Interactive Toxicity of Triclosan and Nano-TiO2 to Green Alga

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Ecotoxicology and Human Environmental Health

Interactive Toxicity of Triclosan and Nano-TiO2 to Green Alga Eremosphaera viridis in Lake Erie: A New Perspective based on Fourier Transform Infrared Spectromicroscopy and Synchrotron-based X-ray Fluorescence Imaging Xiaying Xin, Gordon Huang, Chunjiang An, and Renfei Feng Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.9b03117 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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Environmental Science & Technology

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Interactive Toxicity of Triclosan and Nano-TiO2 to Green Alga Eremosphaera viridis in

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Lake Erie: A New Perspective based on Fourier Transform Infrared Spectromicroscopy

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and Synchrotron-based X-ray Fluorescence Imaging

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Xiaying Xin,a Gordon Huang,a,* Chunjiang Anb, Renfei Fengc

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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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Montreal, Canada H3G 1M8

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c

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

Canadian Light Source, Saskatoon, Saskatchewan, Canada S7N 2 V3

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*Corresponding author: Tel: +1-306-585-4095; Fax: +1-306-585-4855; 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 toxicity of triclosan in the presence of TiO2 P25 to the green alga

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Eremosphaera viridis in Lake Erie. Multiple physicochemical endpoints were conducted to

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perform a comprehensive analysis of the toxic effects of individual and combined pollutants.

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Fourier transform infrared spectromicroscopy and synchrotron-based X-ray fluorescence

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imaging were first documented to be applied to explore the distribution variation of

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macromolecules and microelements in single algal cells in interactive toxicity studies. The

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results were different based on different triclosan concentrations and measurement endpoints.

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Comparing with individual pollutants, the toxicity intensified in lipids, proteins and oxidative

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stress at 1000 and 4000 µg/L triclosan in the presence of P25. There were increases in dry

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weight, chlorophyll content, lipids, and catalase content when cells were exposed to P25 and

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15.625 µg/L triclosan. The toxicity alleviated when P25 interacted with 62.5 and 250 μg/L

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triclosan compared with triclosan-only exposure. The reasons could be attributed to the

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combination of adsorption, biodegradation, and photocatalysis of triclosan by algae and P25,

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triclosan dispersion by increased biomass, triclosan adherency on algal exudates, and triclosan

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adsorption site reduction on algae surface owing to P25’s taking-over. This work provides new

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insights into the interactive toxicity of nanoparticles and personal care products to freshwater

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photosynthetic organisms. The findings can help with risk evaluation for predicting outcomes of

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exposure to mixtures, and prioritize further studies on joint toxicity.

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Keywords: Interactive toxicity, Fourier transform infrared spectromicroscopy imaging,

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Synchrotron-based X-ray fluorescence imaging, Eremosphaera viridis, Lake Erie

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1. INTRODUCTION

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Triclosan (2,4,4’-trichloro-2’-hydrixydupheyl ether) is a broad-spectrum antimicrobial

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compound, incorporated with numerous personal care products including soaps, detergents,

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cleaners, toothpastes, and deodorants.1 The widespread application of triclosan has led to its

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presence in wastewater effluents, biosolids, and receiving surface waters,2 resulting in direct

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exposure to aquatic organisms. Triclosan can inhibit fatty-acid synthesis, denature protein, and

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breakdown chlorophyll pigments of algae.3 Triclosan may cause unexpected health problems or

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environmental risks to higher trophic organisms through food chains.

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On the other hand, titanium dioxide nanoparticles (nano-TiO2) have been widely used as

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ingredients in many commercial products, such as pigments, sunscreens, paints, ointments and

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toothpaste, because of its high brightness, high refractive index, and anti-UV function.4 Global

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demand for TiO2 was 6.00 million tons as valued at US$13.50 billion in 2014, and is expected to

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reach US$17.00 billion in 2020.5 The widespread use of TiO2 could accidentally or inevitably

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make their ways into freshwater ecosystem.4 Once released to the aquatic environment, nano-

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TiO2 may interact with concomitant triclosan vigorously.6 The unique properties of nano-TiO2

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include: (a) high specific surface area, (b) abundant reactive sites on the surface due to a large

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fraction of atoms located on the exterior, and (c) high mobility.7 These could change the state of

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triclosan and further alter its toxicity. The relevant interactive impacts on algae have been rarely

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known.

62



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Toxicological studies have documented that pollutant mixtures exert toxic effects differing from

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individual ones. Although the individual effects of triclosan and nano-TiO2 have been well

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established, the environmental risks of their interactions have not been explored. The previous

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efforts mainly focused on the toxicity, involving organic-organic mixtures, metal-metal mixtures,

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and organic-metal mixtures; however, the toxicology studies regarding the organic-nano metal

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oxides mixtures are very limited. Previous study results are simplex, either enhancing or

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reducing toxicity of organic pollutants to algae when interacting with nano metal oxides. For

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example, Wang6 reported that the toxicity associated with the exposure of florfenicol to

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Chlorella pyrenoidosa was enhanced by the presence of nano-TiO2. Zhang8 found that the co-

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occurrence of hexachlorobenzene and nano-TiO2 led to an antagonist effect of joint toxicity to

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Chlorella pyrenoidosa. Wang9 stated that Suwannee River fulvic acid could significantly

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increase the toxicity of CuO NPs to Microcystis aeruginosa. However, in natural waters, the

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concentrations of pollutants are various, resulted from temporal and spatial variation of

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environmental conditions. The corresponding toxic effects might be varied with changing

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pollutant concentrations. Lu10 indicated that the toxicity of phenanthrene to Artemia salina

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increased by 2% in the presence of 5 mg/L nano-TiO2, but decreased by 24.5% in the presence of

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400 mg/L nano-TiO2. Thus, the toxic effects on algae are variable because of varied pollutant

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concentrations.

