]daphnia was 45.6-59.7% of the respective

323

concentrations determined at 24 oC. Based on these results, our calculation showed that the

324

Cd accumulation predicted from [Ti]daphnia was close to the observed amount (i.e.,

325

PAA-TiO2-NPs served as the carriers of Cd accumulation), as long as endocytosis was not

326

involved in the accumulation of PAA-TiO2-NPs in D. magna (Figure 5). In other words, the

327

endocytosis of the metal-NP complexes and the rapid dissociation of Cd from the NP surfaces

328

during this process were the main cause for the reduced accumulation of Cd at the low Ca

329

concentration.

330

Although the rapid dissociation of Cd from PAA-TiO2-NPs during their endocytosis

331

was verified in the present study, we were unable to directly determine whether dissociation

332

occurred before or after internalization of the Cd-NP complexes. Nevertheless, the latter

333

possibility is more likely considering that at the high Ca concentration the metal-NP 13

ACS Paragon Plus Environment

Environmental Science & Technology

334

complexes also passed through the abdominal areas and were intercepted by the filtering

335

setae before being actively ingested, but no dissociation was observed. There was also no

336

reports of a remarkable liberation of strong metal-binding ligands in the microdomains

337

around the thoracic limbs of D. magna, and Cd adsorption on PAA-TiO2-NPs remained

338

unchanged in the medium in all of the above-described experiments. Once PAA-TiO2-NPs

339

were endocytosed, they concentrated first in endosomes and later in lysosomes, where the pH

340

was in the range of 4.0-5.5.23 A variety of biomolecules (possibly containing sulfhydryl

341

groups) with strong Cd-binding affinity may also be present in these organelles.24 In a

342

preliminary experiment, we observed that supernatants prepared from D. magna tissue

343

homogenates could desorb > 70% of the NP-adsorbed Cd (Supporting Information, Figure

344

S4b). Therefore, the reduced accumulation of Cd in the presence of the well-dispersed

345

PAA-TiO2-NPs could be mainly attributed to: (i) the rapid dissociation of Cd from the NPs in

346

the acidic environment of the lysosomes containing various metal-binding biomolecules, and

347

(ii) the rapid depuration of the metal (i.e., this process was so quick that the metal could

348

hardly have time to interact with intracellular sensitive sites) through exocytosis or other

349

routes of elimination, after the internalization of the metal-NP complexes. A rapid (within a

350

few seconds) completion of the endocytosis-exocytosis cycle (membrane retrieval) has been

351

demonstrated in a variety of cell types25-27 and explains why, in the 48-h experiment,

352

[Cd]daphnia was already much lower than the predicted value after 15 min of exposure. Similar

353

to the present work, a previous study also showed that a substantial amount of Ca in exocrine

354

cells is exported via exocytosis, a process linked to its uptake via endocytosis and the rapid

355

release of endosomal Ca.28

356

Efflux of Cd and PAA-TiO2-NPs. The opposing effects of the well-dispersed

357

PAA-TiO2-NPs and their micrometer-sized aggregates on Cd accumulation were further

358

supported by the distinct pattern of efflux of the metal from D. magna pre-exposed to Cd in

359

the presence or absence of differently-aggregated NPs (Figure 6). Although PAA-TiO2-NPs

360

were depurated at the same rate at high and low Ca concentrations within the first 24 h,

361

significantly (p < 0.05) more NPs were retained in the latter (2.40-4.05% vs. 0.57-1.57%)

362

after 48 h (Figure 6a). Nevertheless, the rapid elimination of differently-aggregated 14

ACS Paragon Plus Environment

Page 14 of 27

Page 15 of 27

Environmental Science & Technology

363

PAA-TiO2-NPs from D. magna implies that the particles will not be readily transferred

364

along the food chain. Similarly, > 90% of the graphene accumulated in D. magna was

365

eliminated within 24-h depuration.29 In the case of Cd, its efflux was independent of [Ca]dis,

366

with an efflux rate constant of 0.0029-0.0033 h-1, when PAA-TiO2-NPs were absent from the

367

pre-exposure medium (Figure 6b). This result is similar to those reported in the literature

368

(0.0021-0.0038 h-1).30, 31 As for the daphnids pre-exposed to both Cd and the NPs at the low

