Mixture Toxicity of Nickel and Microplastics with Different Functional

Oct 11, 2017 - In recent years, discarded plastic has become an increasingly prevalent pollutant in aquatic ecosystems. These plastic wastes decompose...
2 downloads 7 Views 2MB Size
Subscriber access provided by LAURENTIAN UNIV

Article

Mixture toxicity of nickel and microplastics with different functional groups on Daphnia magna Dokyung Kim, Yooeun Chae, and Youn-Joo An Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03732 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

Environmental Science & Technology 1

1 2

Mixture toxicity of nickel and microplastics with different functional groups on Daphnia magna

3 4

Dokyung Kim, Yooeun Chae, and Youn-Joo An*

5 6 7

Department of Environmental Health Science, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea

8 9 10 11 12 13 14 15 16

*

Corresponding author: Youn-Joo An Tel.: +82-2-2049-6090; Fax: +82-2-2201-6295; Email: anyjoo@konkuk.ac.kr

Abbreviations and nomenclature: HOCs, hydrophobic organic chemicals; MP, microplastic; POPs, persistent organic pollutants; PS, microplastic without functional group; PS-COOH, microplastic with COOH functional group

ACS Paragon Plus Environment

Environmental Science & Technology

Page 2 of 28 2

17

ACS Paragon Plus Environment

Page 3 of 28

Environmental Science & Technology 3

18

ABSTRACT

19

In recent years, discarded plastic has become an increasingly prevalent pollutant in aquatic

20

ecosystems. These plastic wastes decompose into microplastics, which not only pose a direct

21

threat to aquatic organisms but also an indirect threat via adsorption of other aquatic

22

pollutants. In this study, we investigated the toxicities of variable and fixed combinations of

23

two types of microplastics [one coated with a carboxyl group (PS-COOH) and the other

24

lacking this functional group (PS)] with the heavy metal nickel (Ni) on Daphnia magna and

25

calculated mixture toxicity using a toxic unit model. We found that toxicity of Ni in

26

combination with either of the two microplastics differed from that of Ni alone. Furthermore,

27

in general, we observed that immobilization of D. magna exposed to Ni combined with PS-

28

COOH was higher than that of D. magna exposed to Ni combined with PS. Collectively, the

29

results of our study indicate that the toxic effects of microplastics and pollutants may vary

30

depending on the specific properties of the pollutant and microplastic functional groups, and

31

further research on the mixture toxicity of various combinations of microplastics and

32

pollutants is warranted.

33

Keywords: complex toxicity; microplastic; functional group; nickel; Daphnia magna

ACS Paragon Plus Environment

Environmental Science & Technology

Page 4 of 28 4

34

1. INTRODUCTION

35

The considerable increase in the production and consumption of plastics over the last few

36

decades has resulted in a marked increase in the disposal of plastics, and some have been

37

discarded into the aquatic environment without being recycled or treated. Discarded plastic

38

that enter aquatic ecosystems is eventually degraded into microplastics (MPs), which are

39

defined as plastics with particle sizes ≤ 5 mm.1 These plastics can be ingested by aquatic

40

organisms, including fish, shellfish, and shrimps, or can adhere to algae and plankton.2,3

41

Several previous studies have observed that MPs are taken up by aquatic organisms and

42

subsequently have adverse effects on their survival and health.3–8 Although the concentrations

43

of microplastics examined in the above-mentioned studies are not comparable with those

44

occurring in the field, the findings of these studies do suggest that microplastics have the

45

potential to cause adverse effects in organisms. Recently, the presence of MPs in oceans and

46

as pollutants in freshwater has become an issue of concern.9–15 Microplastics are flushed

47

indirectly into freshwater bodies from various sources, such as industrial plant and washing

48

machine effluents,15–18 and plastic products are directly discarded into freshwater sources.

49

Numerous studies have confirmed that MPs have spilled into aquatic systems and have

50

subsequently combine with or have adsorbed onto other pollutants such as heavy metals,

51

persistent organic pollutants (POPs), or hydrophobic organic chemicals (HOCs).19–24 Aquatic

52

organisms could ingest MPs that have adsorbed onto other pollutants, which could lead to

53

complex toxicities, and a few studies have investigated this phenomenon. For instance,

54

Besseling et al.25 observed that the transfer of polychlorinated biphenyls (PCBs) to lugworms

55

and the subsequent effects were enhanced with increasing concentrations of plastic. In

56

another study, Browne et al.26 demonstrated the transfer of endocrine-disrupting chemicals

57

(EDCs) into lugworms via adsorption on MPs. Chua et al.27 evaluated the combined toxicity

ACS Paragon Plus Environment

Page 5 of 28

Environmental Science & Technology 5

58

of polybrominated diphenyl ethers (PBDEs) and MPs on marine amphipods, whereas Batel et

59

al.28 monitored the effects of benzo[a]pyrene with MPs on nauplii. Similarly, some studies29,30

60

have determined the toxicity of fluoranthene adsorbed on MPs using marine mussels, and

61

Wardrop et al.31 evaluated the accumulation of PBDEs in rainbow fish. In recent years, four

62

mixture toxicity tests have been conducted to evaluate the effects of heavy metals (copper,

63

silver, chromium, and gold nanoparticle) combined with MPs32–35 on microalgae and marine

64

fish. Thus, several studies have verified that microplastics in aquatic ecosystems combine

65

with other pollutants and that there is a potential likelihood that aquatic organisms will

66

become exposed to these mixed substances. Therefore, further studies on toxicity using such

67

mixed substances are needed.