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Multiple bulk-measurement endpoints have be used to assess toxicity to algae, providing the

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ability to potentially characterize multiple target sites.11 Zhao12 studied the role of Al2O3 in

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altering graphene oxide toxicity to Chlorella pyrenoidosa through algal-growth-inhibition

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assays, physical-damage observations, and biochemical tests for quantifying membrane damage


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and oxidative stress. Tang13 examined the effects of nano-TiO2 and Zinc on the photosynthetic

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capacity and the survival of Anabaena sp. using algal cell densities, chlorophyll fluorescence

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parameters, and reactive oxygen species (ROS) production. However, techniques capable of

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imaging biochemical alteration for single living cells and directly visualizing micro-element

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distribution for single cells are still not available. The traditional way for determining

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macromolecules fails to provide an accurate quantification owing to the lost amount in extraction

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process during in-vitro tests, and is also not able to obtain visualized information for an

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individual cell.14 Previous studies on micro-element distribution only focus on the interaction of

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nano-TiO2 and a specific element.15, 16 Few studies consider multi-element concurrent uptakes in

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the presence of TiO2. Thus, effective techniques with the ability to image multiple bio-molecules

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and micro-elements are desired for helping reveal the mechanism of toxicity.

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The aim of this study is to explore the interactive impacts of nano-TiO2 and triclosan on green

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alga Eremosphaera viridis under visible light using Lake Erie water. The use of natural water has

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more practical significance to real-world toxicity. The interactions of nano-TiO2 and triclosan

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will be investigated to verify whether there exists adsorption or photocatalysis of triclosan, and

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whether this will modify the toxicity of triclosan to an aquatic photosynthetic organism. Multiple

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algal physicochemical endpoints will be quantified to assess the interactive effects caused by co-

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occurrence of triclosan and nano-TiO2, and to examine the mode(s) of the toxicity. Fourier

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transform infrared spectromicroscopy imaging and synchrotron-based X-ray fluorescence

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imaging will be applied to explore the distribution variation of macromolecules and

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microelements in single cells under exposure of triclosan, nano-TiO2 and their combinations.

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This is the first study to systematically explore the potential interactive effects of nanoparticles



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and personal care products on algae. It is also an initial attempt to document the alteration in the

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distribution of macromolecular components and multiple microelements at a single-cell scale in

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toxicity study. This work can help with risk evaluation for predicting outcomes of exposure to

112

mixtures, and prioritize further studies on joint toxicity.

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2. MATERIALS AND METHODS

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2.1 Materials and Characterization

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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. TiO2 (Degussa P25) powder with an average particle size

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of 25nm was purchased from Degussa AG Company (Germany). P25 stock solutions (500 mg/L)

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were prepared in Milli-Q water (Millipore, 18 MΩ·cm); a 30 min-sonication in ultrasonic bath

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was employed before diluting the stock solutions to the desired concentrations. The morphology

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of P25 was examined using a transmission electron microscope (TEM, Hitachi 7600, Japan).

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Hydrodynamic diameter and zeta potential were measured using a Zetasizer Nano ZS (Malvern,

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United Kindom). All other chemicals were of reagent grade or higher.

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All the experiments were conducted using Lake Erie Water (LEW) to access how triclosan and

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P25 would behave in a freshwater matrix. Surface water in Lake Erie was collected and

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immediately transported to our laboratory where it was stored at 4 °C after filtration through a

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0.2 µm-filtration (NALGENE). Triclosan adsorption and P25 photocatalysis experiments were

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described in Supporting Information.


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2.2 Algal culture and toxicity test

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The freshwater green alga Eremosphaera viridis (referred to hereafter as Eremosphaera) was

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obtained from the Canadian Phycological Culture Center (CPCC, University of Waterloo,

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Canada), and cultured in sterile Bold Basal Medium (BBM). It was chosen for this work because

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its large cell diameter (approximately 140 µm) and unicellular growth habit make it very suitable

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for both infrared and X-ray imaging (Figure S1). The algal cells were cultured at 23 ± 1 °C on a

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12-h light and 12-h dark cycle. Light was provided by 40 W white fluorescent lamps (Philips

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F40 T12/DX, Canada). In the exposure experiment, the algae at the logarithmic growth phase

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were collected, washed and then diluted by LEW to the initial concentration of 2 × 105 cells mL-

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1.

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15.625, and zero μg/L, in the absence and presence of 5 mg/L P25. After 120 h, triclosan in

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supernatant was concentrated by solid phase extraction17 and triclosan in algae was extracted

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following Ding’s method.18 Triclosan concentration was measured by using Agilent 1260 liquid

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chromatograph.

The algal cells were exposed to triclosan at concentrations of 4 000, 1000, 250, 62.5, and

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2.3 Cell morphology and ultrastructure observations

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Algal biomass quantification by dry weight determination and Chlorophyll a/b concentrations

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were revealed in Supporting Information. Surface morphology of cells grown for 120h was

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investigated using scanning electron microscopy (SEM). SEM images were obtained using a FE-

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SEM-cold field emission scanning electron microscope (Hitachi SU8010, Japan). Transmission



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electron microscopy (TEM) was also used to investigate cellular ultrastructure. TEM images

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were obtained on a transmission electron microscopy (Hitachi HT7700, Japan). The details of

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SEM and TEM were included in Supporting Information.

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2.4 Elemental distribution for single cells through synchrotron-based X-ray fluorescence

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imaging

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Cells were harvested after 120 h by centrifugation at 2268 g for 15 min. The cells were

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resuspended and centrifuged again. After three times of media washing, the final concentrated

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biomass was frozen in liquid nitrogen and dried in a freeze-drier (Labconco Co., USA) at -50 °C

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for 24 h. Dried samples were used for acquiring micro X-ray fluorescence imaging (XFI) for five

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elements including Ti, Cu, Mn, Ca, Fe and Zn at the VESPERS (very sensitive elemental and

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structural probe employing radiation from a synchrotron) beamline of Canadian Light Source

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(CLS), Saskatoon, Canada. The storage ring of the CLS operates at an energy of 2.9 GeV and a

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current range of 250-150 mA. The features of the VESPERS beamline have already been

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described extensively by Feng.19 Briefly, the beamline consists of a microprobe employing hard

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X-rays in the energy range of 5-30 keV to illuminate a microvolume of samples. The spot size of

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the beam ranges from 2 × 2 µm to 5 × 5 µm. Details were included in Supporting Information.

