materials mitigate mercury uptake and effects on green alga Chlamydomonas reinhardtii in mixture exposure

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NanoTiO

materials mitigate mercury uptake and effects on green alga Chlamydomonas reinhardtii in mixture exposure

LI, Mengting, LIU, Wei, SLAVEYKOVA, Vera

Abstract

The present study examined the effect of titanium dioxide nanoparticles (nanoTiO2) and mercury (Hg) compounds on the green alga, Chlamydomonas reinhardtii. Mixtures containing nanoTiO2 of different primary sizes (5 nm, 15 nm and 20 nm), inorganic Hg (IHg) or monomethyl Hg (CH3Hg+, MeHg) were studied and compared with individual treatments.

Oxidative stress and membrane damage were examined. Stability of nanoTiO2 materials in terms of hydrodynamic size and surface charge as well as Hg adsorption on different nanoTiO2 materials were characterized. The uptake of Hg compounds in the absence and presence of nanoTiO2 was also quantified. Results show that increasing concentrations of nanoTiO2 with different primary size diminished oxidative stress and membrane damage induced by high concentrations of IHg or MeHg, due to the adsorption of Hg on the nanoTiO2 aggregates and consequent decrease of cellular Hg concentrations. The observed alleviation effect of nanoTiO2 materials on Hg biouptake and toxicity was more pronounced for the materials with smaller primary size. IHg adsorbed onto the nanoTiO2 materials to a higher extent [...]

LI, Mengting, LIU, Wei, SLAVEYKOVA, Vera. NanoTiO₂ materials mitigate mercury uptake and effects on green alga Chlamydomonas reinhardtii in mixture exposure. Aquatic Toxicology, 2020, vol. 224, p. 105502

DOI : 10.1016/j.aquatox.2020.105502

Available at:

http://archive-ouverte.unige.ch/unige:136519

Disclaimer: layout of this document may differ from the published version.

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NanoTiO

materials mitigate mercury uptake and effects on green alga Chlamydomonas reinhardtii in mixture exposure

Mengting Li, Wei Liu and Vera I. Slaveykova*

Environmental Biogeochemistry and Ecotoxicology, Department F.-A. Forel for Environmental and Aquatic Sciences, School of Earth and Environmental Sciences, Faculty of Science, and Institute for Environmental Sciences, University of Geneva, Uni Carl Vogt,

66, boulevard Carl-Vogt, CH-1211 Genève 4, Switzerland

Corresponding author: vera.slaveykova@unige.ch Vera I. Slaveykova ORCID ID 0000-0002-8361-2509

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Abstract

The present study examined the effect of titanium dioxide nanoparticles (nanoTiO2) and mercury (Hg) compounds on the green alga, Chlamydomonas reinhardtii. Mixtures containing nanoTiO2 of different primary sizes (5 nm, 15 nm and 20 nm), inorganic Hg (IHg) or monomethyl Hg (CH3Hg⁺, MeHg) were studied and compared with individual treatments.

Oxidative stress and membrane damage were examined. Stability of nanoTiO2 materials in terms of hydrodynamic size and surface charge as well as Hg adsorption on different nanoTiO2

materials were characterized. The uptake of Hg compounds in the absence and presence of nanoTiO2 was also quantified. Results show that increasing concentrations of nanoTiO2 with different primary size diminished oxidative stress and membrane damage induced by high concentrations of IHg or MeHg, due to the adsorption of Hg on the nanoTiO2 aggregates and decrease of cellular Hg concentrations. The observed alleviation effect of nanoTiO2 materials on Hg biouptake and toxicity was more pronounced for the materials with smaller primary size.

IHg adsorbed onto the nanoTiO2 materials to a higher extent than MeHg. The present study highlights that the effects of contaminants are modulated by the co-existing engineered nanomaterials; therefore, it is essential to get a better understanding of their combined effect in the environment.

Keywords: nanoTiO2; mercury; methylmercury; phytoplankton; bioavailability; toxicity, cocktail effect, mixtures

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1 Introduction

Growing use of nanoparticle-enabled materials in everyday-life products raised concerns about their environmental implications and safety. Indeed, increasing evidence demonstrates that if the engineered nanomaterials (ENMs) are released into the environment during their production, use or disposal, there is a possible risk that they cause harm to the organisms living in these environments (Coll et al., 2016; Holden et al., 2016; Nowack et al., 2012). Despite the fact that the environmental implications of ENMs have received extensive attention in recent years, their interactions with other contaminants and the subsequent effects on aquatic microorganisms are still to explore. ENMs possess enhanced physical and chemical properties, and high reactivity because of their smaller size and larger specific surface area compared to their bulk counterparts, favoring the interaction with various biotic and abiotic components, including contaminants in the aquatic environment (Naasz et al., 2018).

