Insights into Long-term Toxicity of Triclosan to Freshwater Green

4 days ago - This study explored the long-term impacts of a pulse disturbance of triclosan on five non-target green algae in Lake Erie. Comprehensive ...
1 downloads 0 Views 508KB Size
Subscriber access provided by EKU Libraries

Ecotoxicology and Human Environmental Health

Insights into Long-term Toxicity of Triclosan to Freshwater Green Algae in Lake Erie Xiaying Xin, Gordon Huang, Chunjiang An, Renata Raina-Fulton, and Harold Weger Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b00259 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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 34

Environmental Science & Technology

1

Insights into Long-term Toxicity of Triclosan to Freshwater Green Algae in

2

Lake Erie

3 4

Xiaying Xin,a Gordon Huang,a,* Chunjiang Anb, Renata Raina-Fultonc, Harold Wegerd

5 6 7 8

a Institute

9

Canada S4S 0A2

for Energy, Environment and Sustainable Communities, University of Regina, Regina,

10

b

11

Canada H3G 1M8

12

c Department

13

d

Department of Building, Civil and Environmental Engineering, Concordia University, Montreal,

of Chemistry and Biochemistry, University of Regina, Regina, Canada S4S 0A2

Department of Biology, University of Regina, Regina, Canada S4S 0A2

14 15 16 17 18

*Corresponding author: Tel: +1-306-5854095; Fax: +1-306-5854855; E-mail:

19

[email protected]

1 ACS Paragon Plus Environment

Environmental Science & Technology

20

Abstract:

21

This study explored the long-term impacts of a pulse disturbance of triclosan on five non-target

22

green algae in Lake Erie. Comprehensive analyses were performed using multiple physiological

23

endpoints at community and subcellular scales. The toxic mechanism of triclosan in a wide range

24

of concentrations was analyzed. The diverse sensitivity of algae species and complex

25

interrelationships among multiple endpoints were revealed. The results showed the taxonomic

26

groups of algae were the key issue for sensitivity difference. High doses of triclosan caused

27

irreversible damage on algae, and environmentally relevant doses initiated either inhibition or

28

stimulation. Smaller cells had higher sensitivity to triclosan, while larger cells had a wider size

29

variation after exposure. Colonial cells were less sensitive than unicells. For chlorophyll, there

30

were better dose-response relationships in Chlorococcum sp., Chlamydomonas reinhardtii CPCC

31

12 and 243 than Asterococcus superbus and Eremosphaera viridis. For chlorophyll fluorescence,

32

Fv/Fm was the most sensitive parameter, and qN was more sensitive than qP. Triclosan showed

33

long-term effects on biochemical components, such as lipids, proteins, and nucleic acids. The

34

findings will be helpful for a systematic and complete assessment of triclosan toxicity in natural

35

waters and the development of appropriate strategies for its risk management.

36

Keywords: Long-term impacts, Triclosan, Green algae, Sensitivity, Multiple endpoints, Lake

37

Erie

38

2 ACS Paragon Plus Environment

Page 2 of 34

Page 3 of 34

39

Environmental Science & Technology

1. Introduction

40 41

Triclosan is an antimicrobial agent used in personal care products, medical devices, athletic

42

clothing and meat packaging in the hope of providing long-lasting antibacterial protection.1

43

Triclosan has been frequently detected in surface water, sediment, biosolids, and aquatic

44

organisms around the world.2 There are also some health issues about triclosan, including

45

antibiotic resistance, endocrine disruption, allergies, and the formation of carcinogenic by-

46

products.3 Triclosan is being increasingly scrutinized due to the growing concerns of its potential

47

harmful effects to human health and the environment. However, the Canadian government has a

48

positive attitude towards triclosan. To date, there is no ban on triclosan containing soaps and

49

handwashes in Canada like that in the United States and European Union. In order to provide a

50

sound scientific basis for policy making, it is expected to obtain more systematic and

51

comprehensive evidence of triclosan toxicity to the ecosystem.