68

In the present study, the toxicity of mixtures of MPs with a heavy metal was assessed in

69

the water flea, Daphnia magna. We selected a heavy metal pollutant and designed the study

70

based on nickel. The characteristics of MPs vary, including their functional groups, sizes, and

71

coating agents. Therefore, the effects of MPs on aquatic organisms may depend on the

72

characteristics of the MPs themselves and may differ when combined with different

73

chemicals.36,37 To evaluate this phenomenon, we selected two plastics, one containing a

74

carboxyl functional group (PS-COOH) and the other lacking this group (PS), and compared

75

the mixture toxicities of the MPs combined with nickel. To evaluate mixture toxicities, we

76

measured the rate of immobilization of water fleas in the presence and absence of

77

microplastics and compared the amount of nickel uptake depending on the type of

78

microplastics. Because we assumed that the nickel cation would be more adsorbed onto PS-

79

COOH, which has a negatively charged functional group, than onto PS, we hypothesized that

80

Daphnia exposed to nickel with PS-COOH would ingest considerably more nickel.

81

Accordingly, we hypothesized that nickel with PS-COOH would show much higher toxicity

82

than nickel with PS.

ACS Paragon Plus Environment

Environmental Science & Technology

Page 6 of 28 6

83

84

85

2. MATERIAL AND METHODS 2.1. Test chemicals

86

Nickel was purchased from Sigma-Aldrich (St. Louis, MO, USA) as nickel chloride

87

(NiCl2, purity 98%), and two types of polystyrene MPs were purchased from Bangs

88

Laboratories, Inc. (Fishers, IN, USA). One of the plastics contained a bound carboxyl group

89

(PS-COOH), whereas the other plastic lacked this bound functional group. Fotopoulou and

90

Karapanagioti38 have demonstrated that virgin microplastics, which do not have a functional

91

group, can have a negative charge due to multiple factors in the aquatic environment. We

92

therefore selected PS-COOH with a negative charge and PS in a virgin state. We assumed that

93

Ni cations would adsorb onto the anionic carboxylate functional group of the MPs (PS-

94

COOH) to a greater extent than onto MPs lacking this functional group (PS). The average

95

diameter of PS and PS-COOH was 201.5 and 191.3 nm in deionized water (DW) and 194.0

96

and 182.7 nm in moderately hard water (MHW)39, respectively. The zeta potentials of the PS

97

in each medium were -31.9 and -31.1 mV in DW and MHW, respectively, whereas those of

98

PS-COOH were -36.8 and -28.0 mV in DW and MHW, respectively. The surface of the MPs

99

was observed using a field emission scanning electron microscope (FE-SEM; S-4300, Hitachi,

100

Japan). Scanning electron microscopy (SEM) images of the MPs are shown in Fig. 1.

101

102

2.2. Test species (D. magna)

103

Daphnia magna specimens were obtained from the National Institute of Environmental

104

Research (NIER, Incheon, Korea). They were cultured in modified MHW at a temperature of

105

21 °C under a 16-h:8-h light:dark cycle. The modified MHW contained cyanocobalamin

ACS Paragon Plus Environment

Page 7 of 28

Environmental Science & Technology 7

106

(vitamin B12; Daejung, Korea) and sodium selenite (Na2SeO3; Kanto Chemical, Japan). Once

107

daily, Daphnia feed was injected with 1 mL of green algae (Chlorella vulgaris) at a

108

concentration of 1 × 108 cells/mL. The Daphnia specimens used in all the experiments were

109

neonates less than 24 h old.

110

111

2.3. Acute toxicity test on D. magna

112

Acute toxicity tests were performed according to the Organization for Economic

113

Cooperation and Development (OECD) guidelines for testing chemicals40 for 48 h at 21 °C

114

under a 16-h:8-h light:dark cycle and without food. Glass vials (diameter 6 mm, height 75

115

mm, and volume 35 mL) were used as test vessels, each containing 10 mL of test solution and

116

five daphnids. A control group was used for all experiments. The control group was exposed

117

to uncontaminated MHW.

118

We initially conducted acute toxicity tests using nickel, PS, and PS-COOH separately.

119

For the Ni toxicity test, a 100 mg/L stock solution was diluted with MHW to concentrations

120

of 1, 2, 3, 4, and 5 mg/L. Before the toxicity tests, we dispersed the MPs by rolling the stock

121

solutions for 30 min using a roller mixer. Thereafter, 100 mg/L stock solutions were prepared

122

for each MP and diluted further with MHW to obtain the final MP working concentrations of

123

1, 5, 10, 20, and 30 mg/L. The 100 mg/L MP stock solution contained approximately 2.6 ×

124

105 particles/mL according to a certificate of analysis of the MPs obtained from Bangs

125

Laboratories (Density: 1.05-1.06 g/mL). After a 48-h exposure to the MPs, test organism

126

abnormalities, including immobilization and changes in morphology, were recorded. To

127

calculate the half-maximal effective concentration (EC50) of the MPs, we conducted

128

additional tests with PS using higher concentrations (20, 40, 60, and 80 mg/L). The test

ACS Paragon Plus Environment

Environmental Science & Technology

Page 8 of 28 8

129

conditions were the same as described above. The Ni and MP tests were conducted with 12

130

and 8 replicates, respectively.

131

132

2.4. Mixture toxicity test on D. magna

133

We also carried out three sets of mixture toxicity tests: (i) variable Ni-fixed MP, (ii) fixed

134

Ni-variable MP, and (iii) variable Ni-variable MP. Before each test, 10-mL samples of the test

135

solutions were prepared in vials with lids, which were continuously shaken at 130 rpm for 24

136

h under a 16-h:8-h light:dark cycle in an incubator (21 °C) to mix the Ni and MP in the MHW.

137

Five D. magna individuals were placed in each of the four replicate vials, and negative (no Ni,

138

no MP) and positive (fixed chemicals only) controls were prepared. The concentrations and

139

detailed experimental procedures for each test are described below.

