Nanocolloids in Natural Water: Isolation, Characterization, and Toxicity

Mar 19, 2018 - Nanocolloids are widespread in natural water systems, but their characterization and ecological risks are largely unknown. Herein, tang...
0 downloads 9 Views 2MB Size
Subscriber access provided by Queen Mary, University of London

Ecotoxicology and Human Environmental Health

Nanocolloids in Natural Water: Isolation, Characterization and Toxicity Shaohu Ouyang, Xiangang Hu, Qixing Zhou, Xiaokang Li, Xinyu Miao, and Ruiren Zhou Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05364 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 19, 2018

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 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 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.

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 42

Environmental Science & Technology

TOC

ACS Paragon Plus Environment

Environmental Science & Technology

1

Nanocolloids in Natural Water: Isolation, Characterization and Toxicity

2 3

Shaohu Ouyang, Xiangang Hu, Qixing Zhou*, Xiaokang Li, Xinyu Miao, Ruiren

4

Zhou

5

Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of

6

Education)/Tianjin Key Laboratory of Environmental Remediation and Pollution

7

Control, College of Environmental Science and Engineering, Nankai University,

8

Tianjin 300350, China

9 10

Corresponding authors: Qixing Zhou, [email protected]

11

Fax, 0086-022-85358121

12

Tel, 0086-022-85358121

13

ABSTRACT

14

Nanocolloids are widespread in natural water systems, but their characterization and

15

ecological risks are largely unknown. Herein, tangential flow ultrafiltration (TFU)

16

was used to separate and concentrate nanocolloids from surface waters. Unexpectedly,

17

nanocolloids were present in high concentrations ranging from 3.7 to 7.2 mg/L in the

18

surface waters of the Harihe River in Tianjin City, China. Most of the nanocolloids

19

were 10-40 nm in size, contained various trace metals and polycyclic aromatic

20

hydrocarbons, and exhibited fluorescence properties. Envelopment effects and

21

aggregation of Chlorella vulgaris in the presence of nanocolloids were observed.

22

Nanocolloids entered cells and nanocolloid-exposed cells exhibited stronger 1

ACS Paragon Plus Environment

Page 2 of 42

Page 3 of 42

Environmental Science & Technology

23

plasmolysis, chloroplast damage and more starch grains than the control cells.

24

Moreover, nanocolloids inhibited the cell growth, promoted reactive oxygen species

25

(ROS), reduce the chlorophyll a content and increased the cell permeability. The

26

genotoxicity of nanocolloids was also observed. The metabolomics analysis revealed

27

a significant (p 100 nm) were filtered through 0.1 µm Millipore Durapore

113

membranes using conventional membrane filtration.26 Second, the ultra-filtration step

114

(in cross flow mode) using a PES module with nominal pore size of 5 kDa to to

115

removal ions and free natural organic matters according to the use guidelines of

116

Vivaflow50 PES module.27 Then, 400 mL of these pre-filtered samples were filtered

117

through the TFU fractionation system at a steady flow rate of 300 mL/min at room

118

temperature (296 K), and a concentrated volume of 20 mL was saved. Subsequently,

119

to purify and remove the free small molecules and ions on the filters, the samples

120

were dialyzed by 6,000-8,000 Dalton MWCO dialysis membranes (B0052, Viskase,

121

USA) in 3 L of doubly distilled water with magnetic stirring for 24 h. Three parallel

122

water samples were prepared for each sampling location.

123

Characterization of the Nanocolloids

124

Characterization of the nanocolloids were provided in the supporting information.

125

C. vulgaris Cultivation and Exposure

126

The characteristics (such as the morphology, size distribution, chemical composition,

127

and optical properties) of the nanocolloids at all the sample locations were similar,

128

which will be described in the results and discussion section. R9 exhibited the highest

129

concentration of nanocolloids and was used in the toxicological experiments. Based

130

on the detected nanocolloid concentrations (Table S2), nanocolloids from R9 were

131

prepared at levels from 0.72 to 36.0 mg/L for the toxicological exposure. C. vulgaris 6

ACS Paragon Plus Environment

Environmental Science & Technology

132

(FACH13-8) was obtained from the Institute of Wuhan Hydrobiology, Chinese

133

Academy of Sciences. C. vulgaris was grown in the blue-green culture medium

134

(BG-11, pH=7.0 ± 0.3, electrical conductivity (EC) =3.70 mS/cm), as previously

135

reported.28,29 The components of the BG-11 medium are presented in Table S3. The

136

growth of C. vulgaris after exposure to the nanocolloids for 96 h was quantified

137

according to the OECD guideline 201 with some slight modifications, as described

138

below.30,31Briefly, the algal cultures were grown in an artificial climate incubator

139

(Shanghai Boxun Medical Biological Instrument Incorporated Company, SPX-300I-C,

140

China) at 25.0 ± 0.5 °C and 80 % humidity. An illumination in the incubator of 10,000

141

LX was provided by daylight lamps under a light/dark regime of 16:8 h. C. vulgaris

142

was exposed to nanocolloid concentrations of 0.0, 0.72, 7.2 and 36.0 mg/L in 250 mL

143

glass flasks containing 100 mL of BG-11. The initial density of the algal cells was

144

approximately 1.1 × 105 cells/mL.

145

Electron Microscopy Observation

146

The surface alteration and cellular ultrastructure of algal cells after exposure to 36.0

147

mg/L of nanocolloids for 96 h were observed by scanning electron microscopy (SEM)

148

and transmission electron microscope (TEM), respectively. The details were presented

149

in the Supporting Information.

150

Toxicological Experiments

151

The algal cells were counted using flow cytometry (CyFLOW Space, Partec,

152

Germany) at 0, 24, 48, 72 and 96 h. The growth inhibition (%) was calculated by

153

subtracting the number of cells in the nanocolloid exposure group from the number of 7

ACS Paragon Plus Environment

Page 8 of 42

Page 9 of 42

Environmental Science & Technology

154

cells in the control group and dividing that number by the number of cells in the

155

control. The growth of the algal cells exposed or not exposed to nanocolloids at 96 h

156

was observed by an inverted fluorescence microscope (IX71, Olympus, Japan) at

157

10×100 magnification with an immersion oil. The concentrations of chlorophyll a

158

were measured using a UV-vis spectrophotometer (UV-2600, SHIMADZU, Japan), as

159

previously described.31 2′,7′-Dichlorodihydrofluorescin diacetate (DCFH-DA) was

160

used as a fluorescence probe to measure the intracellular reactive oxygen species

161

(ROS), as previously described.32,33 Cell permeability of algal cells exposed to the

162

nanocolloids was measured by fluorescein diacetate (FDA) method (details were

163

provided in the Supporting Information). Photocatalytic effects of nanocolloids on

164

algal cell were analyzed based on the method described by Akhavan et al (details were

165

provided in the Supporting Information).34 Genotoxicity (total RNA content and DNA

166

damage) of nanocolloids in algal cells was also studied (details were provided in the

167

Supporting Information).

168

Metabolic Profile

169

Details about the metabolic profile can be found in our previous study32and in the

170

Supporting Information.

171

Data Analyses

172

IBM SPSS 22.0 statistical software was used for the statistical analyses. All

173

experiments were at least performed in triplicate, and the results are presented as the

174

mean ± standard deviation. One-way analysis of variance (ANOVA) with Tukey’s test

175

was employed to analyze the differences. Statistical significance “*” was accepted at a 8

ACS Paragon Plus Environment

Environmental Science & Technology

176

level of p < 0.05. The TEM images of the algal cells were treated with PhotoshopCS6

177

and ImageJ. The thermal map was drawn using MeV 4.8.1 software. The default

178

distance metric for hierarchical clustering (HCL) was the Pearson correlation, and the

179

linkage method selection was achieved through average linkage clustering. Principal

180

component analysis (PCA) and orthogonal partial least-squares discriminant analysis

181

(OPLS-DA) were performed with SIMCA-P 11.5 software. The metabolic pathways

182

were examined with MetaboAnalyst 3.0, according to the Kyoto Encyclopedia of

183

Genes and Genomes (KEGG).