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2.5 Biochemical alteration for single living cells through Fourier transform infrared

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spectromicroscopy imaging

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Cells were harvested after 120 h by centrifugation at 2268 g for 15 min. The biomass was

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carefully resuspended in distilled water and centrifuged again. This process was repeated three

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times. Algal cells were resuspended in 30 μL of D2O, and then loaded onto the optical

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

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

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around the edge of the windows to compensate for evaporation from the sample.14 FTIR

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spectromicroscopy measurements were carried out on beamline 01B1-01(MidIR) at CLS,

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Saskatoon, Canada. A Bruker Vertex 70v interferometer coupled to a Hyperion 3000 IR confocal

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microscope with a 15× objective was applied to acquire the images using a globar (Figure S2).

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Samples were maintained under a dry nitrogen purge to remove CO2 and H2O interference.

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Spectral maps were acquired using transmission mode, 4 cm-1 resolutions with 128 co-additions,

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64×64 pixel mid-infrared FPA detector (Bruker Optics, MA, USA).

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2.6 Oxidative stress

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Intracellular ROS was measured using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA,

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BioVision Incorporated, USA). The details were included in Supporting Information. The

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fluorescence was measured using a BioTek microplate reader (Winooski, VT, US; λex = 485 nm;

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λem = 528 nm). Mitochondrial membrane potential and catalase activity were described in

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Supporting Information.

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2.7 Statistical analysis

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All experiments were carried out at least in 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). Synchrotron-based XFI

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images was generated by using VESPERS GUI to normalize the data relative to the ionization

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chamber values, register the images and produce multi-channel visualizations.20 The images

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were created from the raw data using SigmaPlot Version 12.0 software (Systat software Inc., CA,

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USA). Principal component analysis (PCA) was performed to visualize the correlation of

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relevant responses and their distinctions using SPSS 18.0 (IBM, NY, USA). FTIR spectra maps

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were collected using OPUS 7.2 software (Bruker Optics, MA, USA) over the mid-infrared range

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(4000-900 cm-1). At least 3-5 cells for each sample were selected randomly. The images were

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generated using CytoSpec 2.00.01 software (Cytpspec Inc., MA, USA). Other data processing

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and figure drawing were conducted using Origin Pro 8.0 software (Origin lab Co., MA, USA).

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3. RESULTS AND DISCUSSIONS

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3.1 Effects on cell morphology and ultrastructure

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Experiments on the interaction of triclosan and P25 showed triclosan adsorption by P25 occurred

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but was mild, and the degradation rate of triclosan in visible light was low, no matter it was due

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to photolysis or photocatalysis. Detailed analyses on the interaction of triclosan and P25 were

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revealed in Supporting Information. There is a good dose-response trend for the interactive

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effects of triclosan and P25 on dry weight and chlorophyll a/b content of Eremosphaera. The


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presence of P25 can increase algal biomass and chlorophyll pigments, and further alleviated the

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toxicity of triclosan. However, P25 can cause inhibition in algal biomass and chlorophyll

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pigments when interacting with triclosan in a range from 250 to 4000 µg/L. Coexistence of P25

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and triclosan at 15.625 µg/L could cause stimulated growth on Eremosphaera. Such interaction

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increased the complexity of the toxicity to algae. Detailed analyses on biomass responses (dry

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weight and Chlorophyll a/b) were revealed in Supporting Information.

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The changes on cell surface impacted by triclosan and P25 were observed using SEM (Figure 1).

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SEM images in Figure 1A and B show untreated cells had round shape with intact and smooth

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surface, indicating an uncompromised cell membrane. The exudates released by algae were

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observed around cells. After exposure to 1000 µg/L triclosan, all cells had morphological

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deformity, and aggregated more tightly than untreated cells. The representative single cell shrank

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with some wrinkles on the external surface (Figure 1C and D). In Figure 1E and F, P25

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aggregated on the outside of the cells. Some of them had intact surface, and some had slight

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wrinkles. The co-exposed cells showed aggregation and attachment of cells forming a network-

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like rough surface with altered morphology (Figure 1G and H). Dramatic alterations in cell wall

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including cell surface disruption and shrinkage occurred. Extensive surface irregularity was also

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found, indicating the occurrence of cell wall rupture and degradation. The observed aggregates

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were probably P25-exudate mixture.

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TEM images in Figure S10 are shown to compare internal ultrastructure of cells exposed to

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triclosan with and without P25. The cells exposed to 1000 µg/L triclosan in Figure S10 (A) and

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(B) had thinner cell membrane, less cellular organelles, and more sparse thylakoids than cells in



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the presence of P25 at the same triclosan concentration. The chloroplast membrane disappeared

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and the inner stroma spilled out. The cytoplasm had become vacuolated. However, there was no

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significant ultrastructure damage found in co-exposed cells. The internalization, owing to

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endocytosis, was observed from black-colored particles in cytoplasm in Figure S10 (C) and (D).

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These particles were further analyzed by TEM-EDS. The comparison with the standard lattice

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index of TiO2 showed that these particles were TiO2. It indicated that P25 penetrated the cell

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wall and plasma membrane, and located inside cell. For cells only exposed to P25, the weight

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percent of Ti was 0.021%, while for cells under co-exposure of P25 and 1000 µg/L triclosan, the

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weight percent of Ti was 0.696%. It is known that triclosan can cause algal membrane disruption

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by inhibiting fatty acid synthesis and causing protein aggregation.21 Thus, the damage to cell

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membrane from triclosan aggravated the internalization of P25. The concentration of triclosan

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was shown in Supporting Information.