52 53

Algae are a good indicator for environmental toxicity. As primary producers, algae form the

54

basis of the aquatic food web and any detrimental effect on algae may lead to significant

55

alterations in the rest of the ecosystem.4, 5 It usually takes about 3-5 days to observe detrimental

56

effects on algae in traditional chronic tests.6, 7 But in the natural aquatic environment, algae are

57

often exposed to triclosan for longer time periods. Although a few studies focused on triclosan

58

toxicity to algae over longer time periods, there are still some limitations. For example, Eriksson8

59

investigated the long-term effects of triclosan at 0.316 to 10000 nM on marine periphyton

60

communities in flow-through mesocosms for 17 days. Results showed photosynthesis increased

61

with increasing triclosan concentrations lower than 1000 nM, and community tolerance was

3 ACS Paragon Plus Environment

Environmental Science & Technology

62

observed at 100 nM and higher. Nietch’s9 studied the effects of triclosan on created benthic

63

communities in indoor mesocosms for 56 days. Results found species abundance were varied

64

based on different triclosan doses and resistance significantly increased at doses of 0.5 µg/L and

65

above. Such effects were only measured at the community level and lacked observations from

66

individual algae species. Moreover, these studies have not considered the dynamic change of

67

triclosan amount and whether it still leads to the physicochemical variation of algae.

68 69

Multi-endpoint analyses of triclosan toxicity to algae have been applied with the advantage of

70

characterizing multiple target sites.10-12 Since the dose makes the poison,13 dose-dependent

71

bioassays have also been used to reveal different MoA (mode of action) induced by triclosan.14

72

Besides, species sensitivity to triclosan has been verified to be various, owing to a diversity of

73

taxonomic groups of algae. However, the existing studies still unilaterally investigated the

74

triclosan toxicity. For instance, Pan15 investigated the effects of triclosan on Chlamydomonas

75

reinhardtii through measuring the algal growth, chlorophyll content, lipid peroxidation, and

76

transcription of the antioxidant-related genes as well as biochemical alterations. However there

77

was no comparison between Chlamydomonas reinhardtii and other algae species regarding

78

different toxic responses. Ciniglia16 reported that increasing triclosan concentrations in the range

79

of 0.125-5 mg/L had relevant effects in both chloroplast morphology and dimension on

80

Closterium ehrenbergii. However there was no information about the effects on chlorophyll

81

contents or photosynthesis efficiency, leading to inadequate assessment of toxicity. Moreover,

82

although triclosan had a dose-dependent DNA damage to Closterium ehrenbergii, it was still

83

unknown whether there was any damage on other biochemical alterations.16 Therefore, a

4 ACS Paragon Plus Environment

Page 4 of 34

Page 5 of 34

Environmental Science & Technology

84

comprehensive and systematic exploration of triclosan toxicity to various algae species is

85

required.

86 87

The aim of this study was to explore the long-term impacts of a pulse disturbance of triclosan on

88

five non-target algae species in Lake Erie. Lake Erie water (LEW) can provide sufficient

89

microelements and nutrients for algae cultivation.17 Such rich nutrient condition will not cause

90

algae failure to thrive, and thus will not interfere with the toxic effects of triclosan. Using LEW

91

as a background medium is also associated with natural physical/chemical characteristics as

92

defined by pH, conductivity, dissolved oxygen and DOC.17 It adds more practical significance to

93

real toxicity study. The comprehensive toxic analyses were conducted using multiple

94

physicochemical endpoints at both community and subcellular scales. The toxic mechanism of

95

triclosan under different concentration-based scenarios in the range down to environmentally

96

relevant doses were analyzed. The diverse sensitivity of algae species and complex

97

interrelationships among multiple endpoints were revealed. This study represented a creative

98

thinking to provide a meaningful and valid statistical evaluation of triclosan in a comprehensive

99

manner, using green algae as a representative model. The findings explored various long-term

100

effects on multi-algal species in Canadian natural waters, which can support the environmental

101

risk management for triclosan application.