184

RESULTS AND DISCUSSION

185

Morphology and Size Distribution of the Nanocolloids

186

TEM and AFM were used to observe the morphology of the nanocolloids that were

187

separated from the Haihe River surface water using TFU. The samples from the nine

188

sampling locations exhibited similar morphologies. The physical dimensions of the

189

nanocolloids were between 1.4 and 99.4 nm (Table S2) with an average value of 17.0

190

nm, according to the TEM particle analysis (Figure 1a and Figure S3a). The TEM size

191

distribution histograms (Figure 1b and Figure S3b) showed a similar size distribution

192

in all the surface water samples after the TFU separation and that the majority (more

193

than 90 %) of the observed nanocolloids were smaller than 40 nm. Furthermore, 41.7 %

194

- 63.9 % of the nanocolloids exhibited diameters of approximately 10 to 40 nm. The

195

small colloids (diameters less than 10 nm) accounted for 33.2-51.6 % of the total

196

composition, and the large colloids (diameters from approximately 40 nm to 100 nm)

197

accounted for 2.8 - 6.7 %. The morphologies of the nanocolloids were further 9

ACS Paragon Plus Environment

Page 10 of 42

Page 11 of 42

Environmental Science & Technology

198

characterized by AFM (Figure S4). The AFM images showed that the heights of the

199

nanocolloids ranged from 1.6 to 6.8 nm. The size distribution of nanocolloids in the

200

present work was consistent with the reports for other aquatic systems, in which size

201

fractionation was less than 10 nm or ranged from approximately 4 to 40 nm.35,36

202

Notably, aggregates larger than 150 nm were also observed in the AFM (Figure S4)

203

and TEM (Figure S5) images. The ζ-potentials of the nanocolloid suspensions were

204

approximately 24-30 mV (Table S2), which implied that the suspensions were

205

metastable. The aggregation kinetics of the nanocolloids was obtained using the initial

206

rates of the Dh change with time in Figure S6. The initial sizes from the DLS

207

measurements were 30.9 − 83.9 nm, and they increased to approximately 300 nm at

208

96 h. The similar aggregation phenomenon was also observed in environmental

209

colloids in natural aquatic system, and the aggregation phenomenon was influenced

210

by the surface charges and colloidal components. 37,38 Moreover, EDS was used to

211

analyze the compositions of the nanocolloids. As shown in Figure 1c, the chemical

212

compositions of the nanocolloids included C, N, O, S, Si, Cl, P and metal elements

213

(Mg, Fe, Ca, Al and Cr). The compositions of environmental colloids remained

214

unclear due to the complex and variable sources over time and space.39 In general, the

215

compositions of nanocolloids were the mixture of inorganic matters (e.g., Ca, Fe, Mn

216

and Mg) and organic matters (e.g., humic acid substances and polysaccharides).36,40

217

Optical Properties and Composition of the Nanocolloids

218

The optical properties were characterized through UV-vis and fluorescence spectra.

219

The UV-vis absorption spectra of the nanocolloids were broad with a maximum 10

ACS Paragon Plus Environment

Environmental Science & Technology

220

absorption peak at 280 nm for all the samples (Figure 2a and S7a). The UV absorption

221

at 280 nm represents a π–π* electron transition from aromatic compounds, such as

222

polycyclic aromatic hydrocarbons, benzoic acid, and phenolic arenes with two or

223

more rings.41 The GC-MS analysis confirmed that the nanocolloids contained PAHs,

224

as listed in Table S4. The average total PAH concentration was 50.5 µg/kg. The PAH

225

with the highest concentration was naphthalene, and its average concentration was

226

14.2 µg/kg. As shown in Figure 2b and S7a, the fluorescence spectra (excitation

227

wavelength at 280 nm and emission wavelength at 560 nm) of the nanocolloids were

228

similar to those of humic-like substances.42 FT-IR was performed to identify the

229

presence of functional groups. Figure 2c and S7b clearly show that carboxyl, hydroxyl,

230

ether and amino groups were present in the nanocolloids. The C-H stretching

231

vibrations were centered at 2850 and 2923 cm−1, and weak oxygen-containing groups,

232

such as OH (3250 cm−1) and C-O (1380 cm−1), were also observed, which suggested

233

fatty acids or carboxylic acids were present on the surface of the nanocolloids.43 The

234

strong peaks that correspond to nitrogen-containing functional groups, such as N−H

235

(3240 cm−1), C-N (1360 cm−1), and amide I (1630 cm−1), can be attributed to amino

236

compounds.44 The largest absorption peaks were observed at 1120 cm-1, which are

237

attributed to ethers.45

238

The mean mass concentrations of the nanocolloids from upstream (R1, R2 and R3),

239

midstream (R4, R5 and R6) and downstream (R7, R8 and R9) were 4.05, 3.80 and

240

6.70 mg/L, respectively (Table S2). The highest nanocolloid concentration was 7.2

241

mg/L at R9. Compared with the concentrations of engineered nanoparticles; e.g., Ag 11

ACS Paragon Plus Environment

Page 12 of 42

Page 13 of 42

Environmental Science & Technology

242

nanoparticles or TiO2 nanoparticles are present at pg-ng/L levels in surface water,46

243

the nanocolloids had high concentrations due to their complex components, including

244

various metallic and non-metallic elements, as listed in Table S2 and Figure 1c and

245

2d-f. The mean concentrations of TC, TN and P were 1.2, 0.2 and 0.1 mg/L,

246

respectively (Figure 2d). The mean concentrations of Ca, Mg and Cr were 206.1,

247

138.2 and 39.8 µg/L, respectively (Figure 2e). The concentrations of Al and Fe were

248

23.8 and 39.9 µg/L, respectively. The concentrations of other trace elements, Mn, Zn

249

and Sr, were 1.2, 5.3 and 1.3 µg/L, respectively. The mass concentrations of TC, TN

250

and P accounted for 34.3±4.6 % of the nanocolloids. Nutrient elements, such as Si, O

251

and heavy metals, comprised more than 65 % of the nanocolloid composition (Figure

252

2f).

253

Furthermore, in order to explore the influence of different depth, the characteristics

254

of nanocolloids from deep water samples (4.5 m) at sampling sites R2, R4 and R9

255

were analyzed, as added in Table S5. There were no obvious differences of chemical

256

properties (e.g., pH, EC, size and ζ-potential) and compositions (e.g., TC, TN, P and

257

heavy metals) of nanocolloids between surface water (0.5 m) and deep water (4.5 m).

258

Similar results were observed for silver-based nanoparticles in surface waters.47 The

259

parameters (e.g., morphology, size, stability and chemical composition) of

260

nanomaterials, influence the biological responses to nanomaterials. The results in the

261

present work suggested that the above characteristics of the nanocolloids at all the

262

sample locations were similar. However, the interactions between organisms and the

12

ACS Paragon Plus Environment

Environmental Science & Technology

263

natural nanocolloids remain unclear.19 In the followed sections, the toxicity of

264

nanocolloids on C. vulgaris was studied.