140

We also conducted adsorption experiments to confirm differences in the adsorption of Ni

141

caused by the presence of the functional group. After 24 h of shaking, samples were filtered

142

using filter paper (Whatman 0.45 µm, Whatman, UK) and centrifugal filters (Amicon® Ultra-

143

4, Merck Millipore, USA) to remove MPs with adsorbed Ni. The filtrate was analyzed using

144

an inductively coupled plasma atomic emission spectrophotometer (ICP–AES) system (JY

145

138; Ultrace, Jobin Yvon, France).

146

The variable Ni-fixed MP tests (i) were used to confirm the change in Ni toxicity in the

147

presence of MP. Ni concentrations were the same as those used in the acute toxicity test,

148

whereas the MP concentration was fixed at 5 mg/L, which had no adverse effects on D.

149

magna in the MP acute toxicity tests. After a 48-h exposure, observed immobilization was

150

recorded, and then living specimens were selected for Ni analysis. The D. magna specimens

151

were rinsed several times with distilled water, dried in a 65 °C dry oven, and digested with

ACS Paragon Plus Environment

Page 9 of 28

Environmental Science & Technology 9

152

HNO3 (Duksan Pure Chemical, Korea). Ni in each D. magna specimen was measured by

153

ICP–MS to determine bioaccumulation.

154

Fixed Ni-variable MP tests (ii) were conducted to compare the mixture toxicity of Ni and

155

PS or PS-COOH using MP concentrations of 1, 5, 10, 20, and 30 mg/L and Ni at 3 mg/L

156

based on the results of the acute toxicity tests in which the 3 mg/L treatment was not a no-

157

observed-effect concentration for D. magna in the Ni acute toxicity test (after a 48-h exposure,

158

immobilization was recorded).

159

Furthermore, variable Ni-variable MP tests (iii) were performed to compare the mixture

160

toxicity of Ni and MP at the same concentrations used in each of the respective acute toxicity

161

tests, such as 1 mg/L Ni-1 mg/L MP, 2 mg/L Ni-5 mg/L MP, and 5 mg/L Ni-30 mg/L. These

162

concentrations were selected for comparison of mixture toxicity with tests (i) and (ii).

163

Immobilization was recorded after a 48-h exposure.

164

165

2.5. Statistical analyses

166

To assesses whether the differences between the negative controls and exposure groups

167

were significant, a Dunnett’s test (version 1.5) was used.41 EC50 values were computed using

168

the trimmed Spearman-Karber method.42 A 95% confidence limit was used for all

169

comparisons (p < 0.05). To analyze the mixture effect of Ni and MP, the toxic unit (TU)

170

approach, which determines the toxicity of a mixture by summing the toxic strength of its

171

separate compounds, was used.43 TU values were expressed as concentrations of the mixtures,

172

and their sum was expressed as follows:

173



ΣTUi = ∑  ,

ACS Paragon Plus Environment

Environmental Science & Technology

Page 10 of 28 10

174

where, n is the number of chemicals in the mixture, Ci is the concentration of the individual

175

chemicals, and EC50i is the EC50 of the ith mixture components When the 50% adverse effect

176

(EC50mix) was observed at TU values < 1, the combined effect was interpreted as a

177

synergistic effect. In contrast, when the 50% adverse effect (EC50mix) was observed at TU

178

values > 1, the combined effect was interpreted as an antagonistic effect.

179

180

181

3. RESULTS AND DISCUSSION 3.1. Acute toxicity tests on D. magna

182

Among all acute toxicity tests, no immobilization was observed in the negative controls.

183

The results of the MP acute toxicity test on D. magna are shown in Fig. 2. The effect of PS

184

on D. magna was negligible up to 30 mg/L, which was the highest concentration tested in this

185

study. Immobilization of D. magna with increasing PS concentrations of 1, 5, 10, 20, and 30

186

mg/L was 0%, 0%, 0%, 12.5%, and 7.5%, respectively, whereas the corresponding

187

immobilization induced by PS-COOH was 7.5%, 5%, 10%, 27.5%, and 62.5%. In an

188

additional experiment with PS at high concentration (20, 40, 60, and 80 mg/L), the

189

immobilization rates of D. magna were 12.5, 45, 62.5, and 90%, respectively. At all the MP

190

concentrations tested in the present study, the toxicity of PS-COOH was higher than that of

191

PS. This difference in MP toxicity by functional group corresponds with previous studies

192

using PS-NH2 and PS-COOH, and the higher toxicity of PS-COOH in this study is possibly

193

due to the differences in surface charge.37 Meanwhile, Watt et al.44 confirmed that MPs with

194

different functional groups (COOH, NH2) were found at different locations in crab gills. The

195

authors suggested that the difference in localization may be because the binding capacity

196

within the gill tissue changes depending on the characteristics of the particle surface.

ACS Paragon Plus Environment

Page 11 of 28

Environmental Science & Technology 11

197

Similarly, in this study, PS-COOH appeared to exhibit higher toxicity because MPs with

198

different functional groups have different binding capacities in Daphnia. In the present study,

199

the 48-h EC50 values for PS and PS-COOH were 42.78 and 25.96 mg/mL, respectively (Table.

200

1). The results of the Ni acute toxicity tests on D. magna are shown as dark gray bars in Fig.

201

3. Immobilization values observed in D. magna at increasing Ni exposure concentrations of 1,

202

2, 3, 4, and 5 mg/L were 0%, 3.33%, 25%, 46.67%, and 85%, respectively, whereas the 48-h

203

EC50 value of Ni was 3.85 mg/L.