102 103

2. Materials and methods

104 105

2.1. Chemicals

106

5 ACS Paragon Plus Environment

Environmental Science & Technology

107

Triclosan (purity > 99 %) was purchased from Alfa Aesar (Ward Hill, USA). Its main

108

characteristics are shown in Table S1. All other chemicals were of reagent grade or higher.

109

(maybe you should have something on the buffer used here or in SI materials –you still need to

110

define the pH of buffer and its composition)

111 112

2.2. Algal culture and toxicity test

113 114

LEW, was used in all algae exposure experiments including controls. After collection, LEW was

115

stored at 4 °C until use. Prior to use, LEW was filtered through a sterile filter membrane with 0.2

116

µm pore size to remove bacteria and particulate matter. Freshwater algae Chlamydomonas

117

reinhardtii CPCC 12, Chlamydomonas reinhardtii CPCC 243, Asterococcus superbus, and

118

Eremosphaera viridis were obtained from the Canadian Phycological Culture Center (CPCC,

119

University of Waterloo, Canada). The green microalga, Chlorococcum sp., was isolated from

120

Pasqua Lake in the Qu’Appelle River system (Saskatchewan, Canada). The Chlamydomonas

121

reinhardtii CPCC 243, Asterococcus superbus, and Eremosphaera viridis were cultured in a

122

sterile Bold Basal Medium (BBM). Chlamydomonas reinhardtii CPCC 12 and Chlorococcum sp.

123

were cultured in a sterile High Salt Medium (HSM) and a sterile BG-11 liquid medium,

124

respectively. They are all ideal algae for synchrotron-based FTIR microspectroscopy due to the

125

unicellular structure with a diameter range of 9 – 144 μm (Figure S1). The algae were cultured at

126

23 ± 1 °C on a 12-h light and 12-h dark cycle. Light was provided by 40 W white fluorescent

127

lamps (Philips F40 T12/DX). In the triclosan exposure experiment, the algae at the logarithmic

128

growth phase were collected, washed and then diluted to the initial concentration of 2 × 105 – 8

6 ACS Paragon Plus Environment

Page 6 of 34

Page 7 of 34

Environmental Science & Technology

129

× 105 cells mL-1. The algae was exposed to triclosan at concentrations of 1 000, 154, 23.6, 3.6,

130

0.56, and 0.0862 μg/L, with control flasks that did not contain any chemicals.

131 132

2.3. Cell densities and cell viability

133 134

Optical density (OD) was used to monitor the algae growth.18 After different exposure times (0,

135

2, 7, 18, 29 days), OD of bulk cultures was measured at 680 nm with a Cary-300 double beam

136

UV-visible spectrophotometer (Agilent Technologies, CA, USA). Each sample was measured

137

three times and the average value was used. To observe cell viability on 29 days, 20 μL of the

138

medium-algae mixture was added to a slide, covered with a coverslip. Cell size was measured

139

using a Zeiss Axio Observer Z1 microscope (Zeiss, Birkerød, Denmark). Cell diameter was

140

recorded by approximating a single cell to a spherical cell. Ten cells for each sample were

141

measured and the average value was taken. The pH value was measured on day 29 by a pH meter

142

(Mettler Delta 320, Halstead, UK).

143 144

2.4. Measurement of chlorophyll concentration and chlorophyll fluorescence

145 146

Chlorophyll a (Chl a) and chlorophyll b (Chl b) were measured through a modified method

147

(more details in Supporting Information).19 A Handy PEA fluorometer (Opti-Sciences, MA, UK)

148

was used to measure the photosynthetic activity of the five algae species.20 On day 29, 1 mL

149

mixture was transferred to a 2-mL vial, and were then adapted for 30 mins under room

150

temperature and dark conditions. Six measured parameters include Fo, Fm, Fv/Fm, qP, qN, and

151

Y(PSII), indicating the initial fluorescence level, maximum fluorescence yield, maximum

7 ACS Paragon Plus Environment

Environmental Science & Technology

152

quantum yield, photochemical quenching coefficient, non-photochemical quenching coefficient,

153

and photochemical quantum yield, respectively.