265

Nanocolloid Adsorption, Uptake and Cellular Ultrastructure Damage in Algal

266

Cells

267

Some unknown compounds were observed around the algal cells, as shown by the

268

red arrows in Figure S8. The unknown compounds could be nanocolloids or

269

extracellular secretions. To identify the unknown compounds, fluorescence imaging of

270

the algal cells was performed, as shown in Figure 3. At a 240-280 nm ultraviolet

271

excitation, the control cells emitted a red fluorescence due to the presence of

272

chlorophyll. The nanocolloids emitted a 560 nm blue fluorescence under fluorescence

273

excitation (240-280 nm, Figure 2c). However, the surfaces of the algal cells in the

274

treated group emitted red and blue fluorescence (denoted by the blue arrows in Figure

275

3b), which suggested that the nanocolloids enveloped the algal cells. Moreover, the

276

above results were confirmed by the scanning electron microscopy (SEM) images

277

(Figure 3c-d). Irregular grooves covered the surface of the control cells in Figure 3c

278

and are denoted by black arrows. However, the grooves were not obvious for the cells

279

exposed to 36.0 mg/L of nanocolloids in Figure 3d. Similar envelopment effects for

280

algae cells have also been reported for carbon nanotubes and graphene.32,48,49 In

281

addition, trapping microorganisms or cells within aggregated nanoparticles was

282

another mechanism contributing to the adverse effects of the nanoparticle.50,51

283 284

The TEM image of the control cells showed an intact and clear cellular ultrastructure that included a cell wall, plasma membrane, chloroplast, nucleus and 13

ACS Paragon Plus Environment

Page 14 of 42

Page 15 of 42

Environmental Science & Technology

285

other organelles (Figure 4a-b). However, the structures of the chloroplasts and other

286

organelles were indistinct in the cells that were exposed to the nanocolloids (Figure

287

4c). To investigate whether nanoparticles were internalized, TEM-EDS was a useful

288

and important tools to track label-free nanoparticles in cells.52,53 TEM-EDS was used

289

to compare the differences of cellular ultrastructure between treated and control

290

groups and nanoparticle internalization. Compared with the control cells, some

291

unknown nanoparticles entered the algal cells and gathered in the vicinity of the starch

292

grains after 96 h of exposure, as indicated by the green arrows (Figure 4b and Figure

293

4d). The morphologies of these unknown nanoparticles were observed in the TEM

294

images and were irregular shapes that were similar to the original nanocolloids

295

(diameters ranging from 29.1 to 87.5 nm). In addition, the unknown nanoparticles

296

observed near the starch grains were also investigated by EDS (Figure 4e). Si and Cr

297

were discovered in the nanocolloids and the cells exposed to the nanocolloids. These

298

results implied that the nanocolloids entered the algal cells and mainly gathered in the

299

vicinity of the starch grains. Moreover, three apparent physiological changes (i.e.,

300

plasmolysis, thicker cell walls and more starch grains) occurred in the cells exposed to

301

the nanocolloids, as indicated by the double black arrows, blue arrows and red arrows,

302

respectively, in Figure 4c-d. The analysis of the TEM images (n=20) showed that the

303

ratio of the plasmolysis area to the total cell area was 19.4±3.5 % after exposure to

304

36.0 mg/L of the nanocolloids, and this ratio was significantly higher than that of the

305

control (9.1±2.0 %) (Figure 4f). Shrinkage of the cytoplasm contributed to the

306

plasmolysis, and the metabolic mechanisms of the plasma membrane damage will be 14

ACS Paragon Plus Environment

Environmental Science & Technology

307

explored in the next section. As shown in Figure 4f, the number of starch grains after

308

the nanocolloid exposure was 2.1-fold larger than that in the control. The increase in

309

the number of starch grains in the cells was likely a self-defense strategy to protect

310

themselves from the nanocolloids.54 In addition, the average thickness of the cell wall

311

in the control cells was 49.2 nm, whereas the average cell wall thickness for the algal

312

cells that were exposed to 36.0 mg/L of the nanocolloids was 69.1 nm. The cell wall

313

thickening in the algal cells that was induced by the nanocolloids may be due to

314

upregulation of the cellulose and chitin levels.55,56

315

Cytotoxicity

316

The envelopment or uptake effects of the nanocolloids can inhibit cell division. The

317

algal growth kinetics are shown in Figure S9. Compared with the kinetics of the

318

control, the nanocolloids inhibited cell division from 24 h to 96 h (Figure 4g). The

319

cell numbers after exposure to 0.72, 7.2 and 36 mg/L of the nanocolloids decreased by

320

5.0-7.0 %, 11.0-16.0 % and 16.0-18.0 %, respectively. The nanocolloid inhibition of

321

cell division was concentration dependent. The nanocolloid envelopment of the cells

322

hinders the nutrient exchange between the cell and culture medium, which likely

323

resulted in the algal growth inhibition.57 The nanocolloid inhibition of the algal cell

324

growth was comparable with that of other engineered materials; e.g., TiO2

325

nanoparticles, Ag nanoparticles, carbon nanotubes and graphene significantly inhibit

326

algal growth at concentrations of approximately 0.1-10 mg/L.58-61 Figure 4h shows a

327

significant increase in the ROS level in the nanocolloid-exposed algal cells compared

328

with that in the control. The relative intensities of the oxidative stress in the algal cells 15

ACS Paragon Plus Environment

Page 16 of 42

Page 17 of 42

Environmental Science & Technology

329

exposed to 0.72, 7.2 and 36.0 mg/L of the nanocolloids were 75 %, 80 % and 170.9 %

330

higher than that in the control, respectively. These results were consistent with those

331

for engineered nanomaterials.62,63 The adsorption of nanoparticles induced stress

332

response due to mitochondrial membrane damage, functional impairment and increase

333

of free radical production.64,65 Furthermore, the generation of free radicals affected

334

algal photosynthesis and biosynthesis of chlorophyll.66,67 Similarly, PAHs and heavy

335

metal (e.g., Cr) detected in nanocolloids may directly induce oxidative stress and

336

inhibit biosynthesis of chlorophyll in C. vulgaris by a carrier effect.68 As shown in

337

Figure 4i, compared with that of the control, the chlorophyll a content significantly

338

decreased by 12.5 %, 12.2 % and 21.9 % after exposure to 0.72, 7.2 and 36.0 mg/L of

339

the nanocolloids, respectively. The downregulation of the chlorophyll a biosynthesis

340

was consistent with the damage to the chloroplasts (Figure 4c).

341

As shown in Figure S10, the results of FDA assay were applied to determine cell

342

viability of algae after exposure to nanocolloids for 96 h. There were significant

343

alterations of the cell viability in treated groups compared with the control. Compared

344

with control, the cell viability reduced by 28.9-64.8% in the nanocolloid treated

345

groups. The nanotoxicological mechanisms probably linked to cell wall or membrane

346

damage by nanomaterial sharp edges and the envelopment effects.32,69 Moreover, the

347

cell wall or membrane damage affected algal photosynthesis and biosynthesis of

348

chlorophyll.70 Nanomaterials (e.g., graphene-titanium oxide composite) as

349

photocatalysts worked well to kill the bacteria under solar light irradiation.34 The

350

photocatalytic effects of nanocolloids on algal cells were studied as shown in Figure 16

ACS Paragon Plus Environment

Environmental Science & Technology

351

S11. The percentage of the surviving algal cells was more than 99% under both dark

352

and solar light irradiation conditions, which suggested that the photocatalytic effects

353

of nanocolloids was not obvious under solar light irradiation.

354

Genotoxicity of nanoparticles is a vital mechanism of nanotoxicology. Previous

355

reports suggested that graphene-based nanomaterials induced genetic damage (e.g.,

356

DNA, chromosome damage and RNA efflux) even at low concentration 0.1

357

mg/L.69,71,72 In the present work, the genotoxicity induced by nanocolloids was

358

measured by monitoring total RNA content and DNA fragmentations of algal cells. As

359

shown in Figure S12, there was no significant difference of the total RNA (p > 0.05)

360

between control and treated groups. However, the DNA fragmentation significantly (p

361

< 0.05) increased with the concentration increase of exposed nanocolloids in Figure

362

S13. A very high DNA fragmentation (58.9 ± 1.6 %) was observed for the nanocolloid

363

exposure at the high concentration of 36.0 mg/L.