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3.2 Ti introduction and distribution with association of endogenous elements

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Synchrotron-based XFI is attractive for determining the distribution of elements because this

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multi-element spectroscopy technique allows co-localization studies, with a high sensitivity and

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spatial resolution. Recent development of high brilliance and high energy synchrotrons, coupled

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with advances in focusing optics has brought significant improvement in micrometer probes for

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imaging applications.22 Due to conducting analyses in air, little sample preparation is needed. It

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is also a non-destructive approach, providing the high penetration power of X-ray and short

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collecting time.23 Although synchrotron-based XFI is becoming widely applied in plant and

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animal tissues, there are still few studies on in situ element distribution in algae. The current


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study used synchrotron-based XFI to determine the distribution of Ti and endogenous elements

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in Eremosphaera exposed to triclosan in the absence and presence of P25. Five elements

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including Ti, Fe, Cu, Mn, and Ca were analyzed. Fe is an essential micro-nutrient for algae,

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notably involved in photosynthesis as a redox-active metal. Since chloroplasts contain up to 80%

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of the cellular Fe, its distribution is therefore a good indicator of the chloroplast’s location in the

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observed cell.24 Cu is also essential for photosynthesis and mitochondrial respiration, and

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oxidative stress protection.24 Mn is essential for algal metabolism and development and occurs in

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oxidation states II, III, and IV in approximately 35 enzymes.25 Ca is rich in a few of the electron-

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dense spherical structures, which are presumably polyphosphate bodies containing

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polyphosphate complexed with Ca.26

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Figure 2 presents the images of the distribution of five elements in a single algal cell exposed to

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a series of triclosan in the absence and presence of P25. In Figure 2 (A), the distribution of

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element Fe, Cu, Ca and Mn was very clear and there was no Ti in the untreated cell. Fe and Cu

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located around the periphery of cell. The distribution of Ca and Mn were in the same area, which

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is consistent with Rao’s finding.27 It suggested that elements had specific distribution patterns,

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some being partially or entirely co-localized. When cells were exposed to 250 µg/L triclosan

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(Figure S12 (A)), Cu accumulated more than Fe, and the accumulation of Ca and Mn had been

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inhibited. The inhibition of Mn suggested that protein might be affected by triclosan.28 There was

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still no Ti in the cell. When introducing P25 (Figure 2 (B)), Ti occurred around the cell and the

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concentrations of all other elements decreased. P25 inhibited the accumulation of Ca, Mn, Fe and

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Cu, which is probably because P25 may bind those metal, and thus prevent the uptake by cells.

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As triclosan increased from 15.625 to 1000 μg/L in the presence of P25 (Figure S12 (B) - (D)



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and Figure 2 (B) - (C)), the concentrations of Ti, Fe, Cu and Mn increased. The accumulating

293

amount decreased with the order of Ti > Fe > Cu > Mn. The accumulation of Ca had been totally

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inhibited.

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Cell wall is the primary site for P25 interaction with algal cells and acts as a key barrier to P25

297

uptake.29 When the size of P25 aggregates is smaller than that of the cell wall pore (3-50 nm),30

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P25 can enter the cell by endocytosis, diffusion or the action of carrier proteins.7 However, P25

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aggregates have hydrodynamic diameter much larger than the pore size. The internalization of Ti

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increased with increasing triclosan concentrations. The collection of Ti could be due to the

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damaged cell wall under elevated triclosan concentrations, resulting in more P25 entering the

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cell.31 Although P25 aggregates had larger size at higher triclosan concentrations, Ti

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internalization was more attributed to membrane permeability under triclosan attack than the

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influence of aggregate size. When more P25 and triclosan entered the cells simultaneously, the

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chance to direct contact with cellular inclusion and disturb their function would increase and then

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result in more serious effects on algae.

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The interrelationships of co-localization among five elements under different treatments in

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Eremosphaera were further investigated using PCA. As shown in Figure 3, the distribution of

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Cu, Mn and Ca was closely correlated, because they all located at the positive side of PC1 and

311

were adjacent to each other in untreated cells. Five elements in triclosan exposed cells all located

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at the positive side of PC2 but were totally separated. When cells were exposed to P25, the

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collection of Mn and Ca were correlated because they were close to each other. The same

314

situation happened to the relation between Fe and Cu. Under the interaction of P25 and triclosan,


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the accumulation of Ca, Fe, and Cu were closely related. The distribution of Ti was also getting

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closer to all other elements because the introduction of Ti affected the distribution of other

317

elements. It can be seen that the different treatments could affect the uptake of those endogenous

318

elements and their quantitative relationship, leading to the alteration of micro-element

319

distribution.

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3.3 Imaging quantification of biochemical alteration

322 323

The application of FTIR spectroscopy to study biological tissue usually uses a reflecting

324

microscope with mid-IR light to obtain detailed molecular information.32 Despite of the

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simplicity of FTIR spectromicroscopy method and the extensive characterization of spectral

326

fingerprints, the approach remained uncommon in unicellular living algae study. Micrasterias

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hardyi is the only algae which has been investigated in spatial imaging.33, 34 FTIR

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spectromicroscopy is a robust and reliable method to investigate the chemical composition of

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single living cell in the absence of distinctive morphological features.35 IR imaging can be

330

applied as a complementary method to discover gathered biomolecular information and explore

331

biochemical alteration through distinct absorption bands of chemical groups simultaneously.36

332 333

Lipids and proteins are two main macromolecules being investigated. The dominant absorption

334

features of lipid spectra are in the region 3000-2800 cm-1 and the strong band at 1740 cm-1. Both

335

are assigned to asymmetric and symmetric (CH)n stretching vibrations and ester C=O groups,

336

respectively. The protein spectrum mainly focuses on Amide I (1710-1585 cm-1), which arises



337

primarily from C=O stretching vibration of the peptide backbone. Amide I is particularly

338

sensitive to protein secondary structure.37

339 340

Figure 4 shows the spatial imaging of the distribution of major bands for lipids and proteins in

341

the Mid-IR spectra of Eremosphaera cells. The same single cell was imaged to get the chemical

342

group distribution information for each treatment. With increasing triclosan, the amount of (CH)n

343

became more sparse and were gathered towards to one direction. (CH)n concentrations in only

344

P25-exposed cells were higher than that in untreated cells. When P25 interacted with 15.625

345

µg/L triclosan, (CH)n concentration was higher than that in P25-only exposed cells. When cells

346

were exposed to both triclosan and P25, its distribution seemed more homogeneous compared to

347

the corresponding triclosan exposure. The similar trend occurred for the distribution of C=O, but

348

the impacts seemed less than that of (CH)n. The cell co-exposed to P25 and 4000 µg/L triclosan

349

had blurry periphery, indicating both chemical groups on the cell wall and cell membrane had

350

been seriously damaged. However, Amide I concentrations gradually became less with the

351

elevation of triclosan, but its distribution was homogeneous until exposed to higher levels of

352

triclosan. There was no obvious boundary in cellular periphery observed from the distribution of

353

Amide I. Amide I concentrations in only P25-exposed cells were higher than that in untreated

354

cells. Under the interaction of P25 and triclosan at two highest amounts (1000 and 4000 µg/L),

355

the amount of Amide I decreased obviously, even less than that in triclosan-only exposed cells.