154 155

2.5. Biochemical measurement for single living cell through Synchrotron-based FTIR

156

spectromicroscopy

157 158

Cells were harvested after exposure time of 29 days by centrifugation at 4500 rpm for 15 min.

159

The biomass was carefully resuspended in distilled water and centrifuged again. This process

160

was repeated three times. Algal cells were resuspended in 30 μL of D2O, and then loaded on the

161

optical CaF2 window. The sample was compressed by another CaF2 window, leaving a

162

polymeric spacer between them. This was held in a sample holder, allowing the flow of an

163

aqueous solution around the edge of the windows to compensate for evaporation from the

164

sample.21

165 166

Synchrotron-based FTIR (SR-FTIR) microspectroscopy measurements were carried out at the

167

beamline 01B1-01(MidIR) at the Canadian Light Source, Saskatoon, Canada. A Bruker Vertex

168

70v interferometer coupled to a Hyperion 3000 IR confocal microscope equipped with a liquid

169

nitrogen cooled mercury cadmium telluride (MCT) detector (Bruker Optics, MA, USA) was used

170

to acquire the data using synchrotron-based infrared light (Figure S2). The brilliant synchrotron

171

source could maintain an adequate signal-to-noise ratio even with small apertures. A pair of 36×

172

objectives were used for spectra collection in transmission mode with a 10 × 10 µm point size on

173

the sample, with 512 scan co-added scans, measured over a broad range of 4000 - 800 cm-1

174

wave-numbers.

8 ACS Paragon Plus Environment

Page 8 of 34

Page 9 of 34

Environmental Science & Technology

175 176

2.6. Determination of triclosan concentration

177 178

A 10 mL culture solution from the algae treatments with 154 and 1000 g/L of initial triclosan

179

was sampled at 0, 6, 12, 18, 24 and 29 days, respectively. All algae treatments including triclosan

180

blank were sampled on day 29. The aliquots taken from the culture solution were centrifuged at

181

12,000 rpm for 10 min. Triclosan in supernatant was concentrated by solid phase extraction22 and

182

triclosan in algae was extracted following Ding’s method.23 Triclosan concentration was

183

analyzed using Agilent 1260 liquid chromatograph equipped with a diode array detector (Santa

184

Clara, CA, USA). A ZORBAZ XDB-C18 column (250 × 4.6 mm, 5 µm, Agilent) was used for

185

the analysis, with the oven temperature of 40 ˚C. The injection volume was 50 µL. The mobile

186

phase consisting of 70:30 v:v% acetonitrile:water with flow rate of 0.8 mL/min. The wavelength

187

used for detection was 214 nm.

188 189

2.7. Statistical analysis

190 191

All experiments were carried out in at least triplicate. The quantitative data were expressed as

192

mean and standard deviation/standard error. If the homoscedasticity assumptions of the data

193

were satisfied, LSD tests were further conducted to analyze the statistical significance among

194

individual treatments using SPSS 18.0 (SPSS, IL, USA) (p < 0.05). The principal component

195

analysis (PCA) was performed to visualize the correlation of relevant responses and their

196

distinctions using SPSS 18.0. SR-FTIR data were collected using OPUS 7.2 software (Bruker

197

Optics, MA, USA). At least 10 cells for each sample were selected randomly to generate average

9 ACS Paragon Plus Environment

Environmental Science & Technology

198

spectra. Background spectra were taken for every sample to compensate for atmospheric

199

alteration and synchrotron ring current changes. Raw spectra were baseline-corrected using the

200

automatic baseline correction algorithm.24 Other data processing and figure drawing were

201

conducted using Origin Pro 8.0 software (Origin lab Co., Northhampton, USA).