364

Metabolic Disturbance Contribution to the Phytotoxicity

365

Approximately 160−190 peaks in each sample were analyzed using GC−MS with a

366

derivatization preparation, and 47 metabolites were identified. The relative levels of

367

the metabolites in the control and nanocolloid-exposed groups are presented using

368

heat maps, and the samples were divided into two clusters by using an HCL analysis:

369

the control and nanocolloid-exposed clusters (Figure 5a). The nanocolloid-exposed

370

cluster was divided into two sub-clusters, nanocolloids 0.72/nanocolloids 7.2 and

371

nanocolloids 36.0, by PCA (Figure S14). As shown in Figure S15, the 47 identified

372

metabolites included amino acids, carbohydrates, fatty acids, small molecule acids, 17

ACS Paragon Plus Environment

Page 18 of 42

Page 19 of 42

Environmental Science & Technology

373

alcohols, alkanes, urea and lipids. Moreover, ANOVA with Tukey’s test suggested that

374

the levels of the amino acids and fatty acids were significantly different in the control

375

and exposure groups.

376

Furthermore, changes in the metabolic pathways provided new insights into the

377

biological responses to nanocolloids. As shown in Figure 5b, the glycine, serine,

378

threonine, alanine, aspartate and glutamate metabolisms were downregulated. The cell

379

division inhibition was proposed to be associated with the amino acids and other

380

nitrogen-containing compounds.59,73 Plasmolysis was one of the most remarkable

381

phenomena induced by the nanomaterials. The nanocolloids reduced the levels of

382

alanine, aspartate and glutamate in the algal cells. The downregulation of alanine,

383

aspartate and glutamate reduced the intracellular protein content, which can lead to a

384

decrease in the osmotic pressure in the cytoplasm.74 The plasma membrane shrinkage

385

is associated with a low osmotic pressure.75 As shown in Figure 5c, after nanocolloid

386

exposure, the fatty acid, arginine, proline and inositol phosphate metabolisms were

387

upregulated. Furthermore, OPLS-DA modeling was conducted to explore the

388

relationships between the biological endpoints (such as ROS and chlorophyll a

389

content) and the metabolic disturbance. The positive coefficient CS (CoeffCS) and the

390

VIP (variable importance in the projection) were calculated. As shown in Figure S16,

391

23 of the 47 metabolites exhibited a positive CoeffCS, which indicated that these

392

metabolites had significant positive contributions to the ROS levels. While the

393

remaining 24 metabolites had negative contributions. The metabolites with VIP

394

values >1 (e.g., palmitic acid, butanedioic acid and ethanol) (Figure S17) are labeled 18

ACS Paragon Plus Environment

Environmental Science & Technology

395

with a red “*” and suggested significant positive contributions to the ROS levels. For

396

example, the upregulation of ethanol, which plays a regulatory role in aerobic and

397

anaerobic metabolism, is related to ROS generation in mitochondria complexes I and

398

III and results in cytotoxicity.76,77 The relationships between the chlorophyll a content

399

and the metabolic disturbance were shown in Figure S18 and S19. The metabolites

400

(e.g., palmitic acid, stearic acid and 1,3-propanediol) with VIP values >1 had

401

significant positive contributions to the decrease in chlorophyll a. The upregulation of

402

palmitic acid and stearic acid results in damage to the cell plasma membranes and

403

inhibition of the electron transport in photosynthesis.78,79 Moreover, the increase in

404

palmitic acid and stearic acid may damage the chloroplast by disintegrating

405

phycobilin from the thylakoid membrane.78 Furthermore, the specificity of

406

metabolites correlated to ROS formation or chlorophyll a synthesis was analyzed. The

407

metabolites with a VIP value rank of top ten contributed to ROS formation or

408

chlorophyll a synthesis were presented in Figure S20. L-5-oxoproline and lactic acid

409

were the certain metabolites correlated to the ROS formation and the decrease in

410

chlorophyll a, respectively. L-5-oxoproline was a sign of increased glutathione

411

turnover in response to oxidative stress.80 Lactic acid affected xylose metabolism and

412

pentose phosphate metabolism, which generated nicotinamide adenine dinucleotide

413

phosphate (NADPH) and pentose phosphates.81,82 NADPH and pentose phosphates

414

played a vital role in synthesis of chlorophyll a.83 The above results suggest that the

415

metabolic analysis can provide new insights into the toxicological mechanisms of

416

nanocolloids. 19

ACS Paragon Plus Environment

Page 20 of 42

Page 21 of 42

Environmental Science & Technology

417

Environmental Implications

418

The complexity, heterogeneity and high concentrations of colloids in aquatic

419

environments, especially nanocolloids with a high activity, may create potential

420

threats to human health and ecological environments. However, information on the

421

characterization and environmental implications of nanocolloids is largely unavailable.

422

In this study, we used TFU with other techniques (e.g., TEM-EDS, AFM, FT-IR,

423

GC-MS and ICP-OES) to fractionate and characterize nanocolloids from surface

424

waters. Changing pH, ionic strength, and other compositions of blue-green culture

425

medium will affect the growth of C. vulgaris. In the reveal contaminated environment,

426

the characteristics like pH, ionic strength, and presence of other size fractions of

427

nanocolloids would affect nanotoxicity.84 The various PAHs and heavy metals in

428

nanocolloids also present potential risks and deserve attention in water treatment and

429

ecological risk evaluations. The toxicological study indicated that the nanocolloids

430

enveloped and entered cells, inhibited cell division, and induced oxidative stress and

431

cell ultrastructure damage. The metabolomics analysis screened the specific

432

metabolites and metabolic pathways that contribute to the above adverse effects.

433

Nanocolloids deserve more attention due to their high exposure concentrations and

434

observable toxicity, e.g., the effects of natural organic matter and light irradiation on

435

the environmental behavior and risks of nanocolloids. The quantitative method to

436

track and quantify the distribution of nanocolloids in biological matrices is

437

unavailable due to the complex compositions of nanocolloids. Thus, the toxic effects

438

of nanocolloids on microcosm assays and higher order test organism (such as mass 20

ACS Paragon Plus Environment

Environmental Science & Technology

439

transfer by food chains) are failed to realize. In future work, more attention should be

440

paid to quantitative analysis and field scenarios (e.g., environmental behavior,

441

seasonal variation, microcosm assays and higher order organism toxic test) for

442

nanocolloids.

443 444

ASSOCIATED CONTENT

445

Supporting Information Available

446

Tables S1- S5, and Figures S1-S20 for sampling locations, schematics of the

447

tangential-flow ultrafiltration, BG-11 medium, nanocolloid characterizations,

448

cytotoxicity and metabolic analysis.

449 450

AUTHOR INFORMATION

451

Correspondence author

452

*E-mail: [email protected] (Q.Z.). Phone: +86-022-23507800. Fax:

453

+86-022-66229562.

454

NOTES

455

The authors declare no competing financial interest.

456 457

ACKNOWLEDGEMENTS

458

This work was financially supported by the National Natural Science Foundation of

459

China (grant nos. 21722703, 31770550 and 21577070), the Ministry of Education

460

(People’s Republic of China) as an innovative team rolling project (grant no. 21

ACS Paragon Plus Environment

Page 22 of 42

Page 23 of 42

Environmental Science & Technology

461

IRT_17R58), a 111 program (grant no. T2017002), and special funds for basic

462

scientific research services of central colleges and universities.