356

The cell size was observed to become smaller with increasing triclosan concentrations. Thus,

357

lipids and proteins had been found to be reduced with increasing triclosan concentrations.

358

Proteins had less toxic impacts from triclosan/P25 co-exposure. P25 alleviated the molecular


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toxicity of triclosan below 1000 µg/L, but intensified the effects when interacting triclosan

360

beyond 1000 µg/L.

361 362

It is known that triclosan can affect multiple cytoplasmic and membrane targets.38 Triclosan can

363

not only act on the gene fabI to block the incorporation from acetate into lipids to inhibit the

364

synthesis of fatty acids and lipids39, but also specifically inhibit enoyl-acyl carrier protein

365

reductase to stop the final and regulatory step in fatty acid synthase cycle.40 Thus, triclosan can

366

block fatty acid and lipid synthesis. Phospholipid fatty acids can be regarded as another reliable

367

predictors of algal biomass besides chlorophyll a.41 The variation of major bands for fatty acids

368

is consistent with that of chlorophyll content. The inhibition and stimulation of fatty acids

369

represent the amount change in available photochemical energy in response to the fluctuation in

370

chlorophyll pigment.42 Moreover, triclosan can change protein secondary structure to cause

371

protein aggregation.21 Amide I is directly related to the backbone conformation of protein

372

secondary structure.43 The frequency of the Amide I band is responsive for different hydrogen-

373

bonding environments about α-helix and β-sheet conformations.44 In detail, the breakdown of α-

374

helices structure and the elevation of β-sheets structure suggest that severe oxidative

375

stress renders the cell to be unable to synthesize aromatic amino acids with triclosan exposure.45

376 377

It is interesting to find P25 at 5mg/L stimulated lipid and protein syntheses of Eremosphaera in

378

visible light. Chen’s study46 verified this by reporting that the soluble protein content in the low

379

dosage groups (1 and 10 mg/L nano-TiO2) of Chlamydomonas reinhardtii was higher than that in

380

the untreated groups. It may be due to the stimulation of de nova protein synthesis, which plays

381

an important role in cell adaptation to nano-TiO2 exposure.46 The interaction of P25 and high



382

levels of triclosan caused synergetic effects on lipids. It is probably because membrane

383

disruption was initially resulted from triclosan attack, leading to the formation of pores to make

384

the membrane more permeable to internalise P25,47 and then the disruption was enhanced by the

385

interaction of triclosan and P25 inside the cell. Initiated lipid peroxidation by P25 can further

386

lead to inactivation of enzymes and proteins, and finally result in cell death.48 Once within the

387

cell, nano-anatase may cause autophagy and change the structures of pepsin and trypsin, leading

388

to decreased enzyme activity.49, 50 P25 can also convert protein secondary structure from an α-

389

helix to a β-sheet, leading to its dis-configuration.51 Moreover, since the variation of interactive

390

conditions affects the uptake of micro-elements, it would also have an influence on element-

391

related enzymes. For example, ferritin, as an iron-storage protein, plays an important role in

392

sequestering or releasing Fe on demand.52 Fe collection would affect ferritin synthesis. SOD

393

enzymatic system was comprised of Mn-SOD, Fe-SOD, and CuZn-SOD.53 The uptake limitation

394

of Mn, Fe, Cu, and Zn would cause downregulation of SOD enzyme. Thus, there are many direct

395

and indirect factors influencing protein amount under co-exposure.

396 397

3.4 Responses of oxidative stress

398 399

It is hypothesized that the toxicity was dependent on ROS derived from triclosan, internalization

400

of P25, and their interaction. We examined the responses of the mitochondrion membrane and

401

antioxidant defense enzyme to intracellular ROS generation in Eremosphaera. Responses of

402

mitochondrion membrane potential were revealed in Supporting Information.

403


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404

Generally, the exposure of contaminants could often result in increased production of ROS. ROS

405

is a by-product of metabolism, which is important for cell normal functioning, such as chemical

406

defense against predators and oxidizing or reducing toxic metals.54, 55 However, when present in

407

excess, ROS can react with biomolecules to modify or destroy their inherent function, or make

408

other exudates more toxic by oxidizing them.56 In Figure 5, when cells exposed to triclosan at

409

15.625 and 62.5 µg/L, ROS production increased insignificantly compared to that in untreated

410

cells. As triclosan concentrations increased above 250 µg/L, there was decreasing ROS

411

production, but it was still significantly higher than that in untreated cells. Cells exposed to P25

412

had 163% ROS of untreated cells. In the presence of P25, ROS production had similar dose-

413

response relationship with that in triclosan-only exposure, but had significant lower value than

414

that in the absence of P25 when triclosan concentration was between 250 and 1000 µg/L.

415 416

The trend in ROS production was similar with that in chlorophyll content, mitochondrial

417

membrane potential and the distribution variation of lipid- and protein-related functional groups.

418

Firstly, in algae, ROS are normally produced by the leakage of reactive electrons from electron

419

transport activities occurring in the chloroplast, mitochondria and plasma membrane.57 When

420

there is an imbalance between ROS production and the cellular antioxidant defense mechanisms,

421

oxidative stress increases, leading to mitochondrial malfunction, chloroplast disruption,

422

apoptosis and even cell death.58 Thus, ROS production had a positive correlation with

423

chlorophyll pigment and mitochondrial polarization. Secondly, triclosan at 15.625 and 62.5 µg/L

424

had little effects on ROS production, indicating hormetic effects on lipids and proteins had no

425

direct relations with ROS. When triclosan went beyond 250 µg/L, ROS production significantly

426

increased compared to control, which was consistent with the inhibition of biomolecules. The



427

increase in ROS was associated with oxidative stress, which would result in damage of lipids and

428

proteins.59 Membrane lipids can be oxidized by excess hydroxyl radical. Enzyme activity and

429

ATP production can be disrupted, leading to cell apoptosis finally.60 Proteins can be oxidized by

430

ROS reactions through amino acid modification, peptide chain fragmentation, electrical charge

431

alteration, and ultimate enzyme function inactivation.61

432 433

There are some reasons for the variation of ROS production. On the one hand, triclosan can