202 203

3. Results and Discussions

204 205

The U.S. Food & Drug Administration banned the use of triclosan as an antiseptic ingredient for

206

over-the-counter consumer wash products in 2016.25 The European Union passed a similar ban in

207

2107.26 In Canada, however, triclosan can still be contained in cosmetics, non-prescription drugs

208

and natural health products with a limit much higher than that in Australia and Japan.27 In reality,

209

adverse outcomes triggered by long-term and low-level exposure to triclosan are more common.

210

Therefore, the present study aimed to explore a pulse disturbance on non-target multiple algae

211

species created by triclosan under a long-term exposure. It was also designed to reveal the toxic

212

mechanism for different concentration-based scenarios using multiple physiobiological endpoints.

213 214

3.1. Measured triclosan concentrations

215 216

The dynamic change of triclosan in different algae treatment concentrations (1000 and 154 µg/L)

217

was monitored during the exposure period, and the results are given in Figure S3. Triclosan in

218

the blank samples during the exposure period was not detected. There was a trend that the

219

concentration of triclosan decreased rapidly on the first 6 days, and then gradually decreased on

220

the remaining days. The change of concentration of triclosan with Chlorococcum sp. was the

10 ACS Paragon Plus Environment

Page 10 of 34

Page 11 of 34

Environmental Science & Technology

221

highest among all species. The remaining percentage of triclosan for other species were in the

222

range of 54.03 to 69.72%, and 56.80 to 76.39% at the end of the exposure period with treatment

223

concentrations of 1000 and 154 µg/L, respectively. The final average percentages of triclosan in

224

all treatments were in the range of 37.31 to 77.51% (Figure S4). Most algae except

225

chlorococcum sp. were all exposed to more than a half of the original triclosan concentrations

226

over the 29-day period. The existing triclosan is sufficient to induce multiple responses on algae.

227

The loss of triclosan could be attributed to photodegradation, species-based bio-adsorption,

228

bioaccumulation and biodegradation.15 Since algae were exposed in a relatively closed system,

229

the remaining triclosan and its degradation products could still affect algae cells, resulting in

230

various long-term effects.

231 232

3.2. Biomass variation analyses

233 234

There are several variables used in the evaluation of toxicity to algae. The biomass-type endpoint

235

is preferred by many eco-toxicologists, because algal biomass is considered to be stable,

236

comparable, and eco-logically relevant.28 In Figure S5, the dose-dependent growth of

237

Chlorococcum sp. gradually occurred after day 18, while others had such trend after day 7. The

238

growth rates of Asterococcus superbus and Chlamydomonas reinhardtii CPCC 12 exposed to 1

239

000 µg/L triclosan went down lower than the starting point at the end of the test, indicating the

240

complete cell collapse under such high-level exposure. Under other lower concentrations, their

241

growth on the 29th day increased much more than the starting biomass. Figure 1 shows the

242

concentration-inhibition rate curve on day 29, EC25 were 3.15, 16.75, 0.38, 0.58 and 4468.21

243

µg/L, for Chlorococcum sp., Asterococcus superbus, Chlamydomonas reinhardtii CPCC 12,

11 ACS Paragon Plus Environment

Environmental Science & Technology

244

Chlamydomonas reinhardtii CPCC 243, and Eremosphaera viridis, respectively. When exposed

245

to 1000 µg/L triclosan, all algae species had the highest inhibition rates. With decreasing

246

exposure doses of triclosan, the inhibition rates gradually reduced. Chlamydomonas reinhardtii

247

CPCC 12 was the most sensitive species, while Eremosphaera viridis was the least sensitive. It

248

indicated that different concentrations of triclosan caused diverse toxic effects on different algae

249

species, and the inhibition would be alleviated as time went on. Lawrence29 reported an increase