204 205

3.2. Mixture toxicity tests on D. magna

206

Analysis of Ni removal from medium in the presence of MPs indicated that the

207

difference in adsorption to the two types of MP was not significant. The removal rates of Ni

208

were 97.2%, 97.9%, 100.6%, 100.1%, and 99.7% compared to no MP condition for

209

increasing PS concentrations (5, 10, 30, 50, and 100 PS mg/L, respectively). The removal

210

rates of Ni for PS-COOH were 96.3%, 103.0%, 99.7%, 98.4%, and 97.4%, respectively. As

211

MP concentration increased, it was not confirmed that more Ni was adsorbed. There was no

212

statistically significant difference in the concentration of nickel adsorbed on the MP based on

213

the type of MP. Turner et al.23 measured the amount of Ni adsorbed on a plastic pellet over

214

time finding that little was adsorbed as was confirmed in this study. However, it has been

215

shown that more Ni adsorbs on the beached pellets than on virgin pellets. On the other hand,

216

Holmes21 et al. found that trace metals can be detected in plastic pellets collected from

217

England beaches with some comprised of Ni up to 562 µg/g.

218

Figure 3 includes the results of the mixture Ni and MP toxicity tests on D. magna

219

[variable Ni-fixed PS test (i)]. Immobilization values observed in D. magna in this test were

220

2.5%, 12.5%, 12.5%, 30%, and 62.5% for increasing Ni concentrations (1, 2, 3, 4, and 5

ACS Paragon Plus Environment

Environmental Science & Technology

Page 12 of 28 12

221

mg/L, respectively), whereas values for the variable Ni-fixed PS-COOH were 7.5%, 12.5%,

222

62.5%, 55%, and 75%, respectively. Immobilization values of 7.5% and 2.5% were observed

223

in the negative controls (no Ni, no MP) for PS and PS-COOH, respectively. The 48 h-EC50

224

values for the variable Ni-fixed PS and variable Ni-fixed PS-COOH tests were 4.67 and 3.14

225

mg/L, respectively (Table. 1). These results showed that the immobilization rate observed

226

following the variable Ni-fixed PS-COOH treatments was higher than that observed in the Ni

227

acute toxicity tests at all Ni concentrations, whereas immobilization in the variable Ni-fixed

228

PS tests did not differ significantly from that of the Ni only acute toxicity test. This PS

229

mixture toxicity result is consistent with previous studies on population growth inhibition of

230

the microalgae Tetraselmis chuii.32 Davarpanah et al.32 evaluated the toxicity of copper on

231

these marine microalgae in the presence of MP (polyethylene). They confirmed that the

232

toxicity of copper decreased when MP was present; however, this effect was not statistically

233

significant. In particular, the percentage immobilization observed in the 3 mg/L Ni with PS-

234

COOH treatment was significantly higher than that in the Ni-only exposure group. However,

235

because there was no difference in Ni adsorption by the MPs, the difference in mixture

236

toxicity was not due to adsorption but due to toxicity of the MPs, which was dependent on the

237

respective functional groups, as has been described previously in the literature.37,44

238

The results of ICP-MS analysis of Ni bioaccumulation in D. magna indicated that

239

individual water fleas ingested more nickel in the presence of an MP (Fig. 4). Although there

240

is also the possibility of excretion of Ni by D. magna through digestion in this procedure, we

241

attempted to compare Ni accumulation according to presence and type of MPs. Therefore, we

242

excluded the effects of depuration. Ni accumulation was generally higher in D. magna in the

243

variable Ni-fixed MP tests than it was in the Ni only acute toxicity tests, except at a Ni

244

concentration of 5 mg/L. Although not statistically significant, similar trends were observed,

245

with that of the D. magna immobilization tests. Similarly, Khan et al.33 confirmed that silver

ACS Paragon Plus Environment

Page 13 of 28

Environmental Science & Technology 13

246

uptake increased in the zebrafish intestine when MPs were present. Based on these results, we

247

assume that in the variable Ni-fixed MPs tests, the presence of MPs induced higher

248

immobilization rates due to the increase in Ni intake and accumulation in D. magna.

249

However, at the highest Ni concentration (5 mg/L), the Ni concentrations in D. magna and

250

percentage D. magna immobilization in the presence of MPs were lower than those observed

251

in the Ni acute toxicity test at that concentration, although these differences were not

252

statistically significant. We were unable to establish the reason for this observation because of

253

the lack of surviving D. magna at a Ni concentration of 5 mg/L.

254

In the fixed Ni-variable PS tests (ii), the percentage immobilization of D. magna at

255

increasing concentrations of PS and PS-COOH (1, 5, 10, 20, and 30 mg/L) was 12.5%, 10%,

256

30%, 60%, and 87.5% and 12.5%, 25%, 60%, 75%, and 77.5%, respectively (Fig. 5).

257

Immobilization in the negative controls (no Ni, no MP) for PS and PS-COOH was 20% and

258

17.5%, respectively. The 48-h EC50 values of the fixed Ni-variable PS and fixed Ni-variable

259

PS-COOH treatments were 17.72 and 10.63 mg/L, respectively (Table 1). At low

260

concentrations, the percentage immobilization induced by PS and PS-COOH was slightly

261

lower than that induced in the negative controls. Comparing the two MP treatments, the

262

immobilization rate of D. magna exposed to Ni combined with PS-COOH was higher than

263

that of the D. magna exposed to Ni combined with PS at concentrations of 5, 10, and 20

264

mg/L. Furthermore, this observed difference was statistically significant at 5 and 10 mg/L.

265

Therefore, we inferred that the toxicity of Ni combined with PS-COOH was higher than that

266

of Ni combined with PS, which is attributable to the fact that PS-COOH binds more strongly

267

to Ni than does PS. Treatment with MP at 30 mg/L in the mixture toxicity tests showed that

268

percentage immobilization was higher than that in the Ni and MP acute toxicity tests.

269

Therefore, these results indicate that the toxicity of Ni increased concentration dependently in

ACS Paragon Plus Environment

Environmental Science & Technology

Page 14 of 28 14

270

the presence of MPs.

271

In the variable Ni-variable PS tests (iii), the percentage immobilization induced in D.