463 464

REFERENCES

465

(1) Zhou, Z.; Stolpe, B.; Guo, L.; Shiller, A. M. Colloidal size spectra, composition

466

and estuarine mixing behavior of DOM in river and estuarine waters of the northern

467

Gulf of Mexico. Geochim. Cosmochim. Ac. 2016, 181, 1-17.

468

(2) Avilov. S.; Lamon, L.; Hristozov, D.; Marcomini, A. Improving the prediction of

469

environmental fate of engineered nanomaterials by fractal modelling. Environ. Int.

470

2017, 99, 78-86.

471

(3) Patchin, E. S.; Anderson, D. S.; Silva, R. M.; Uyeminami, D. L.; Scott, G. M.;

472

Guo, T.; Van Winkle, L. S.; Pinkerton, K. E. Size-dependent deposition, translocation,

473

and microglial activation of inhaled silver nanoparticles in the rodent nose and brain.

474

Environ. Health Persp. 2016, 124 (12), 1870-1875.

475

(4) Gosens, I.; Cassee, F. R.; Zanella, M.; Manodori, L.; Brunelli, A.; Costa, A. L.;

476

Bokkers, B. G.; de Jong, W. H.; Brown, D.; Hristozov, D. Organ burden and

477

pulmonary toxicity of nano-sized copper (II) oxide particles after short-term

478

inhalation exposure. Nanotoxicology 2016, 10 (8), 1084-1095.

479

(5) Peijnenburg, W. J. G. M.; Baalousha, M.; Chen, J.; Chaudry, Q.; Kammer, F. v. d.;

480

Kuhlbusch, T. A. J.; Nickel, C.; Quik, J. T. K.; Renkerg, M.; Koelmans, A. A. A

481

review of the properties and processes determining the fate of engineered

22

ACS Paragon Plus Environment

Environmental Science & Technology

482

nanomaterials in the aquatic environment. Crit. Rev. Env. Sci. Tec. 2015, 45 (19),

483

2084-2134.

484

(6) Farré, M.; Sanchís, J.; Barceló, D. Analysis and assessment of the occurrence, the

485

fate and the behavior of nanomaterials in the environment. Trends Anal. Chem. 2011,

486

30 (3), 517-527.

487

(7) Morrison, M. A.; Benoit, G. Investigation of conventional membrane and

488

tangential flow ultrafiltration artifacts and their application to the characterization of

489

freshwater colloids. Environ. Sci. Technol. 2004, 38 (24), 6817-6823.

490

(8) Trefry, J. C.; Monahan, J. L.; Weaver, K. M.; Meyerhoefer, A. J.; Markopolous,

491

M. M.; Arnold, Z. S.; Wooley, D. P.; Pavel, I. E. Size selection and concentration of

492

silver nanoparticles by tangential flow ultrafiltration for SERS-based biosensors. J.

493

Am. Chem. Soc. 2010, 132 (32), 10970-10972.

494

(9) Pansare, V. J.; Tien, D.; Thoniyot, P.; Prud’homme, R. K. Ultrafiltration of

495

nanoparticle colloids. J. Membrane Sci. 2017, 538 (Supplement C), 41-49.

496

(10) Zhi, L.; Qu, M.; Ren, M.; Zhao, L.; Li, Y.; Wang D. Graphene oxide induces

497

canonical Wnt/β-catenin signaling-dependent toxicity in Caenorhabditis elegans.

498

Carbon 2017, 113, 122-131.

499

(11) Zhao, Y.; Wu, Q.; Wang, D. An epigenetic signal encoded protection mechanism

500

is activated by graphene oxide to inhibit its induced reproductive toxicity in

501

Caenorhabditis elegans. Biomaterials 2016, 79, 15-24.

502

(12) Jeannet, N.; Fierz, M.; Schneider, S.; Künzi, L.; Baumlin, N.; Salathe, M.;

503

Burtscher, H.; Geiser, M. Acute toxicity of silver and carbon nanoaerosols to normal 23

ACS Paragon Plus Environment

Page 24 of 42

Page 25 of 42

Environmental Science & Technology

504

and cystic fibrosis human bronchial epithelial cells. Nanotoxicology 2016, 10 (3),

505

279-291.

506

(13) Wilke, C. M.; Tong, T.; Gaillard, J.-F.; Gray, K. A. Attenuation of microbial

507

stress due to nano-Ag and nano-TiO2 interactions under dark conditions. Environ. Sci.

508

Technol. 2016, 50 (20), 11302-11310.

509

(14) Papageorgiou, I.; Brown, C.; Schins, R.; Singh, S.; Newson, R.; Davis, S.; Fisher,

510

J.; Ingham, E.; Case, C. P. The effect of nano- and micron-sized particles of cobalt–

511

chromium alloy on human fibroblasts in vitro. Biomaterials 2007, 28 (19),

512

2946-2958.

513

(15) Gunawan, C.; Teoh, W. Y.; Marquis, C. P.; Amal, R. Cytotoxic origin of

514

copper(II) oxide nanoparticles: comparative studies with micron-sized particles,

515

leachate, and metal salts. ACS Nano 2011, 5 (9), 7214-7225.

516

(16) Wagner, S.; Gondikas, A.; Neubauer, E.; Hofmann, T.; von der Kammer, F. Spot

517

the difference: Engineered and natural nanoparticles in the environment-release,

518

behavior, and fate. Angew. Chem. Int. Edit. 2014, 53 (46), 12398-12419.

519

(17) Petosa, A. R.; Jaisi, D. P.; Quevedo, I. R.; Elimelech, M.; Tufenkji, N.

520

Aggregation and deposition of engineered nanomaterials in aquatic environments:

521

role of physicochemical interactions. Environ. Sci. Technol. 2010, 44 (17),

522

6532-6549.

523

(18) Bakshi, S.; He, Z. L.; Harris, W. G. Natural nanoparticles: Implications for

524

environment and human health. Crit. Rev. Env. Sci. Tec. 2015, 45 (8), 861-904.

525

(19) Moore, M. N. Do nanoparticles present ecotoxicological risks for the health of 24

ACS Paragon Plus Environment

Environmental Science & Technology

526

the aquatic environment? Environ. Int. 2006, 32 (8), 967-976.

527

(20) Gomes, T.; Xie, L.; Brede, D.; Lind, O.-C.; Solhaug, K. A.; Salbu, B.; Tollefsen,

528

K. E. Sensitivity of the green algae chlamydomonas reinhardtii to gamma radiation:

529

Photosynthetic performance and ROS formation. Aquat. Toxicol. 2017, 183, 1-10.

530

(21) Dauda, S.; Chia, M. A.; Bako, S. P. Toxicity of titanium dioxide nanoparticles to

531

Chlorella vulgaris Beyerinck (Beijerinck) 1890 (Trebouxiophyceae, Chlorophyta)

532

under changing nitrogen conditions. Aquat Toxicol. 2017, 187, 108-114.

533

(22) Liu, Y.; Wang, Z.; Yan, K.; Wang, Z.; Torres, O. L.; Guo, R.; Chen, J. A new

534

disposal method for systematically processing of ceftazidime: the intimate coupling

535

UV/algae-algae treatment. Chem. Eng. J. 2017, 314, 152-159.

536

(23) Ahmad, F.; Yao, H.; Zhou, Y.; Liu, X. Toxicity of cobalt ferrite (CoFe2O4)

537

nanobeads in Chlorella vulgaris: interaction, adaptation and oxidative stress.

538

Chemosphere 2015, 139, 479-485.

539

(24) Qian, X.; Liang, B.; Liu, X.; Liu, X.; Wang, J.; Liu, F.; Cui, B. Distribution,

540

sources, and ecological risk assessment of polycyclic aromatic hydrocarbons in

541

surface sediments from the Haihe River, a typical polluted urban river in Northern

542

China. Environ. Sci. Pollut. R. 2017, 24 (20), 17153-17165.