369

Ca concentration, their depuration of Cd followed a trend similar to that seen in daphnids

370

pre-exposed to Cd only. This phenomenon suggested that Cd uptake in the form of free ions

371

had a major contribution to [Cd]daphnia under this condition, despite the fact that most Cd was

372

adsorbed on the well-dispersed PAA-TiO2-NPs in the pre-exposure medium and should have

373

been able to enter the daphnids together with the NPs. Nevertheless, greater Cd elimination

374

was achieved when PAA-TiO2-NPs were applied in the pre-exposure medium with a [Ca]dis

375

of 0.2 mM. The efflux rate constant thus obtained was 0.006 h-1. It seems that the

376

well-dispersed PAA-TiO2-NPs, especially those accumulated by passive drinking, may still

377

have some inductive effects on Cd efflux. When the daphnids were pre-exposed to both the

378

metal and the NP aggregates at the high Ca concentration, their elimination of Cd followed a

379

biphasic pattern similar to that of the NPs, with only 2.4% retained by the end of the

380

experiment. The similarity in the elimination of Cd and PAA-TiO2-NP aggregates supports

381

our hypothesis of a close relationship between these two pollutants during their

382

accumulation by the daphnids.

383

In addition to the determination of the efflux kinetics of PAA-TiO2-NPs, our study also

384

tried to identify the routes of NP elimination in D. magna. The contribution of the different

385

routes to the elimination of both PAA-TiO2-NPs and Cd was Ca-dependent (Figure 6c). At a

386

high vs. a low ambient Ca, greater proportions of Cd and Ti were contained in the feces and

387

less in the molts or dissolved phases. There was no consistent impact of Ca on the

388

elimination of PAA-TiO2-NPs and Cd via daphnid reproduction. Moreover, these two

389

pollutants were mostly lost into the dissolved phase (64.1-80.7%), with much lower amounts

390

(3.3-15.9%) of both occurring in molts, neonates, and feces. Excretion into water is also a

391

dominant route (> 80%) of Cd efflux from D. magna pre-exposed to Cd-containing algal 15

ACS Paragon Plus Environment

Environmental Science & Technology

392

food particles.30, 31 Nevertheless, that most of the eliminated PAA-TiO2-NPs ended up in the

393

dissolved phase (< 0.22 µm) even at the high Ca concentration—when PAA-TiO2-NPs

394

formed micrometer-sized aggregates unable to pass through a 0.22-µm filter—was

395

surprising and suggested that the PAA-TiO2-NPs became more hydrophilic after their

396

accumulation in D. magna. How this changes the behavior, effects, and fate of both

397

pollutants warrants further investigation.

398

Overall, Ca was used in the present study to manipulate the aggregation of

399

PAA-TiO2-NPs. We then demonstrated the ability of PAA-TiO2-NPs to alleviate Cd toxicity

400

to D. magna at both low and high Ca concentrations, albeit by very different mechanisms. At

401

the high Ca concentration, PAA-TiO2-NPs formed micrometer-sized aggregates that were

402

actively ingested by D. magna. Although Cd accumulation was induced under this condition,

403

it was concentrated in the daphnid gut together with the NPs and the toxicity was thus

404

relieved. At the low Ca concentration, PAA-TiO2-NPs were well dispersed, with particle sizes

405

still in the nano-range. These NPs were taken up mainly by endocytosis and passive drinking,

406

which resulted in their accumulation throughout D. magna, especially in abdominal areas and

407

the gut. In this case, Cd was also distributed throughout the daphnid and accumulation was

408

considerably impaired because of the metal’s rapid dissociation from the NP surfaces during

409

endocytosis of the metal-NP complexes. The opposing effects of the well-dispersed

410

PAA-TiO2-NPs vs. their micrometer-sized aggregates on Cd accumulation in D. magna may

411

likely be extended to other NPs, trace metals, and organisms. Moreover, particles with

412

different sizes may very well have completely different effects on metal bioaccumulation.

413

These observations should be taken into account in evaluations of the environmental risks of

414

NPs and other particles.