272

magna was 0%, 15%, 30%, 75%, and 100% for the respective increasing treatment

273

concentrations, whereas the corresponding values of the variable Ni-variable PS-COOH tests

274

were 10%, 22.5%, 80%, 97.5%, and 97.5% (Fig. 6). Immobilization in the negative controls

275

was 0% under both test conditions. The immobilization rates of D. magna exposed to PS-

276

COOH were generally higher than those of D. magna exposed to PS. Furthermore, these

277

results were similar to those of the other mixture toxicity tests in this study in that

278

immobilization observed in D. magna exposed to Ni combined with PS-COOH was higher

279

than that of D. magna exposed to Ni combined with PS at intermediate concentrations, which

280

were used in this study.

281

282

3.3. Comparison of the 48-h EC₅₀mix values of mixture toxicity tests

283

In the mixture toxicity tests of Ni combined with PS, the EC50mix values were 1.33

284

(variable Ni-fixed PS), 1.16 (fixed Ni-variable PS), and 1.07 (variable Ni-variable PS) mg/L

285

(Table 2). For the mixture toxicity tests of Ni combined with PS-COOH, the EC50mix values

286

were 0.96 (variable Ni-fixed PS-COOH), 1.05 (fixed Ni-variable PS-COOH), and 0.78

287

(variable Ni-variable PS-COOH) mg/L. All EC50mix values for Ni mixed with PS were higher

288

than 1.00, whereas those of Ni mixed PS-COOH were slightly higher or less than 1.00. In all

289

tests, the 95% confidence intervals of each test did not include 1.00; however, the EC50mix

290

values were very close to 1.00. Therefore, we determined that when combined with Ni, PS

291

had a slight antagonistic effect on toxicity, whereas PS-COOH had a slight synergistic effect.

292

ACS Paragon Plus Environment

Page 15 of 28

Environmental Science & Technology 15

293

3.4. Environmental implications

294

This study aimed to evaluate the effects of two MPs on Ni toxicity in the water flea D.

295

magna and to compare the mixture toxicity of Ni combined with two types of MPs (PS and

296

PS-COOH), a real phenomenon in the aquatic environment. Overall, we discovered that the

297

toxicity of Ni in combination with either of the two MPs changed compared with that of Ni

298

alone. Compared to the Ni acute toxicity test, the toxicity to D. magna exposed to Ni in

299

combination with PS was mostly lower, whereas toxicity to D. magna exposed to Ni in

300

combination with PS-COOH tended to be higher. Furthermore, the adverse effect on

301

immobilization of D. magna exposed to Ni combined with PS-COOH was generally higher

302

than that of D. magna exposed to Ni combined with PS. Calculation of mixture toxicity using

303

a TU model based on these results revealed that PS had a slight antagonistic effect on Ni

304

toxicity, whereas the effect of PS-COOH in combination with Ni was slightly synergistic.

305

These results revealed that the effects of MPs and other pollutants may be altered by the

306

specific characteristics of the pollutants and the functional group of the MPs. However, in

307

contrast to our hypothesis that Ni (cation) may be adsorbed onto PS-COOH to a greater

308

extent than onto PS due to the presence of the COOH- (anion) group, the difference in the

309

amount of Ni adsorbed on PS and PS-COOH was not significant. This could be attributed to

310

the fact that nickel is not a substance that adsorbs, much unlike a hydrophobic substance.

311

Therefore, it is important to conduct further detailed investigations on the mechanisms of the

312

binding capacity of MPs according to their functional groups and the higher toxicity of Ni in

313

combination with PS-COOH than with PS. Moreover, we confirmed that the effects of MPs

314

and pollutants may vary depending on the specific properties of the pollutant and MP

315

functional groups. Even though the concentrations of nickel and microplastics tested in this

316

study were higher than the concentrations of substances that can exist in the environment, this

ACS Paragon Plus Environment

Environmental Science & Technology

Page 16 of 28 16

317

study confirmed that the toxicity of one substance to organism can change when in the

318

presence of another substance. This result indicates the possibility that toxicity may change

319

when aquatic organisms are exposed to other substances with microplastics and even that

320

toxicity trends may vary for the same substance depending on the functional group and

321

characteristics of the microplastics. Therefore, further research on mixture toxicity using

322

various MPs and pollutants is warranted.

323

324

ACKNOWLEDGMENTS

325

This research was supported by the Basic Science Research Program through the

326

National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and

327

Future Planning (2016R1A2B3010445). This study was funded by the Graduate School of

328

Specialization for managing information of chemical risk, and supported by Konkuk

329

University Researcher Fund in 2017. The authors thank the Korean Basic Science Institute

330

for performing FE-SEM, ICP-AES and ICP-MS analyses.

331

332

REFERENCES (327)

333 334

(1)

Browne, M. a; Galloway, T.; Thompson, R. Microplastic-an emerging contaminant of potential concern? Integr. Environ. Assess. Manag. 2007, 3 (4), 559–566.

335 336

(2)

Derraik, J. G. . B. The pollution of the marine environment by plastic debris: a review. Mar. Pollut. Bull. 2002, 44 (9), 842–852.

337 338 339

(3)

Browne, M. A.; Dissanayake, A.; Galloway, T. S.; Lowe, D. M.; Thompson, R. C. Ingested microscopic plastic translocates to the circulatory system of the mussel, Mytilus edulis (L.). Environ. Sci. Technol. 2008, 42 (13), 5026–5031.

340 341

(4)

Kaposi, K. L.; Mos, B.; Kelaher, B. P.; Dworjanyn, S. A. Ingestion of microplastics has limited impact on a marine larva. Environ. Sci. Technol. 2014, 48, 1638.

342 343

(5)

Au, S. Y.; Bruce, T. F.; Bridges, W. C.; Klaine, S. J. Responses of Hyalella azteca to acute and chronic microplastic exposures. Environ. Toxicol. Chem. 2015, 34 (11),

ACS Paragon Plus Environment

Page 17 of 28

Environmental Science & Technology 17

344

2564–2572.