543

(25) Cao, Z.; Wang, Y.; Ma, Y.; Xu, Z.; Shi, G.; Zhuang, Y.; Zhu, T. Occurrence and

544

distribution of polycyclic aromatic hydrocarbons in reclaimed water and surface water

545

of Tianjin, China. J. Hazard. Mater. 2005, 122 (1), 51-59.

546

(26) Hernandez, L. M.; Yousefi, N.; Tufenkji, N. Are there nanoplastics in your

547

personal care products? Environ. Sci. Technol. Let. 2017, 4 (7), 280-285. 25

ACS Paragon Plus Environment

Page 26 of 42

Page 27 of 42

Environmental Science & Technology

548

(27) Chen, Y.; Ren, C.; Ouyang, S.; Hu, X.; Zhou, Q. Mitigation in multiple effects of

549

graphene oxide toxicity in zebrafish embryogenesis driven by humic acid. Environ.

550

Sci. Technol. 2015, 49 (16), 10147-10154.

551

(28) Cam, N.; Benzerara, K.; Georgelin, T.; Jaber, M.; Lambert, J.-F.; Poinsot, M.;

552

Skouri-Panet, F.; Cordier, L. Selective uptake of alkaline earth metals by

553

Cyanobacteria forming intracellular carbonates. Environ. Sci. Technol. 2016, 50 (21),

554

11654-11662.

555

(29) Schwab, F.; Bucheli, T. D.; Camenzuli, L.; Magrez, A.; Knauer, K.; Sigg, L.;

556

Nowack, B. Diuron sorbed to carbon nanotubes exhibits enhanced toxicity to

557

chlorella vulgaris. Environ. Sci. Technol. 2013, 47 (13), 7012-7019.

558

(30) Tatsi, K.; Turner, A.; Handy, R. D.; Shaw, B. J. The acute toxicity of thallium to

559

freshwater organisms: implications for risk assessment. Sci. Total Environ. 2015, 536

560

(Supplement C), 382-390.

561

(31) Deng, X.-Y.; Gao, K.; Zhang, R.-C.; Addy, M.; Lu, Q.; Ren, H.-Y.; Chen, P.; Liu,

562

Y.-H.; Ruan, R. Growing Chlorella vulgaris on thermophilic anaerobic digestion

563

swine manure for nutrient removal and biomass production. Bioresour. Technol. 2017,

564

243 (Supplement C), 417-425.

565

(32) Ouyang, S.; Hu, X.; Zhou, Q. Envelopment-internalization synergistic effects and

566

metabolic mechanisms of graphene oxide on single-cell Chlorella vulgaris are

567

dependent on the nanomaterial particle size. ACS Appl. Mater. Inter. 2015, 7 (32),

568

18104-18112.

26

ACS Paragon Plus Environment

Environmental Science & Technology

569

(33) Juganson, K; Mortimer, M.; Ivask, A.; Pucciarelli, S.; Miceli, C.; Orupõld, K.;

570

Kahru, K. Mechanisms of toxic action of silver nanoparticles in the protozoan

571

Tetrahymena thermophila: from gene expression to phenotypic events. Environ.

572

Pollut. 2017, 225, 481-489.

573

(34) Akhavan, O.; Ghaderi, E.; Rahimi, K. Adverse effects of graphene incorporated

574

in TiO2 photocatalyst on minuscule animals under solar light irradiation. J. Mater.

575

Chem. 2012, 22 (43), 23260-23266.

576

(35)Stolpe, B.; Guo, L.; Shiller, A. M.; Aiken, G. R. Abundance, size distributions

577

and trace-element binding of organic and iron-rich nanocolloids in Alaskan rivers, as

578

revealed by field-flow fractionation and ICP-MS. Geochim. Cosmochim. Ac. 2013,

579

105, 221-239.

580

(36) Stolpe, B.; Guo, L.; Shiller, A. M.; Hassellöv, M. Size and composition of

581

colloidal organic matter and trace elements in the Mississippi River, Pearl River and

582

the northern Gulf of Mexico, as characterized by flow field-flow fractionation. Mar.

583

Chem. 2010, 118 (3), 119-128.

584

(37) Philippe, A.; Schaumann, G. E. Interactions of dissolved organic matter with

585

natural and engineered inorganic colloids: a review. Environ. Sci. Technol. 2014, 48

586

(16), 8946-8962.

587

(38) Petosa, A. R.; Jaisi, D. P.; Quevedo, I. R.; Elimelech, M.; Tufenkji, N.

588

Aggregation and deposition of engineered nanomaterials in aquatic environments:

589

Role of physicochemical interactions. Environ. Sci. Technol. 2010, 44 (17),

590

6532-6549. 27

ACS Paragon Plus Environment

Page 28 of 42

Page 29 of 42

Environmental Science & Technology

591

(39) Meesters, J. A. J.; Quik, J. T. K.; Koelmans, A. A.; Hendriks, A. J.; van de Meent,

592

D. Multimedia environmental fate and speciation of engineered nanoparticles: A

593

probabilistic modeling approach. Envion. Sci. Nano 2016, 3 (4), 715-727.

594

(40) Hartland, A.; Fairchild, I. J.; Lead, J. R.; Zhang, H.; Baalousha, M. Size,

595

speciation and lability of NOM–metal complexes in hyperalkaline cave dripwater.

596

Geochim. Cosmochim. Ac. 2011, 75 (23), 7533-7551.

597

(41) Stępień, M.; Gońka, E.; Żyła, M.; Sprutta, N. Heterocyclic nanographenes and

598

other polycyclic heteroaromatic compounds: synthetic routes, properties, and

599

applications. Chem. Rev. 2017, 117 (4), 3479-3716.

600

(42) Jacquin, C.; Lesage, G.; Traber, J.; Pronk, W.; Heran, M. Three-dimensional

601

excitation and emission matrix fluorescence (3DEEM) for quick and

602

pseudo-quantitative determination of protein- and humic-like substances in full-scale

603

membrane bioreactor (MBR). Water Res. 2017, 118 (Supplement C), 82-92.

604

(43) Jasim, D. A.; Ménardmoyon, C.; Bégin, D.; Bianco, A.; Kostarelos, K. Tissue

605

distribution and urinary excretion of intravenously administered chemically

606

functionalized graphene oxide sheets. Chem. Sci. 2015, 6(7), 3952- 3964.

607

(44) Yang, H.; Yang, S.; Kong, J.; Dong, A.; Yu, S. Obtaining information about

608

protein secondary structures in aqueous solution using Fourier transform IR

609

spectroscopy. Nat. Protoc. 2015, 10 (3), 382.

610

(45) Wang, L.; Ge, L.; Rufford, T. E.; Chen, J.; Zhou, W.; Zhu, Z.; Rudolph V. A

611

comparison study of catalytic oxidation and acid oxidation to prepare carbon

612

nanotubes for filling with Ru nanoparticles. 2011, Carbon 49(6), 2022-2032. 28

ACS Paragon Plus Environment

Environmental Science & Technology

613

(46) Laborda, F.; Bolea, E.; Jiménez-Lamana, J. Single particle inductively coupled

614

plasma mass spectrometry for the analysis of inorganic engineered nanoparticles in

615

environmental samples. TrEAC-Trends Environm. Analy. Chem. 2016, 9, 15-23.

616

(47) Li, L.; Stoiber, M.; Wimmer, A.; Xu, Z.; Lindenblatt, C.; Helmreich, B.; Schuster,

617

M. To what extent can full-scale wastewater treatment plant effluent influence the

618

occurrence of silver-based nanoparticles in surface waters? Environ. Sci. Technol.