415



ACKNOWLEDGEMENTS

416

We thank Dr. Qiaoguo Tan and three anonymous reviewers for their instructive

417

comments on this paper. The financial support to A. J. Miao by Chinese public science and

418

technology research funds projects of ocean (201505034) and the National Natural Science

419

Foundation of China (41271486, 41001338, and 21237001) made this work possible.

420



SUPPORTING INFORMATION 16

ACS Paragon Plus Environment

Page 16 of 27

Page 17 of 27

Environmental Science & Technology

421

Additional figures showing the time related variation of the survival rate of Daphnia

422

magna, its change with Cd bioaccumulation, Cd adsorption on PAA-TiO2-NPs during the

423

24-h toxicity experiment, and the effects of both pH and D. magna tissue homogenates on Cd

424

adsorption are included. This material is available free of charge on the ACS Publications

425

Website.

426 427

17

ACS Paragon Plus Environment

Environmental Science & Technology

428



429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470

(1) 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. (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) Kahru, A.; Dubourguier, H. C. From ecotoxicology to nanoecotoxicology. Toxicology 2010, 269, (2-3), 105-119. (4) Zhang, W.; Miao, Y.; Lin, K.; Chen, L.; Dong, Q.; Huang, C. Toxic effects of copper ion in zebrafish in the joint presence of CdTe QDs. Environ. Pollut. 2013, 176, 158-164. (5) Sun, H.; Zhang, X.; Zhang, Z.; Chen, Y.; Crittenden, J. Influence of titanium dioxide nanoparticles on speciation and bioavailability of arsenite. Environ. Pollut. 2009, 157, (4), 1165-1170. (6) Tan, C.; Wang, W. X. Modification of metal bioaccumulation and toxicity in Daphnia magna by titanium dioxide nanoparticles. Environ. Pollut. 2014, 186, 36-42. (7) Rosenfeldt, R. R.; Seitz, F.; Schulz, R.; Bundschuh, M. Heavy metal uptake and toxicity in the presence of titanium dioxide nanoparticles: a factorial approach using Daphnia magna. Environ. Sci. Technol. 2014, 48, (12), 6965-6972. (8) Basiruddin, S. K.; Saha, A.; Pradhan, N.; Jana, N. R. Advances in coating chemistry in deriving soluble functional nanoparticle. J. Phys. Chem. C 2010, 114, (25), 11009-11017. (9) 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. (10)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. (11) 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. (12) Tan, L. Y.; Huang, B.; Xu, S.; Wei, Z. B.; Yang, L. Y.; Miao, A. J. TiO2 nanoparticle uptake by the water flea Daphnia magna via different routes is calcium-dependent. Environ. Sci. Technol. 2016, 50, (14), 7799-7807. (13) 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. (14) Tsui, M. T. K.; Wang, W. X. Maternal transfer efficiency and transgenerational toxicity of methylmercury in Daphnia magna. Environ. Toxicol. Chem. 2004, 23, (6), 1504-1511. (15) Laforsch, C.; Tollrian, R. A new preparation technique of daphnids for Scanning Electron Microscopy using hexamethyldisilazane. Arch. Hydrobiol. 2000, 149, (4), 587-596. (16) Tan, Q. G.; Wang, W. X. Acute toxicity of cadmium in Daphnia magna under different calcium and pH conditions: importance of influx rate. Environ. Sci. Technol. 2011, 45, (5),