345 346 347

(6)

Avio, C. G.; Gorbi, S.; Regoli, F. Experimental development of a new protocol for extraction and characterization of microplastics in fish tissues: First observations in commercial species from Adriatic Sea. Mar. Environ. Res. 2015, 111, 18–26.

348 349 350

(7)

Van Cauwenberghe, L.; Claessens, M.; Vandegehuchte, M. B.; Janssen, C. R. Microplastics are taken up by mussels (Mytilus edulis) and lugworms (Arenicola marina) living in natural habitats. Environ. Pollut. 2015, 199, 10–17.

351 352 353 354

(8)

Nobre, C. R.; Santana, M. F. M.; Maluf, A.; Cortez, F. S.; Cesar, A.; Pereira, C. D. S.; Turra, A. Assessment of microplastic toxicity to embryonic development of the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea). Mar. Pollut. Bull. 2015, 92 (1–2), 99–104.

355 356 357

(9)

Moore, C. J.; Lattin, G. L.; Zellers, A. F. Quantity and type of plastic debris flowing from two urban rivers to coastal waters and beaches of Southern California. Rev. Gestão Costeira Integr. 2011, 11 (1), 65–73.

358 359 360

(10)

Eriksen, M.; Mason, S.; Wilson, S.; Box, C.; Zellers, A.; Edwards, W.; Farley, H.; Amato, S. Microplastic pollution in the surface waters of the Laurentian Great Lakes. Mar. Pollut. Bull. 2013, 77 (1–2), 177–182.

361 362 363

(11)

Free, C. M.; Jensen, O. P.; Mason, S. A.; Eriksen, M.; Williamson, N. J.; Boldgiv, B. High-levels of microplastic pollution in a large, remote, mountain lake. Mar. Pollut. Bull. 2014, 85 (1), 156–163.

364 365 366 367

(12)

Lechner, A.; Keckeis, H.; Lumesberger-Loisl, F.; Zens, B.; Krusch, R.; Tritthart, M.; Glas, M.; Schludermann, E. The Danube so colourful: A potpourri of plastic litter outnumbers fish larvae in Europe’s second largest river. Environ. Pollut. 2014, 188, 177–181.

368 369 370

(13)

Sadri, S. S.; Thompson, R. C. On the quantity and composition of floating plastic debris entering and leaving the Tamar Estuary, Southwest England. Mar. Pollut. Bull. 2014, 81 (1), 55–60.

371 372 373 374

(14)

Wagner, M.; Scherer, C.; Alvarez-Muñoz, D.; Brennholt, N.; Bourrain, X.; Buchinger, S.; Fries, E.; Grosbois, C.; Klasmeier, J.; Marti, T.; et al. Microplastics in freshwater ecosystems: what we know and what we need to know. Environ. Sci. Eur. 2014, 26 (1), 12.

375 376 377

(15)

Eerkes-Medrano, D.; Thompson, R. C.; Aldridge, D. C. Microplastics in freshwater systems: A review of the emerging threats, identification of knowledge gaps and prioritisation of research needs. Water Res. 2015, 75, 63–82.

378 379 380

(16)

Browne, M. A.; Crump, P.; Niven, S. J.; Teuten, E. L.; Tonkin, A.; Galloway, T.; Thompson, R. C. Accumulations of microplastic on shorelines worldwide: sources and sinks. Environ. Sci. Technol. 2011, 45, 9175–9179.

381 382 383

(17)

Leslie, H. A.; van Velzen, M. J. M.; Vethaak, A. D. IVM Institute for Environmental Studies Microplastic survey of the Dutch environment. Microplastic Surv. Dutch Enviroment 2013, 476 (September).

ACS Paragon Plus Environment

Environmental Science & Technology

Page 18 of 28 18

384 385 386

(18)

Rocha-Santos, T.; Duarte, A. C. A critical overview of the analytical approaches to the occurrence, the fate and the behavior of microplastics in the environment. Trac-Trends Anal. Chem. 2015, 65, 47–53.

387 388 389

(19)

Mato, Y.; Isobe, T.; Takada, H.; Kanehiro, H.; Ohtake, C.; Kaminuma, T. Plastic resin pellets as a transport medium for toxic chemicals in the marine environment. Environ. Sci. Technol. 2001, 35 (2), 318–324.

390 391

(20)

Fries, E.; Zarfl, C. Sorption of polycyclic aromatic hydrocarbons (PAHs) to low and high density polyethylene (PE). Environ. Sci. Pollut. Res. 2012, 19 (4), 1296–1304.

392 393

(21)

Holmes, L. A.; Turner, A.; Thompson, R. C. Adsorption of trace metals to plastic resin pellets in the marine environment. Environ. Pollut. 2012, 160 (1), 42–48.

394 395

(22)

Lee, H.; Shim, W. J.; Kwon, J. H. Sorption capacity of plastic debris for hydrophobic organic chemicals. Sci. Total Environ. 2014, 470–471, 1545–1552.

396 397

(23)

Turner, A.; Holmes, L. A. Adsorption of trace metals by microplastic pellets in fresh water. Environ. Chem. 2015, 12 (5), 600–610.

398 399 400

(24)

Brennecke, D.; Duarte, B.; Paiva, F.; Caçador, I.; Canning-Clode, J. Microplastics as vector for heavy metal contamination from the marine environment. Estuar. Coast. Shelf Sci. 2016, 178, 189–195.

401 402 403

(25) Besseling, E.; Wegner, A.; Foekema, E. M.; van den Heuvelgreve, M. J.; Koelmans, A. A. Effects of microplastic on fitness and PCB bioaccumulation by the Lugworm Arenicola marina (L.). Environ. Sci. Technol. 2013, 47 (1), 593–600.

