Insights into Long-term Toxicity of Triclosan to Freshwater Green

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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

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Environmental Science & Technology

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

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Lake Erie

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

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a Institute

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Canada S4S 0A2

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

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b

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Canada H3G 1M8

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c Department

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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

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

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[email protected]

1 ACS Paragon Plus Environment


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Abstract:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Erie

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1. Introduction

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

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

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

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

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

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

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

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

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

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

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comprehensive evidence of triclosan toxicity to the ecosystem.

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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was no comparison between Chlamydomonas reinhardtii and other algae species regarding

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

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

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

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

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

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


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

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required.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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risk management for triclosan application.

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2. Materials and methods

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2.1. Chemicals

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

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

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

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define the pH of buffer and its composition)

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2.2. Algal culture and toxicity test

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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2.3. Cell densities and cell viability

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

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

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

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

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

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

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

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

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(Mettler Delta 320, Halstead, UK).

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2.4. Measurement of chlorophyll concentration and chlorophyll fluorescence

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

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

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

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

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

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



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

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and photochemical quantum yield, respectively.

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

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spectromicroscopy

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

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

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

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

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

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

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sample.21

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

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

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

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

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

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

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

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

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wave-numbers.


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2.6. Determination of triclosan concentration

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

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

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

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

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

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

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

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

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

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used for detection was 214 nm.

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2.7. Statistical analysis

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

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

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

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

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

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

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



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

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

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

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

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3. Results and Discussions

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

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

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

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

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

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

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

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

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3.1. Measured triclosan concentrations

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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various long-term effects.

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3.2. Biomass variation analyses

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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different sensitivities of algae species, or the different MoA.

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3.3. Cell size and pH variation analyses

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

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

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

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

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

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


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

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

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

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

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

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would become smaller due to cell shrinking.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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3.4. Photopigment variation and chlorophyll fluorescence analyses

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

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

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

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

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

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

Insights into Long-term Toxicity of Triclosan to Freshwater Green

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