619

2016, 50 (12), 6327-6333.

620

(48) Schwab, F.; Bucheli, T. D.; Lukhele, L. P.; Magrez, A.; Nowack, B.; Sigg, L.;

621

Knauer, K. Are carbon nanotube effects on green algae caused by shading and

622

agglomeration? Environ. Sci. Technol. 2011, 45 (14), 6136-6144.

623

(49) Hu, X.; Gao, Y.; Fang, Z. Integrating metabolic analysis with biological

624

endpoints provides insight into nanotoxicological mechanisms of graphene oxide:

625

from effect onset to cessation. 2016, Carbon 109, 65-73.

626

(50) Hashemi, E.; Akhavan, O.; Shamsara, M.; Rahighi, R.; Esfandiar, A.; Tayefeh, A.

627

R. Cyto and genotoxicities of graphene oxide and reduced graphene oxide sheets on

628

spermatozoa. RSC Advances 2014, 4 (52), 27213-27223.

629

(51) Akhavan, O.; Ghaderi, E.; Esfandiar, A. Wrapping bacteria by graphene

630

nanosheets for isolation from environment, reactivation by sonication, and

631

inactivation by near-infrared irradiation. J. Phys. Chem. B 2011, 115 (19), 6279-6288.

632

(52) Mendes, R. G.; Mandarino, A.; Koch, B.; Meyer, A. K.; Bachmatiuk, A.; Hirsch,

633

C.; Gemming, T.; Schmidt, O. G.; Liu, Z.; Rümmeli, M. H. Size and time dependent

634

internalization of label-free nano-graphene oxide in human macrophages. Nano 29

ACS Paragon Plus Environment

Page 30 of 42

Page 31 of 42

Environmental Science & Technology

635

Research 2017, 10 (6), 1980-1995.

636

(53) Wang, Z.; Li, J.; Zhao, J.; Xing, B. Toxicity and internalization of CuO

637

nanoparticles to prokaryotic alga Microcystis aeruginosa as affected by dissolved

638

organic matter. Environ. Sci. Technol. 2011, 45 (14), 6032-6040.

639

(54) Rizwan, M.; Ali, S.; Qayyum, M. F.; Ok, Y. S.; Adrees, M.; Ibrahim, M.;

640

Zia-Ur-Rehman, M.; Farid, M.; Abbas, F. Effect of metal and metal oxide

641

nanoparticles on growth and physiology of globally important food crops: a critical

642

review. J. Hazard. Mater. 2017, 322, 2-16.

643

(55) Domozych, D. S.; Sørensen, I.; Sacks, C.; Brechka, H.; Andreas, A.; Fangel, J.

644

U.; Rose, J. K. C.; Willats, W. G. T.; Popper, Z. A. Disruption of the microtubule

645

network alters cellulose deposition and causes major changes in pectin distribution in

646

the cell wall of the green alga, Penium margaritaceum. J. Exp. Bot. 2014, 65 (2),

647

465-479.

648

(56) Xing, K.; Liu, Y.; Shen, X.; Zhu, X.; Li, X.; Miao, X.; Feng, Z.; Peng, X.; Qin, S.

649

Effect of O-chitosan nanoparticles on the development and membrane permeability of

650

verticillium dahliae. Carbohyd. Polym. 2017, 165 (Supplement C), 334-343.

651

(57) Perreault, F.; Bogdan, N.; Morin, M.; Claverie, J.; Popovic, R. Interaction of gold

652

nanoglycodendrimers with algal cells (chlamydomonas reinhardtii) and their effect on

653

physiological processes. Nanotoxicology 2012, 6 (2), 109-120.

654

(58) Taylor, C.; Matzke, M.; Kroll, A.; Read, D. S.; Svendsen, C.; Crossley, A. Toxic

655

interactions of different silver forms with freshwater green algae and cyanobacteria

30

ACS Paragon Plus Environment

Environmental Science & Technology

656

and their effects on mechanistic endpoints and the production of extracellular

657

polymeric substances. Environ. Sci. Nano 2016, 3(2), 396-408.

658

(59) Yue, Y.; Li, X.; Sigg, L.; Suter, M. J.; Pillai, S.; Behra, R.; Schirmer, K.

659

Interaction of silver nanoparticles with algae and fish cells: A side by side comparison.

660

J. Nanobiotecg. 2017, 15 (1), 16.

661

(60) Hu, X.; Ouyang, S.; Mu, L.; An, J.; Zhou, Q. Effects of graphene oxide and

662

oxidized carbon nanotubes on the cellular division, microstructure, uptake, oxidative

663

stress, and metabolic profiles. Environ. Sci. Technol. 2015, 49 (18), 10825-10833.

664

(61) Chen, R.; Zhao, L.; Bai, R.; Liu, Y.; Han, L.; Xu, Z., Chen, F.; Autrup H.; Long

665

D.; Chen C. Silver nanoparticles induced oxidative and endoplasmic reticulum

666

stresses in mouse tissues: implications for the development of acute toxicity after

667

intravenous administration. Toxicol. Res. 2016, 5(2), 602-608.

668

(62) Dimkpa, C.; White, J. C.; Elmer, W. H.; Gardea-Torresdey, J. L. Nanoparticle

669

and ionic zn promote nutrient loading of sorghum grain under low npk fertilization. J.

670

Agric. Food Chem. 2017, 65(39), 8552-8559.

671

(63) Barrios, A. C.; Rico, C. M.; Trujilloreyes, J.; Medinavelo, I. A.; Peraltavidea, J.

672

R.; Gardea-Torresdey, J. L. Effects of uncoated and citric acid coated cerium oxide

673

nanoparticles, bulk cerium oxide, cerium acetate, and citric acid on tomato plants. Sci

674

Total Environ. 2016, 563-564, 956-964.

675

(64)Huerta-García, E.; Pérez-Arizti, J. A.; Márquez-Ramírez, S. G.;

676

Delgado-Buenrostro, N. L.; Chirino, Y. I.; Iglesias, G. G.; López-Marure, R. Titanium

677

dioxide nanoparticles induce strong oxidative stress and mitochondrial damage in 31

ACS Paragon Plus Environment

Page 32 of 42

Page 33 of 42

Environmental Science & Technology

678

glial cells. Free Radical Bio. Med. 2014, 73, 84-94.

679

(65) Singh, R. P.; Ramarao, P. Cellular uptake, intracellular trafficking and

680

cytotoxicity of silver nanoparticles. Toxicology Letters 2012, 213 (2), 249-259.

681

(66) Perreault, F.; Popovic, R.; Dewez, D. Different toxicity mechanisms between

682

bare and polymer-coated copper oxide nanoparticles in Lemna gibba. Environ. Pollut.

683

2014, 185, 219-227.

684

(67) Caballero-Díaz, E.; Pfeiffer, C.; Kastl, L.; Rivera-Gil, P.; Simonet, B.; Valcárcel,

685

M.; Jiménez-Lamana, J.; Laborda, F.; Parak, W. J. The toxicity of silver nanoparticles

686

depends on their uptake by cells and thus on their surface chemistry. Part. Part. Syst.

687

Char. 2013, 30 (12), 1079-1085.

688

(68) Subashchandrabose, S. R.; Wang, L.; Venkateswarlu, K.; Naidu, R.; Megharaj,

689

M. Interactive effects of PAHs and heavy metal mixtures on oxidative stress in

690

Chlorella sp. MM3 as determined by artificial neural network and genetic algorithm.

691

Algal Research 2017, 21, 203-212.

692

(69) Akhavan, O.; Ghaderi, E. Toxicity of graphene and graphene oxide nanowalls

693

against bacteria. ACS Nano 2010, 4 (10), 5731-5736.