250

in algal biomass occurred under 4 weeks’ triclosan stress, however the biomass did not reached

251

the same level as that under the unexposed control. Our study also presented a trend towards

252

biomass resilience and recovery with increased exposure time. Moreover, when Eremosphaera

253

viridis was exposed to 0.56 and 0.0862 µg/L of triclosan, a stimulation of growth rates was

254

observed compared with that in control. This was also similar with the study of Pomati30, in

255

which there was a growth stimulation of phytoplankton populations under low doses of triclosan

256

(below 10 μg/L). Such a different performance at low levels of triclosan was likely due to the

257

different sensitivities of algae species, or the different MoA.

258 259

3.3. Cell size and pH variation analyses

260 261

Besides biomass variation, the toxicity also presents impacts on cell size, as shown in Figure S6.

262

The normal average diameter of Asterococcus superbus was 30-35 µm, reducing 48.05 % when

263

exposed to 1 000 µg/L triclosan. The size of Chlorococcum sp. was 8-17 µm in diameter and

264

reduced 16.23 % when exposed to 1 000 µg/L triclosan. Eremosphaera viridis had the largest

265

cell size with 144 µm in diameter and reduced its size by 4.23 % when exposed to 1 000 µg/L

266

triclosan. For Chlamydomonas reinhardtii CPCC 12, intact cells had disappeared when exposed

12 ACS Paragon Plus Environment

Page 12 of 34

Page 13 of 34

Environmental Science & Technology

267

to 1 000 µg/L triclosan and the size could not be measured. Triclosan had little effects on the cell

268

size of Chlamydomonas reinhardtii CPCC 12 and 243 (10 µm in diameter) at all exposure doses.

269

As the smallest algae, they kept their size consistent to untreated cells, whereas the others had

270

various variation on cell size. It is likely because larger cells have more membrane-structured

271

components, which are easier to be attacked by triclosan. When membranes are damaged, cells

272

would become smaller due to cell shrinking.

273 274

A relationship exists between cell size and cell growth. There is a negative correlation between

275

cell size and maximum specific growth rates.31 Larger cells with spherical shape usually have

276

lower growth rate, because they would have longer diffusion pathways from the surface and less

277

competitive to nutrient uptake and growth, compared with smaller cells.32 The sensitivity to a

278

toxicant can be well predicted based on the cellular surface/volume ratio, in which the smaller

279

size is corresponding to the higher sensitivity.33 When exposed to triclosan, Chlamydomonas

280

reinhardtii CPCC 12 and 243, with the smallest size, indeed presented higher growth sensitivity.

281

As the largest algae with the smallest surface/volume ratio, Eremosphaera viridis had the lowest

282

growth sensitivity. As for Chlorococcom sp., even though it has a smaller cell size, a less

283

sensitive growth to triclosan was observed, which was likely due to its colonial property. The

284

colonial-kind growth form enabled the exchange of metabolites among neighbours, in some of

285

which had division of labor among the cells to speed up the biotransformation of triclosan.32

286

During the agglomeration, Chlorococcom sp. could escape the constraints of size-dependent

287

growth and achieve higher growth rates than unicells with a similar size under triclosan exposure.

288

pH variation analysis is included in Supporting Information (Figure S7).

289

13 ACS Paragon Plus Environment

Environmental Science & Technology

290

3.4. Photopigment variation and chlorophyll fluorescence analyses

291 292

Triclosan is known to exhibit multiple toxic effects on photosynthetic process, including

293

uncoupling of oxidative phosphorylation, inhibition of non-photochemical quenching and

294

damage in photopigments.34 In our current study, the changes of chlorophyll a/b concentrations

295

and chlorophyll fluorescence parameters were not consistent. As shown in Figure S8, for

296

chlorophyll a/b, there was a good trend of dose-response for Chlorococcum sp., Chlamydomonas

297

reinhardtii CPCC 12 and 243. Chl a and b contents showed significant decrease (p