ENM - contaminant interactions and consequences for the modulation of contaminants uptake and toxicity upon combined exposure have been reviewed previously (Canesi et al., 2015;

Naasz et al., 2018). Reports on co-exposures vary from reduced to increased effects compared to individual compounds, as well as no effect on co-contaminant uptake and toxicity.

NanoTiO2 reduced growth inhibitory effect of Cd on alga Chlamydomonas reinhardtii (Yang et al., 2012a; Yang et al., 2012b; Yu et al., 2018a), and Cr (VI) toxicity to Scenedesmus obliquus; however, nanoAl2O3 had no such alleviation effect (Dalai et al., 2014). CdSe/ZnS quantum dots (QDs) significantly decreased intracellular Cu and Pb in walled, but increased them in wall-less strains of C. reinhardtii (Worms et al., 2012). The same QDs increased Cu and Pb contents in the metal-resistant bacterium Cupriavidus metallidurans during short-term exposure (Slaveykova et al., 2013) and Cd toxicity to C. reinhardtii (Yu et al., 2018a). NanoAg had no significant influence on the Cd toxicity to green alga C. reinhardtii (Yu et al., 2018b).

By contrast, nanoTiO2 greatly promoted Cd uptake in the protozoa Tetrahymena thermophila

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(Yang et al., 2014). NanoTiO2 enhanced the effect of Cd on algal growth despite a decrease of Cd²⁺ in the exposure medium (Hartmann et al., 2010). NanoTiO2 augmented As(III) and As(V) accumulation by cyanobacterium Microcystis aeruginosa and green alga Scenedesmus obliquus in As species and algal species - dependent way (Luo et al., 2018). Overall, the above examples demonstrated that the ENMs could modify the effects of toxic metals on various unicellular organisms in several ways and pointed out the necessity of better understanding of both ENM - contaminant and ENM - organism interactions.Among the different toxic metals, Cd has received the most attention because of its great toxicity. No similar studies exists for mercury (Hg) compounds, despite the fact that Hg is a priority toxicant of global concern, which bioconcentrates, and biomagnifies in aquatic food webs (Driscoll et al., 2013). Besides, the accumulation of Hg by phytoplankton represents one of the main entry points of Hg into food webs (Le Faucheur et al., 2014). In addition, Hg compounds were reported to affect the growth, photosynthesis, and nutrient metabolism, as well as to induce oxidative stress and membrane damage in green alga (Beauvais-Flück et al., 2016; Beauvais-Flück et al., 2017, 2018).

The present study aimed to provide deeper insights into the effects of mixtures containing toxic metals and ENMs to phytoplankton. The specific focus was on the uptake, oxidative stress and membrane damage to green alga C. reinhardtii exposed to mixtures containing inorganic Hg (IHg) or monomethyl Hg (CH3Hg⁺, MeHg) and nanoTiO2. To our knowledge, this is the first study on the effects of nanoTiO2 and IHg/MeHg in mixtures on freshwater microalgae. We hypothesized that both IHg and MeHg will be adsorbed onto the nanoTiO2, which will result in a decrease of the cellular concentrations of IHg or MeHg and thus alleviation of the oxidative stress and membrane damage. The hypothesis is based on previous knowledge showing that nanoTiO2 tends to form micrometric aggregates (Allouni et al., 2009; Brunelli et al., 2013),

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which can not pass through the pores of algal cell walls (Chen et al., 2016; Zouzelka et al., 2016).

2 Materials and methods

2.1 Chemicals and materials

Two mercury compounds HgCl2 (IHg) and CH3HgCl (MeHg), and three types of nanoTiO2

with different primary sizes were chosen in this study. Powdered nanoscale TiO2 particles with different compositions and primary sizes (anatase 5 nm (A5), anatase 15 nm (A15) and anatase/rutile 20 nm (AR20)) were purchased from Nanostructured & Amorphous Materials Inc, USA. Their characteristics are given in Table S1. Stock suspensions of 2.0 g·L^-1 nano TiO2

were prepared by dispersing nanoparticles in ultrapure water (Millipore, Darmstadt, Germany) and applying sonication for 10 min (50WL^-1 at 40 kHz). Further, 10 min sonication was conducted immediately before dosing.