434

trigger ROS formation at short time exposure, which seems to overwhelm the detoxifying

435

mechanisms.62 Thus, cells exposed to triclosan had more ROS than untreated cells. However,

436

with the increasing triclosan concentration, a large amount of cell death caused significant

437

decrease in cell density, leading to reduced intracellular ROS. On the other hand, TiO2 are

438

usually not photoreactive in visible light, but it can still generate hydroxyl radicals even with a

439

low level.63 Rutile phase in P25 generally contributed more ROS formation than anatase phase in

440

visible light.64 Due to sufficient surface area for ROS production, it is possible to affect cellular

441

processes with the occurrence of small amount of P25 intake.65 Thus, P25-exposed cells had

442

higher ROS than untreated ones (data not shown). What’s more, the interaction of P25 and

443

triclosan led to the significant decrease of ROS beyond 250 µg/L triclosan. The results are also

444

consistent with other endpoints such as dry weight, cell morphology and ultrastructure. Thus, the

445

toxicity of triclosan to Eremosphaera was alleviated in the presence of P25. This could be

446

attributed to the combined effects of adsorption, biodegradation, and photocatalysis of triclosan

447

by algae and P25, the dispersion of triclosan effects on individual cells due to growth

448

stimulation, the adherency of triclosan by the aggregation of the exudates from algae, and the


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449

reduction of triclosan adsorption position on algae surface owing to P25’s take-over. Responses

450

of catalase were revealed in Supporting Information.

451 452

4. ENVIRONMENTAL SIGNIFICANCE

453 454

A variety of nanoparticles and personal care products are commonly produced and used in

455

industry and consumer products. They will inevitably be discharged into aquatic environment,

456

leading to various mixtures. Nano-TiO2 and triclosan are extensively used, and may exist in

457

natural environment simultaneously. The presence of nano-TiO2 would influence the exposure

458

and bioavailability of triclosan, and consequently alter the toxicity of triclosan to aquatic

459

organisms.

460 461

Our study explored that the interactive effects of nano TiO2 and triclosan on algal multiple

462

endpoints under visible light using natural water. This is the first attempt to explore the

463

environmental risk from the coexistence of nanoparticle and personal care products to algae. The

464

toxicity levels were varied based on triclosan concentrations and measurement endpoints.

465

Synergetic effects occurred under the interaction of P25 and 1000 and 4000 μg/L triclosan,

466

leading to significantly decreased amount of lipids, proteins and seriously affected oxidative

467

stress compared to individual exposure. The membrane damage, resulted from high-level

468

triclosan, facilitated P25 to enter the cell and thus intensify the toxicity. Significant increases in

469

dry weight, chlorophyll pigments, lipids, and catalase content occurred when cells were exposed

470

to both P25 and 15.625 μg/L triclosan, leading to hormesis from low-dose exposure.

471

Antagonistic effects existed when P25 interacted with either 62.5 or 250 μg/L triclosan. This



472

could be due to the combination of adsorption, biodegradation, and photocatalysis of triclosan by

473

algae and P25, the dispersion of triclosan effects by increased biomass, the adherency of

474

triclosan on the exudates of algae, and the reduction of triclosan-adsorption sites on algae surface

475

owing to P25’s taking-over. The exudates might be extracellular polymeric substances (EPS),

476

which plays an important role in the bioavailability of nanoparticles. EPS can act as a barrier

477

against direct physical contact of triclosan with cells. This work is the first documented research

478

in joint toxicity to investigate distribution variation in macromolecular components and multiple

479

microelements at a single-cell scale.

480 481

It is worth noting that the current laboratory-based toxicological studies may over-estimate or

482

under-estimate the real toxicity level of nanoparticle or emerging pollutants in natural waters.

483

Firstly, various interactive effects in complex aquatic environment may alleviate the toxicity of

484

pollutants. There are many factors contributing to antagonistic effects, such as surface

485

adsorption, photodegradation, exposure sequence, natural organic matter addition, and EPS

486

complexation. In natural waters, the toxicity to algae has comprehensive consequences due to

487

complexities in doses of nanoparticle and emerging pollutants, and conditions of environmental

488

media, exposure time and their interactions. Further work should be done to explore the joint

489

toxicity of various pollutant combinations by providing robust methodologies for better

490

toxicology assessment on algae. Secondly, the phenomenon of stimulation needs more

491

consideration than inhibition. Stimulation normally occurs at low exposure levels. In fact,

492

pollutants commonly exist in aquatic environment at low levels, and their joint effects might

493

increase biomass, resulting in algae bloom. The complex components in water sometimes may

494

act as nutrient supplement, or may cause hormesis for algae. The stimulation could also


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495

imbalance the ecosystem and destroy biodiversity, resulting in blooming specific species and

496

thus inhibiting the others’ growth or disrupting their physiological function. Moreover, the light

497

in nature could not be as bright as lab UV at all times. Such less energy light may cause less

498

photocatalytic effects from nanoparticles; this visible light-related effect is ubiquitous, but is

499

usually neglected. Our study has paid special attention to this gap. Last but not the least, higher

500

trophic aquatic organisms might take up different pollutants simultaneously, resulting in

501

enhanced bioavailability and bioaccumulation of multi-pollutant mixtures. Due to those

502

organisms’ complicated physiological functions, the joint toxicity may be more complex, and

503

may lead to a larger disturbance to the ecosystem, which merits further exploration.

504 505

SUPPORTING INFORMATION

506 507

The narrative section shows the methods for triclosan adsorption and P25 photocatalysis

508

experiments, determination of algal dry weight and Chlorophyll a/b concentrations, sample

509

preparation for SEM and TEM, details for synchrotron-based X-ray fluorescence imaging,

510

quantification for ROS generation, mitochondrial dysfunction, and catalase activity.

511

The narrative section also shows results and discussions for verification of triclosan adsorption

512

and P25 photocatalysis, dry weight analysis, chlorophyll a/b analysis, variation of triclosan

513

concentrations, and responses of mitochondrion membrane potential and catalase activity.