REFERENCES

18

ACS Paragon Plus Environment

Page 18 of 27

Page 19 of 27

471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511

Environmental Science & Technology

1970-1976. (17) Clifford, M.; McGeer, J. C. Development of a biotic ligand model to predict the acute toxicity of cadmium to Daphnia pulex. Aquat. Toxicol. 2010, 98, (1), 1-7. (18) Penttinen, S.; Kostamo, A.; Kukkonen, J. V. K. Combined effects of dissolved organic material and water hardness on toxicity of cadmium to Daphnia magna. Environ. Toxicol. Chem. 1998, 17, (12), 2498-2503. (19) Zhang, X.; Sun, H.; Zhang, Z.; Niu, Q.; Chen, Y.; Crittenden, J. C. Enhanced bioaccumulation of cadmium in carp in the presence of titanium dioxide nanoparticles. Chemosphere 2007, 67, (1), 160-166. (20) Lavrentjeva, I. F.; Beim, A. M. Study of physiological processes in Daphnia by means of fluorochromes. Gidrobiol. Zh. 1978, 14, (2), 99-102. (21) 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. (22) Chang, E.; Thekkek, N.; Yu, W. W.; Colvin, V. L.; Drezek, R. Evaluation of quantum dot cytotoxicity based on intracellular uptake. Small 2006, 2, (12), 1412-1417. (23) Iversen, T. G.; Skotland, T.; Sandvig, K. Endocytosis and intracellular transport of nanoparticles: present knowledge and need for future studies. Nano Today 2011, 6, (2), 176-185. (24) Docter, D.; Westmeier, D.; Markiewicz, M.; Stolte, S.; Knauer, S. K.; Stauber, R. H. The nanoparticle biomolecule corona: lessons learned - challenge accepted? Chem. Soc. Rev. 2015, 44, (17), 6094-6121. (25) 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. (26) Artalejo, C. R.; Henley, J. R.; McNiven, M. A.; Palfrey, C. H. Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP, and dynamin but not clathrin. PNAS 1995, 92, (18), 8328-8332. (27) Mansvelder, H. D.; Kits, K. S. The relation of exocytosis and rapid endocytosis to calcium entry evoked by short repetitive depolarizing pulses in rat melanotropic cells. J. Neurosci. 1998, 18, (1), 81-92. (28) Gerasimenko, J. V.; Tepikin, A. V.; Petersen, O. H.; Gerasimenko, O. V. Calcium uptake via endocytosis with rapid release from acidifying endosomes. Cur. Biol. 1998, 8, (24), 1335-1338. (29) Guo, X.; Dong, S.; Petersen, E. J.; Gao, S.; Huang, Q.; Mao, L. Biological uptake and depuration of radio-labeled graphene by Daphnia magna. Environ. Sci. Technol. 2013, 47, (21), 12524-12531. (30) Tan, Q. G.; Wang, W. X. The influences of ambient and body calcium on cadmium and zinc accumulation in Daphnia magna. Environ. Toxicol. Chem. 2008, 27, (7), 1605-1613. (31) Guan, R.; Wang, W. X. Dietary assimilation and elimination of Cd, Se, and Zn by Daphnia magna at different metal concentrations. Environ. Toxicol. Chem. 2004, 23, (11), 2689-2698.

512 513 19

ACS Paragon Plus Environment

Environmental Science & Technology

514

Figure legends

515

Figure 1. Variations in the survival rate of Daphnia magna with total concentration of

516

ambient Cd ([Cd]T) at low (0.2 mM) and high (2.0 mM) Ca concentrations in the presence

517

(+Ti) and absence of 4 mg-Ti/L PAA-TiO2-NPs. Dashed lines are the simulated

518

dose-response curves based on the logistic model. Data are mean ± SD (n = 3).

519

Figure 2. Variations in bioaccumulated Cd ([Cd]daphnia) with total concentration of ambient

520

Cd ([Cd]T) at (a) low (0.2 mM) and (b) high (2.0 mM) Ca concentrations in the presence (+Ti)

521

and absence of 4 mg-Ti/L PAA-TiO2-NPs. (c) Variations in the D. magna PAA-TiO2-NP

522

content ([Ti]daphnia) at the different Cd concentrations (0, 10, 20, 50, 100, 200, and 300 µg/L

523

for treatment A to G) in toxicity tests at low and high Ca concentrations in the presence of 4

524

mg-Ti/L PAA-TiO2-NPs. Dashed and solid lines are the linear regression between [Cd]T and

525

[Cd]daphnia or the predicted value of [Cd]daphnia (0.2+Ti-pre and 2+Ti-pre), respectively. Data

526

are mean ± SD (n = 3).

527

Figure 3. Results of the 48-h accumulation experiment showing the variations in (a) the

528

PAA-TiO2-NP content in D. magna ([Ti]daphnia) and (b) bioaccumulated Cd ([Cd]daphnia) as a

529

function of exposure time at low (0.2 mM) and high (2 mM) Ca concentrations. Solid lines in

530

(b) represent the variation of the predicted value of [Cd]daphnia with exposure time at low and

531

high Ca concentrations (0.2-pre and 2-pre, respectively). Data are the mean ± SD (n =3).