HgCl2 and CH3HgCl standard solutions (1.0 g·L^-1) were bought from Sigma-Aldrich, Buchs, Switzerland. All the glassware was soaked in 10% HNO3 (EMSURE, Merck, Darmstadt, Germany) followed by two 10% HCl acid baths (EMSURE, Merck, Darmstadt, Germany) for at least 24 hours in each bath, then thoroughly rinsed with ultrapure water (MilliQ Direct, Merck, Darmstadt, Germany) and dried under a laminar flow hood.

2.2 Exposure of C. reinhardtii to mercury compounds, nanoTiO2, and their mixtures

Wild-type C. reinhardtii (CPCC11, Canadian Phycological Culture Centre, Waterloo, Canada) were grown axenically at 20.2 ± 0.5°C, 115 rpm and 110 μmol·phot·m^-2·s^-1. Algal cells were pre-cultured in 4×diluted Tris-Acetate-Phosphate medium (pH 7.0 ± 0.2, Table S2) (Harris, 2013) until mid-exponential growth, centrifuged (10 min, 1300 g), rinsed and re-suspended

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(~10⁶ cells·mL^-1) in the exposure medium. The exposure medium consisted of 8.2×10^-4 M CaCl2·2H2O, 3.6×10^-4 M MgSO4·7H2O, 2.8×10^-4 M NaHCO3, 1.0×10^-4 M KH2PO4 and 5.0×10^-6 M NH4NO3, pH 7.0±0.1,ionic strength 2.75 mM. The medium was spiked with IHg or MeHg (10^-9, 5×10^-7 M), nanoTiO2 (20, 200 mg·L^-1) or their mixtures (Table S3). The biological responses were followed over a 2 h exposure period corresponding to the attainment of the steady state for Hg uptake by algae (Fig. S1). Cells exposed in the absence of Hg compounds and nanoTiO2 were used as control. Bioassays were performed under sterile conditions and on three biological replicates.

2.3 Physicochemical characterization of nanoTiO2 in exposure medium

Z-average hydrodynamic diameter, size distribution and electrophoretic mobility (EPM) of the three nanoTiO2 materials in exposure medium were measured after 2 h by a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK) at 20°C, using disposable polystyrene cuvettes (Malvern). EPM was converted to zeta potential through the

Smoluchovski’s theory. Both Z-average hydrodynamic diameter corresponding to the intensity weighted mean hydrodynamic diameter of the whole particle distribution and the distributions were registered. Results were the means of three sample measurements, nine runs for each, performed with freshly prepared samples on separate days. The size and morphology of the nanoTiO2 suspended in the exposure medium were observed at different concentrations by TEM (FEI Tecnai™ G2 Sphera, FEI Company, USA) operating at 200 kV.

NanoTiO2 materials were dispersed into exposure medium at 20 and 200 mg·L^-1, and 5 µL of the obtained suspensions were deposited on the carbon coated copper grid and then dried at room temperature for the observation.

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2.4 Determination of the sorption of Hg compounds on nanoTiO2

To study the sorption of mercury compounds on nanoTiO2, suspensions containing 20 and 200 mg L^-1 nanoTiO2 were prepared in test medium. Mercury was then added into the nanoTiO2

suspensions to concentrations of 10^-9 and 5×10^-7 M. To enable comparison between the results in different tests, the sorption experiments were performed under the same conditions as algal culture exposure (20.2 ± 0.5°C, 115 rpm and 110 μmol·phot·m^-2·s^-1). At 2 h, 1.5 mL of the nanoTiO2 suspensions were centrifuged twice for 10 min at 12,000 g using a high-speed centrifuge (Optima XL-80K Ultra Centrifuge, Beckman Coulter Inc.). Then 1 mL of the collected supernatants were used to measure mercury concentrations by a MERX-T®

Automated Total Hg Analytical System (Brooks Rand Instruments, Seattle, WA, USA). The amount of the adsorbed IHg or MeHg was calculated as the difference between initial IHg or MeHg concentrations and those measured in the supernatant at 2 h, and expressed as adsorbed fraction. Results demonstrated an attainment of a plateau of the amount of the adsorbed mercury species to nanoTiO2 after 30 min (Fig. S2).