404 405 406

(26)

Browne, M. A.; Niven, S. J.; Galloway, T. S.; Rowland, S. J.; Thompson, R. C. Microplastic moves pollutants and additives to worms, reducing functions linked to health and biodiversity. Curr. Biol. 2013, 23 (23), 2388–2392.

407 408 409

(27)

Chua, E. M.; Shimeta, J.; Nugegoda, D.; Morrison, P. D.; Clarke, B. O. Assimilation of polybrominated diphenyl ethers from microplastics by the marine amphipod, Allorchestes compressa. Environ. Sci. Technol. 2014, 48 (14), 8127–8134.

410 411 412 413

(28)

Batel, A.; Linti, F.; Scherer, M.; Erdinger, L.; Braunbeck, T. Transfer of benzo[a]pyrene from microplastics to Artemia nauplii and further to zebrafish via a trophic food web experiment: CYP1A induction and visual tracking of persistent organic pollutants. Environ. Toxicol. Chem. 2016, 35 (7), 1656–1666.

414 415 416

(29)

Karami, A.; Romano, N.; Galloway, T.; Hamzah, H. Virgin microplastics cause toxicity and modulate the impacts of phenanthrene on biomarker responses in African catfish (Clarias gariepinus). Environ. Res. 2016, 151, 58–70.

417 418 419 420

(30)

Paul-Pont, I.; Lacroix, C.; Fernández, G. C.; Hégaret, H.; Lambert, C.; Le Goïc, N.; Frère, L.; Cassone, A.-L.; Sussarellu, R.; Fabioux, C.; et al. Exposure of marine mussels Mytilus spp. to polystyrene microplastics: Toxicity and influence on fluoranthene bioaccumulation. Environ. Pollut. 2016, 216, 724–737.

421 422 423

(31)

Wardrop, P.; Shimeta, J.; Nugegoda, D.; Morrison, P. D.; Miranda, A.; Tang, M.; Clarke, B. O. Chemical pollutants sorbed to ingested microbeads from personal care products accumulate in fish. Environ. Sci. Technol. 2016, 50 (7), 4037–4044.

ACS Paragon Plus Environment

Page 19 of 28

Environmental Science & Technology 19

424 425 426

(32)

Davarpanah, E.; Guilhermino, L. Single and combined effects of microplastics and copper on the population growth of the marine microalgae Tetraselmis chuii. Estuar. Coast. Shelf Sci. 2015, 167, 269–275.

427 428 429

(33)

Khan, F. R.; Syberg, K.; Shashoua, Y.; Bury, N. R. Influence of polyethylene microplastic beads on the uptake and localization of silver in zebrafish (Danio rerio). Environ. Pollut. 2015, 206, 73–79.

430 431 432 433

(34)

Luís, L. G.; Ferreira, P.; Fonte, E.; Oliveira, M.; Guilhermino, L. Does the presence of microplastics influence the acute toxicity of chromium(VI) to early juveniles of the common goby (Pomatoschistus microps)? A study with juveniles from two wild estuarine populations. Aquat. Toxicol. 2015, 164, 163–174.

434 435 436

(35)

Ferreira, P.; Fonte, E.; Soares, M. E.; Carvalho, F.; Guilhermino, L. Effects of multistressors on juveniles of the marine fish Pomatoschistus microps: Gold nanoparticles, microplastics and temperature. Aquat. Toxicol. 2016, 170, 89–103.

437 438 439

(36)

Rochman, C. M.; Hoh, E.; Hentschel, B. T.; Kaye, S. Long-term field measurement of sorption of organic contaminants to five types of plastic pellets: Implications for plastic marine debris. Environ. Sci. Technol. 2013, 47 (3), 1646–1654.

440 441 442 443

(37) Della Torre, C.; Bergami, E.; Salvati, A.; Faleri, C.; Cirino, P.; Dawson, K. A.; Corsi, I. Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus. Environ. Sci. Technol. 2014, 48 (20), 12302–12311.

444 445

(38)

Fotopoulou, K. N.; Karapanagioti, H. K. Surface properties of beached plastic pellets. Mar. Environ. Res. 2012, 81, 70–77.

446 447 448 449

(39)

Weber, C. I. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms; Weber, C. I., Ed.; Environmental Monitoring Systems Laboratory, Office of Research and Development, US Environmental Protection Agency: Cincinnati, Ohio, 1991.

450 451

(40)

OECD. OECD guideline for the testing of chemicals. Test no.202 “Daphnia sp., Acute Immobilisation Test.” 2004, No. April, 1–12.

452 453

(41)

Dunnett, C. W. A Multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 1955, 50 (272), 1096–1121.

454 455 456

(42)

Hamilton, M. A.; Russo, R. C.; Thurston, R. V. Trimmed spearman- karber method for estimating median lethal concentrations in toxicity biossays. Environ. Sci. Technol. 1977, 11 (1), :714-719.

457 458 459

(43)

Pape-Lindstrom, P.; Lydy, M. Synergistic toxicity of atrazine and organophosphate insecticides contravenes the response addition mixture model. Environ. Toxicol. … 1997, 16 (11), 2415–2420.

460 461 462

(44)

Watts, A. J. R.; Urbina, M. A.; Goodhead, R.; Moger, J.; Lewis, C.; Galloway, T. S. Effect of microplastic on the gills of the shore crab Carcinus maenas. Environ. Sci. Technol. 2016, 50 (10), 5364–5369.