532

Figure 4. Distribution of Ca (a-d), Cd (e-h), and PAA-TiO2-NPs (i-l) in Daphnia magna

533

exposed to Cd (100 µg/L) with (a, b, e, f, i, and j) or without (c, d, g, h, k, and l) the addition

534

of PAA-TiO2-NPs (4 mg-Ti/L) for 24 h at low (b, f, j, d, h, and l) and high (a, e, i, c, g, and k)

535

Ca levels, as determined by synchrotron-radiation-based micro X-ray fluorescence

536

spectroscopy (µXRF). White arrows indicate the gut (GT) or abdominal area (AD) of D.

537

magna.

538

Figure 5. Linear correlation between the measured content of Cd accumulated in D. magna

539

([Cd]daphnia) and its predicted value based on the bioaccumulation of PAA-TiO2-NPs in a 6-h

540

uptake experiment in which D. magna was exposed to Cd (10 µg/L) and PAA-TiO2-NPs (4

541

mg-Ti/L) at low (0.2 mM) and high (2.0 mM) Ca levels and ambient temperatures of 4 oC 20

ACS Paragon Plus Environment

Page 20 of 27

Page 21 of 27

Environmental Science & Technology

542

(0.2-4 and 2-4) and 24 oC (0.2-24 and 2-24), respectively. Data are the mean ± SD (n =3).

543

Figure 6. Proportion of (a) PAA-TiO2-NPs and (b) Cd retained in D. magna pre-exposed to

544

Cd (10 µg/L) with (+Ti) or without the addition of PAA-TiO2-NPs (4 mg-Ti/L) at low (0.2

545

mM) and high (2.0 mM) Ca levels, respectively, during a 6-d depuration period. (c) The

546

distribution of PAA-TiO2-NPs (0.2-Ti and 2-Ti) and Cd (0.2-Cd and 2-Cd) depurated from D.

547

magna in the dissolved phase (excretion), molts, neonates, and feces at low (0.2 mM) and

548

high (2.0 mM) Ca concentrations, as determined in the efflux experiment. Data are the mean

549

± SD (n =3).

550

21

ACS Paragon Plus Environment

Environmental Science & Technology

Figure 1

survival rate (%)

551

100 0.2 2 0.2+Ti 2+Ti

50

0 1 552 553

10 100 [Cd]T (µg/L)

22

ACS Paragon Plus Environment

Page 22 of 27

Page 23 of 27

554

Environmental Science & Technology

Figure 2

[Cd]daphnia (µg/g-dw)

1000

0.2 0.2+Ti 0.2+Ti-pre

aa

2 2+Ti 2+Ti-pre

b

500

0 1000

b

500

0

[Ti]daphnia (mg-Ti/g-dw)

0

555

20

100 200 [Cd]T (µg/L)

300

0.2 2

c

10

0

A

B

C D E treatments

F

G

556

23

ACS Paragon Plus Environment

Environmental Science & Technology

Figure 3

[Cd]daphnia (µg/g-dw)

[Ti]daphnia (mg-Ti/g-dw)

557

40

20

0 80

b

40

0 0

558

a

0.2 0.2-pre 2 2-pre

10

20

30

40

exposure time (h)

559

24

ACS Paragon Plus Environment

50

Page 24 of 27

Page 25 of 27

560

Environmental Science & Technology

Figure 4

561 562

25

ACS Paragon Plus Environment

Environmental Science & Technology

Figure 5 predicted value of [Cd]daphnia (µg/g-dw)

563

1000

0.2-24 0.2-4

100 10 1 1

564

2-24 2-4

10 100 measured value of [Cd]daphnia (µg/g-dw)

565

26

ACS Paragon Plus Environment

1000

Page 26 of 27

Page 27 of 27

566

Environmental Science & Technology

Figure 6

Ti retained (%)

100

0.2 0.2+Ti 2 2+Ti

10 1 0.1

a

Cd retained (%)

100

10

1 b 0 25 50 75 100 125 150 depuration time (h)

* * es

0

*

fe c

m ol tin g ne * on at es

*

ex cr et

567

0.2-Ti 2-Ti 0.2-Cd 2-Cd

50

io n

relative contribution to total efflux (%)

100 c

568 569

27

ACS Paragon Plus Environment