694

(70) Tao, X.; Yu, Y.; Fortner, J. D.; He, Y.; Chen, Y.; Hughes, J. B. Effects of

695

aqueous stable fullerene nanocrystal (C60) on Scenedesmus obliquus: Evaluation of

696

the sub-lethal photosynthetic responses and inhibition mechanism. Chemosphere 2015,

697

122, 162-167.

698

(71) Akhavan, O.; Ghaderi, E.; Akhavan, A. Size-dependent genotoxicity of graphene

699

nanoplatelets in human stem cells. Biomaterials 2012, 33 (32), 8017-8025. 32

ACS Paragon Plus Environment

Environmental Science & Technology

700

(72) Akhavan, O.; Ghaderi, E.; Emamy, H.; Akhavan, F. Genotoxicity of graphene

701

nanoribbons in human mesenchymal stem cells. Carbon 2013, 54, 419-431.

702

(73) Foflonker, F.; Price, D. C.; Qiu, H.; Palenik, B.; Wang, S.; Bhattacharya, D.

703

Genome of the halotolerant green alga picochlorum sp. reveals strategies for thriving

704

under fluctuating environmental conditions. Environ. Microbiol. 2015, 17 (2),

705

412-426.

706

(74) Miller, M. S.; Lay, W. K.; Elcock, A. H. Osmotic pressure simulations of amino

707

acids and peptides highlight potential routes to protein force field parameterization. J.

708

Phys. Chem. B 2016, 120 (33), 8217-8229.

709

(75) Zhang, Y.; Jiang, W.-L.; Xu, R.-X.; Wang, G.-X.; Xie, B. Effect of short-term

710

salinity shock on unacclimated activated sludge with pressurized aeration in a

711

sequencing batch reactor. Sep. Sci. Technol. 2017, 178, 200-206.

712

(76) Wen, Z.; Liu, Z.; Hou, Y.; Liu, C.; Gao, F.; Zheng, Y.; Chen, F. Ethanol induced

713

astaxanthin accumulation and transcriptional expression of carotenogenic genes in

714

haematococcus pluvialis. Enzyme Microb. Technol. 2015, 78, 10-17.

715

(77) Bailey, S. M.; Pietsch, E. C.; Cunningham, C. C. Ethanol stimulates the

716

production of reactive oxygen species at mitochondrial complexes I and III. Free

717

Radical Bio. Med. 1999, 27 (7), 891-900.

718

(78) Wu, J.-T.; Chiang, Y.-R.; Huang, W.-Y.; Jane, W.-N. Cytotoxic effects of free

719

fatty acids on phytoplankton algae and cyanobacteria. Aquat. Toxicol. 2006, 80 (4),

720

338-345.

33

ACS Paragon Plus Environment

Page 34 of 42

Page 35 of 42

Environmental Science & Technology

721

(79) Venediktov, P. S.; Krivoshejeva, A. A. The mechanisms of fatty-acid inhibition

722

of electron transport in chloroplasts. Planta 1983, 159 (5), 411-414.

723

(80) Emmett, M. Acetaminophen toxicity and 5-oxoproline (pyroglutamic acid): a tale

724

of two cycles, one an ATP-depleting futile cycle and the other a useful cycle. Clin. J

725

Am. Soc. Nephrol. 2014, 9 (1), 191-200.

726

(81) Ma, K.; Hu, G.; Pan, L.; Wang, Z.; Zhou, Y.; Wang, Y.; Ruan, Z.; He, M. Highly

727

efficient production of optically pure l-lactic acid from corn stover hydrolysate by

728

thermophilic Bacillus coagulans. Bioresource Technol. 2016, 219, 114-122.

729

(82)Wasylenko, T. M.; Ahn, W. S.; Stephanopoulos, G. The oxidative pentose

730

phosphate pathway is the primary source of NADPH for lipid overproduction from

731

glucose in Yarrowia lipolytica. Metab. Eng. 2015, 30, 27-39.

732

(83) Beale, S. I. Green genes gleaned. Trends Plant Sci. 2005, 10 (7), 309-312.

733

(84) Ma, S.; Zhou, K.; Yang, K.; Lin, D. Heteroagglomeration of oxide nanoparticles

734

with algal cells: effects of particle type, ionic strength and pH. Environ. Sci. Technol.

735

2015, 49 (2), 932-939.

736 737 738 739 740 741 742 34

ACS Paragon Plus Environment

Environmental Science & Technology

743

Figure Captions

744

Figure 1. Characterization of the nanocolloids from the upstream (R2), midstream (R4)

745

and downstream (R9) of the Haihe River in Tianjin City, China. a, Representative

746

TEM images (n=20); b, nanocolloid size analysis from the TEM images (n=20); c,

747

EDS spectrum of the nanocolloids.

748 749

Figure 2. Optical properties and composition analysis of the nanocolloids. a,

750

Fluorescence spectra of the nanocolloids in samples R2, R4 and R9; b, FT-IR spectra

751

of the nanocolloids in samples R2, R4 and R9; c, average concentrations of TC, TN

752

and P; d, average concentrations of the metals measured by ICP-OES; f, relative

753

abundances of TC, TN and P, Si, O, metals and other unidentified components in the

754

nanocolloids.

755 756

Figure 3. Nanocolloid envelopment of algal cells at 96 h. SEM images of the algal

757

cells in the absence (a) and presence of 36.0 mg/L of the nanocolloids (b).

758

Fluorescence microscopy of algal cells in the absence (c) or presence of 36.0 mg/L of

759

the nanocolloids (d). The blue, black and red arrows indicate the envelopment

760

phenomenon of the nanocolloids.

761 762

Figure 4. Adverse effects in the algal cells exposed to nanocolloids at 96 h. a–f,

763

Damage to the cellular ultrastructure by the nanocolloids at 96 h. TEM images (n=20)

764

of the cells: a-b, control without nanocolloid exposure; c-d, 36.0 mg/L nanocolloid 35

ACS Paragon Plus Environment

Page 36 of 42

Page 37 of 42

Environmental Science & Technology

765

exposure; Cw, cell wall; Pm, plasma membrane; S, starch grain; Chl, chloroplast; Pc,

766

pyrenoid center. The black and red “*” denote significant differences at p < 0.05 (n=3)

767

for the control and nanocolloid groups, respectively. The green arrows denote the

768

uptake of nanocolloids. The red and blue arrows denote the increases in the starch

769

grains and cell wall thickness, respectively. The double black arrows denote

770

plasmolysis; e, EDS spectrum of nanocolloid uptake by the algal cells; f, statistical

771

analysis of the TEM images (n=20); g, cell division after nanocolloid exposure for 24,

772

48, 72 and 96 h; h, relative ROS levels; i, concentrations of chlorophyll a.

773 774

Figure 5. Metabolic analysis of the control and nanocolloid-exposed groups at 96 h. a,

775

Heat map of all the identified metabolites; b, downregulated metabolic pathways after

776

nanocolloid exposure; c, upregulated metabolic pathways after nanocolloid exposure.

777

36

ACS Paragon Plus Environment

Environmental Science & Technology

778 779 780

Figure 1.

781 782

37

ACS Paragon Plus Environment

Page 38 of 42

Page 39 of 42

Environmental Science & Technology

783 784

Figure 2.

785 786 787 788 789 790 791 792 793 794 795

38

ACS Paragon Plus Environment

Environmental Science & Technology

796 797

Figure 3.

798 799 800 801 802 803 804 805

39

ACS Paragon Plus Environment

Page 40 of 42

Page 41 of 42

Environmental Science & Technology

806 807

Figure 4.

808 809

40

ACS Paragon Plus Environment

Environmental Science & Technology

810 811

Figure 5

41

ACS Paragon Plus Environment

Page 42 of 42