514

Figures show the image of green alga Eremosphaera; images of Canadian light source, Mid-IR

515

equipment, and workstation on VESPERS beamline; TEM micrograph of P25; hydrodynamic

516

diameter and zeta potential of P25 aggregates in the presence of different triclosan

517

concentrations; ATR-FTIR spectra for verification of triclosan adsorption onto P25; degradation



518

rate of triclosan in the absence and presence of P25; biomass dry weight; chlorophyll a/b content;

519

TEM images of Eremosphaera; triclosan quantification; partial results for distribution variation

520

of multi-elements in an individual algal cell; mitochondrial depolarization; and catalase

521

activities. Tables show the characteristics of triclosan and the ratios of triclosan concentration

522

variation in nonaqueous phase.

523 524

ACKNOWLEDGEMENT

525 526

This research was supported by the Natural Science and Engineering Research Council of

527

Canada (NSERC), the Canada Foundation for Innovation (CFI) and the Canada Research Chairs

528

Program (CRC). Research described in this paper was performed at the Canadian Light Source

529

(CLS), which is supported by the CFI, NSERC, University of Saskatchewan, Government of

530

Saskatchewan, Western Economic Diversification Canada, National Research Council Canada,

531

and Canadian Institutes of Health Research. The authors are thankful to colleagues of the Mid

532

Infrared Beamline and the VESPERS Beamline at the CLS for providing support in the related

533

measurement and analysis. The authors are grateful to Dr. Harold Weger, Dr. Andrew Cameron,

534

and Joshua R. K. Yoneda from Department of Biology at University of Regina for providing

535

experiment support and valuable comments. The authors are particularly grateful to the editors

536

and the anonymous reviewers for their insightful comments and suggestions.

537


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(40) Levy, C. W.; Roujeinikova, A.; Sedelnikova, S.; Baker, P. J.; Stuitje, A. R.; Slabas, A. R.; Rice, D. W.; Rafferty, J. B. Molecular basis of triclosan activity. Nature 1999, 398, (6726), 383. (41) Napolitano, G. E. The realationship of lipids with light and chlorophyll measurements in freshwater algae and periohyton1. J. Phycol. 1994, 30, (6), 943-950. (42) Stigum, V. M. The effect of light and temperature on lipid production in microalgae. 2012, 1-58. (43) Kong, J.; Yu, S. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim. Biophys. Sin. 2007, 39, (8), 549-559. (44) Miller, L. M.; Bourassa, M. W.; Smith, R. J. FTIR spectroscopic imaging of protein aggregation in living cells. Biochim. Biophys. Acta Biomembr. 2013, 1828, (10), 2339-2346. (45) Benov, L.; Fridovich, I. Why Superoxide Imposes an Aromatic Amino Acid Auxotrophy on Escherichia coli the transketolase connection. J. Biol. Chem. 1999, 274, (7), 4202-4206. (46) Chen, L.; Zhou, L.; Liu, Y.; Deng, S.; Wu, H.; Wang, G. Toxicological effects of nanometer titanium dioxide (nano-TiO2) on Chlamydomonas reinhardtii. Ecotoxicol. Environ. Safety 2012, 84, 155-162. (47) Melegari, S. P.; Perreault, F.; Costa, R. H. R.; Popovic, R.; Matias, W. G. Evaluation of toxicity and oxidative stress induced by copper oxide nanoparticles in the green alga Chlamydomonas reinhardtii. Aquat. Toxicol. 2013, 142, 431-440. (48) Pinto, E.; Sigaud‐kutner, T. C.; Leitao, M. A.; Okamoto, O. K.; Morse, D.; Colepicolo, P. Heavy metal–induced oxidative stress in algae1. J. Phycol. 2003, 39, (6), 1008-1018. (49) Pérez-Pérez, M. E.; Lemaire, S. D.; Crespo, J. L. ROS and autophagy in plants and algae. Plant Physiol. 2012, 160, 156-164. (50) Wang, W.-R.; Zhu, R.-R.; Xiao, R.; Liu, H.; Wang, S.-L. The electrostatic interactions between nano-TiO2 and trypsin inhibit the enzyme activity and change the secondary structure of trypsin. Biol. Trace Elem. Res. 2011, 142, (3), 435-446. (51) Xu, Z.; Liu, X.-W.; Ma, Y.-S.; Gao, H.-W. Interaction of nano-TiO2 with lysozyme: insights into the enzyme toxicity of nanosized particles. Environ. Sci. Pollut. Res. 2010, 17, (3), 798-806. (52) Zhao, L.; Zhang, H.; Wang, J.; Tian, L.; Li, F.-F.; Liu, S.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L.; White, J. C.; Huang, Y. C60 Fullerols Enhance Copper Toxicity and Alter the Leaf Metabolite and Protein Profile in Cucumber. Environ. Sci. Technol. 2019. 53, (4), 2171-2180. (53) Corpas, F. J.; Fernandez-Ocana, A.; Carreras, A.; Valderrama, R.; Luque, F.; Esteban, F. J.; Rodríguez-Serrano, M.; Chaki, M.; Pedrajas, J. R.; Sandalio, L. M. The expression of different superoxide dismutase forms is cell-type dependent in olive (Olea europaea L.) leaves. Plant Cell Physiol. 2006, 47, (7), 984-994. (54) Tillmann, U.; John, U.; Cembella, A. On the allelochemical potency of the marine dinoflagellate Alexandrium ostenfeldii against heterotrophic and autotrophic protists. J. Plankton Res. 2007, 29, (6), 527-543. (55) Lesser, M. P. Oxidative stress in marine environments: biochemistry and physiological ecology. Annu. Rev. Physiol. 2006, 68, 253-278. (56) Marshall, J.-A.; Hovenden, M.; Oda, T.; Hallegraeff, G. M. Photosynthesis does influence superoxide production in the ichthyotoxic alga Chattonella marina (Raphidophyceae). J. Plankton Res. 2002, 24, (11), 1231-1236. (57) Foyer, C. H.; Lopez‐Delgado, H.; Dat, J. F.; Scott, I. M. Hydrogen peroxide‐and glutathione‐associated mechanisms of acclimatory stress tolerance and signalling. Physiol. Plant. 1997, 100, (2), 241-254. (58) Hess, F. D. Light-dependent herbicides: an overview. Weed Sci. 2000, 48, (2), 160-170. (59) Shi, K.; Gao, Z.; Shi, T.-Q.; Song, P.; Ren, L.-J.; Huang, H.; Ji, X.-J. Reactive oxygen speciesmediated cellular stress response and lipid accumulation in oleaginous microorganisms: the state of the art and future perspectives. Front. Microbiol. 2017, 8, 793. (60) Halliwell, B.; Gutteridge, J. M. Free radicals in biology and medicine. Oxford University Press, USA: 2015. (61) Davies, K. J. Protein damage and degradation by oxygen radicals. I. general aspects. J. Biol. Chem. 1987, 262, (20), 9895-9901. 27 ACS Paragon Plus Environment