463

ACS Paragon Plus Environment

Environmental Science & Technology

Page 20 of 28 20

464 465

Table 1. Toxicity values [48-h half-maximal effective concentration (EC50)] of Ni and microplastics (MPs) against Daphnia magna

466

Values (mg/L) EC50 (95% confidence limits) Ni only 3.85 (3.83–3.87) PS only 42.78 (42.38–43.17) PS-COOH only 25.96 (25.75-26.17) Variable Ni + 5 mg/L PS 4.67 (4.65–4.69) Variable Ni + 5 mg/L PS-COOH 3.14 (3.12–3.17) 3 mg/L Ni + Variable PS 17.72 (17.72–17.89) 3 mg/L Ni + Variable PS-COOH 10.63 (10.44–10.82) Trimmed Spearman–Karber method; 95% confidence limits, p < 0.05. Test

467

ACS Paragon Plus Environment

Page 21 of 28

Environmental Science & Technology 21

468 469

Table 2. Forty-eight-hour half-maximal effective concentration-adverse effect (EC50mix) values of Ni and microplastic (MP) mixtures on Daphnia magna

EC50mix Value 95% confidence interval Variable Ni + 5 mg/L PS 1.33 1.32–1.34 3 mg/L Ni + Variable PS 1.16 1.15–1.16 Variable Ni + Variable PS 1.07 1.07–1.08 Variable Ni + 5 mg/L PS-COOH 0.96 0.95–0.97 3 mg/L Ni + Variable PS-COOH 1.05 1.04–1.05 Variable Ni + Variable PS-COOH 0.78 0.78–0.79 Toxic unit model; Pape-Lindstrom and Lydy, 1997, Trimmed Spearman–Karber method; 95% confidence limits, p < 0.05. Test

470 471 472

ACS Paragon Plus Environment

Environmental Science & Technology

Page 22 of 28 22

473 474 475

Fig. 1. Field emission scanning electron microscopy (FE-SEM) images of microplastics (MPs) without (PS, left) and with (PS-COOH, right) a COOH functional group. Scale bar = 500 nm.

ACS Paragon Plus Environment

Page 23 of 28

Environmental Science & Technology 23

Immobilization of D.magna (%)

100

Variable MP

478 479 480 481

*

80

60

#

*

40

*

20

0 MP

476 477

#

Variable PS Variable PS-COOH

1

0

2

1

3

5

4

10

5

20

6

30

Concentration (mg/L) Fig. 2. Immobilization of Daphnia magna exposed to microplastics (MPs) for 48-h MP acute toxicity tests; PS (left) and PS-COOH (right). *p < 0.05 and #p < 0.05 compared to the control group and to each other, respectively. Eight replicates were run for each test, and error bars show standard deviations.

ACS Paragon Plus Environment

Environmental Science & Technology

Page 24 of 28

Immobilization of D.magna (%)

24

120

Variable Ni-Fixed MP

100

484 485 486 487 488

*

#

* *

80

* *

*

60 40 20 0 Ni MP

482 483

Variable Ni+5 mg/L PS Ni only Variable Ni+5 mg/L PS-COOH

0 5

1 5

2 5

3 5

4 5

5 5

Concentration (mg/L) Fig. 3. Immobilization of Daphnia magna exposed to Ni and microplastics (MPs) for 48-h Ni acute toxicity (middle) and variable Ni-fixed MPs combined toxicity tests; PS (left) and PSCOOH (right). *p < 0.05 and #p < 0.05 compared to the control group and to each other, respectively. Twelve and eight replicates were run for Ni acute toxicity and each combined toxicity test, respectively. Error bars show standard deviations.

ACS Paragon Plus Environment

Page 25 of 28

Environmental Science & Technology

Ni concentration in D. magna (ppb)

25

5.5 Variable Ni+5 mg/L PS Ni only Variable Ni+5 mg/L PS-COOH

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 0

489 490 491 492 493 494 495 496

1

2

3

4

5

Ni concentration (mg/L) Fig. 4. Accumulation of Ni in Daphnia magna exposed to Ni and microplastics (MPs) for 48h Ni acute toxicity tests (middle) and variable Ni-fixed MPs combined toxicity tests; MPs without (PS, left) and with (PS-COOH, right) a COOH functional group. Measured by inductively coupled plasma mass spectrometry (ICP-MS, n = 4 replicates). Detection limit and limit of quantization concentrations were 0.0071 and 0.0236 µg/L, respectively. Error bars show standard deviations.

497

ACS Paragon Plus Environment

Environmental Science & Technology

Page 26 of 28 26

Immobilization of D.magna (%)

120

Fixed Ni-Variable MP Ni 3 mg/L+Variable PS Ni 3 mg/L+Variable PS-COOH

100

500 501 502 503 504

* *

*

# 80

* 60

# 40 20 0 Ni MP

498 499

*

1

3 0

2

3 1

3

3 5

4

3 10

5

3 20

6

3 30

Concentration (mg/L) Fig. 5. Immobilization of Daphnia magna exposed to Ni and microplastics (MPs) for 48-h fixed Ni-variable MPs combined toxicity tests; MPs without (PS, left) and with (PS-COOH, right) a COOH functional group. *p < 0.05 and #p < 0.05 compared to the control group and to each other, respectively. Eight replicates were run for each test, and error bars show standard deviations.

505

ACS Paragon Plus Environment

Page 27 of 28

Environmental Science & Technology 27

Immobilization of D.magna (%)

120

Variable Ni-Variable MP

100

508 509 510 511 512

*

*

*

*

*

80 60

* 40

#

*

20 0 Ni MP

506 507

#

#

Variable Ni+Variable PS Variable Ni+Variable PS-COOH

0 0

1 1

2 5

3 10

4 20

5 30

Concentration (mg/L) Fig. 6. Immobilization of Daphnia magna exposed to Ni and microplastics (MPs) for 48-h variable Ni-variable MPs combined toxicity tests; MPs without (PS, left) and with (PSCOOH, right) a COOH functional group. *p < 0.05 and #p < 0.05 compared to the control group and to each other, respectively. Eight replicates were run for each test, and error bars show standard deviations.

ACS Paragon Plus Environment

Environmental Science & Technology

171x78mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 28 of 28