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699 700 701 702 703


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TOC

ACS Paragon Plus Environment


Figure 1. SEM images of Eremosphaera. (A) (B) The untreated control; (C) (D) Cells exposed to 1000 µg/L triclosan; (E) (F) Cells exposed to 5 mg/L P25; (G) (H) Cells co-exposed to 5 m g/L P25 and 1000 µg/L triclosan.


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Ti

10.55

1e-4 2e-4 3e-4 4e-4 5e-4 6e-4 7e-4 8e-4

10.60

10.50

10.45

10.40

10.55

10.50

10.45

-55.15

-55.10

-55.05

-55.20

Horizontal position, mm

-55.15

-55.10

-55.20

Vertical position, mm

10.50

10.45

10.55

10.50

10.40 -55.15

-55.10

-55.05

Ti

-55.20

-55.15

-55.10

-55.05


Fe

8.60

8.55

8.65

8.60

8.55

8.50

8.50

8.60

8.55

8.50 -63.82-63.80-63.78-63.76-63.74-63.72-63.70-63.68


Ca

1e-4 2e-4 3e-4 4e-4 5e-4 6e-4

8.70

8.65

8.60

8.55


Mn

8.0e-5 1.0e-4 1.2e-4 1.4e-4 1.6e-4 1.8e-4 2.0e-4 2.2e-4 2.4e-4

8.70



8.65

-63.82-63.80-63.78-63.76-63.74-63.72-63.70-63.68

-63.82-63.80-63.78-63.76-63.74-63.72-63.70-63.68

1e-4 2e-4 3e-4 4e-4 5e-4 6e-4 7e-4 8e-4

8.70


8.65

Cu 1e-4 2e-4 3e-4 4e-4 5e-4 6e-4 7e-4 8e-4

8.70


1e-4 2e-4 3e-4 4e-4 5e-4

8.70

8.50

8.65

8.60

8.55

8.50

-63.82-63.80-63.78-63.76-63.74-63.72-63.70-63.68

-63.82-63.80-63.78-63.76-63.74-63.72-63.70-63.68



Cu

Fe

Ti 1e-4 2e-4 3e-4 4e-4 5e-4

8.10 8.08 8.06 8.04 8.02 8.00 7.98

8.12

8.12 1e-4 2e-4 3e-4 4e-4 5e-4 6e-4 7e-4 8e-4

8.10


8.12

8.08 8.06 8.04 8.02 8.00

8.06 8.04 8.02 8.00

-73.86 -73.84 -73.82 -73.80 -73.78 -73.76 -73.74

-73.86 -73.84 -73.82 -73.80 -73.78 -73.76 -73.74


8.08

7.98

7.98

-73.86 -73.84 -73.82 -73.80 -73.78 -73.76 -73.74

1e-4 2e-4 3e-4 4e-4 5e-4 6e-4 7e-4 8e-4

8.10



-55.05

10.45



Ca 8.12

8.06 8.04 8.02 8.00 7.98

8.0e-5 1.0e-4 1.2e-4 1.4e-4 1.6e-4 1.8e-4 2.0e-4 2.2e-4 2.4e-4

8.10


8.08


Mn

8.12

0.0004 0.0005 0.0006

8.10


-55.10

8.0e-5 1.0e-4 1.2e-4 1.4e-4 1.6e-4 1.8e-4 2.0e-4 2.2e-4 2.4e-4

10.60


2.0e-4 4.0e-4 6.0e-4 8.0e-4 1.0e-3 1.2e-3

10.55

-55.20


-55.15


Mn

10.40


10.45


10.60

(C)

10.50

-55.05

Ca

(B)

10.55

10.40

10.40 -55.20

1e-4 2e-4 3e-4 4e-4 5e-4 6e-4 7e-4 8e-4

10.60



Cu

Fe 1e-4 2e-4 3e-4 4e-4 5e-4

10.60


(A)

8.08 8.06 8.04 8.02 8.00 7.98

-73.86 -73.84 -73.82 -73.80 -73.78 -73.76 -73.74


-73.86 -73.84 -73.82 -73.80 -73.78 -73.76 -73.74


Figure 2. Investigation of the distribution of multi-elements in an individual algal cell. (A) The untreated control; (B) Cells co-exposed to 5 mg/L P25 and 15.625 µg/L triclosan; (C) Cells coexposed to 5 mg/L P25 and 1000 µg/L triclosan.



Figure 3. Principal component analysis (PCA) biplots for the distribution of five elements (Ti, Fe, Cu, Ca, Mn) in untreated cells, only 1000 μg/L triclosan exposed cells, only P25 exposed cells, as well as P25 and 1000 μg/L triclosan co-exposed cells.


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Figure 4. Investigation of the distribution of major bands for lipids and protein in an individual algal cell. (A) 3000-2800 cm-1 C-H stretch in acyl from fatty acids/lipids; (B) ~1740 cm-1 C=O in ester and ester fatty acids; (C) 1724-1585 cm-1 C=O stretch of Amide I.



Relative ROS level (% of control)

220 200

Without P25 Co-exposure with P25

*** *

180

*** *

160 140

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*** *

** **

120 100 80 0

200

400

600 800 1000 Triclosan concentration (μg/L)

4000

Figure 5. ROS generation of algal cells exposed to triclosan in the absence and presence of 5 mg/L P25 (n=3). Note: “*” represents results that were significantly different from the untreated control (p < 0.05); “**” represents results that were significantly different from the control only exposed to 5 mg/L P25 (p < 0.05). “***” represents results that were significantly different in treatments between in the absence and in the presence of 5 mg/L P25 (p < 0.05).


Interactive Toxicity of Triclosan and Nano-TiO2 to Green Alga

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