2.5 Determination of mercury uptake to C. reinhardtii

The uptake of IHg or MeHg to unicellular alga C. reinhardtii was determined by measurement of the total cellular Hg (IHg+MeHg) concentrations. After 2 h exposure, 30 mL of algal suspensions were centrifuged by the density gradient centrifugation method using 40% and 100% sucrose, as described in details in the accompanying MethodsX paper. Algal suspensions exposed to IHg or MeHg in the absence or presence of nanoTiO2 were thus treated, as well as the negative controls (no IHg, MeHg or nanoTiO2). Algal cells were efficiently separated from the nanoTiO2 aggregates, retrieved from the middle layer of the gradient containing 40%

sucrose and 100% sucrose, washed and centrifuged (5 min, 1300 g). Freeze-dried samples were weighed using an analytical balance (QUINTIX35-1S, Sartorius Lab Instruments GmbH & Co.

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KG, ± 0.00001 g). Samples were then introduced in the Advanced Hg Analyzer AMA 254 (Altec s.r.l., Czech Republic)to determine the amount of the total accumulated Hg (THg). The accuracy of the measurements was checked by analyzing the certified reference material (CRM) MESS-3 Reference Materials (100 ± 0.1% recovery). The amount of the THg accumulated by algal cells was expressed in mg·kg^-1 dry weight of algal biomass. The concentrations of THg in exposure medium were measured by using a MERX^® Automated Total Mercury Analytical System (Brooks Rand Instruments, Seattle, WA, USA). Detection limit was 0.03 ngTHg·L^-1. The accuracy of THg measurements by MERX^® was tested by analyzing the CRM ORMS-5 (116.0 ± 3.5% recovery). The measured concentrations of IHg corresponding to the nominal concentrations of 10^-9 and 5×10^-7 M were (0.89 ± 0.01) ×10^-9 and (8.49 ± 0.59) ×10^-7 M, respectively. For nominal concentration of 10^-9 and 5×10^-7 M MeHg the measured concentrations were (0.90 ± 0.01) ×10^-9 and (4.79 ± 0.23) ×10^-7 M, respectively.

2.6 Flow cytometry measurements

To evaluate the effect of nanoTiO2, IHg or MeHg and their mixtures on C. reinhardtii, algal membrane integrity and oxidative stress were determined by flow cytometry (FCM).

Measurements were performed with a BD Accuri C6 flow cytometer equipped with a CSampler (BD Biosciences, San Jose, CA). 488-nm argon excitation laser and fluorescence detection channels with band pass emission filters at 530 ± 15 nm (FL1), 585 ± 20 nm (FL2) and a long pass emission filter for >670 nm (FL3) were used. Data acquisition and analysis were performed with the BD Accuri C6 Software 264.15. The primary threshold was set to 20 000 events on FSC-H. Information on algal cell number and chlorophyll fluorescence (in FL3) were obtained in a single run after 2 h incubation. Algal cells were discriminated from nanoTiO2

particles applying the gating strategy described in Fig. S3.

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Enhanced cellular reactive oxygen species (ROS) generation and membrane permeability alteration were examined at 2 h using the fluorescent probes CellROX® green (Life Technologies Europe B.V., Zug, Switzerland) and propidium iodide (PI) (Sigma–Aldrich, Buchs, Switzerland), respectively. Staining procedures and gating strategies were the same as in our previous study (Cheloni et al., 2014). Briefly, 5 μM CellROX® green and 7 μM PI were employed to 500 μL aliquots of each test replicate separately for 30 minutes incubation in the dark with no intermediate washing steps. Positive and negative controls were run for each stain:

the unexposed algae were used as negative control, while algae exposed to 5 mM H2O2 for 30 min were used as positive control for CellROX® green stain, and alga incubated at 90°C for 15 minutes were used as positive control for PI stain. Gates designed to assess the percentage of cells experiencing oxidative stress and membrane damages are given in Fig. S4.

2.7 Statistical analysis

All the results are reported as mean and standard deviation (SD), calculated from three independent experiments. Prior to statistical analysis, all data were tested for normality of distribution by the Shapiro-Wilk test. One-way Analysis of Variance (ANOVA) was performed to test for significant differences between the different treatments by Statistic 6.0. A p-value

<0.05 was considered statistically significant. The Tukey Honestly Significant Difference (Tukey HSD) was performed as a post-hoc test.

3 Results and Discussion

3.1 Characterization of nanoTiO2 in exposure medium

Measured Z-average hydrodynamic diameters indicated that the studied nanoTiO2 rapidly aggregated in the test medium (Fig. 1 A-C). A5 formed aggregates larger than A15 and AR20.

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There was no significant difference between the size of the aggregates formed by A15 and AR20 (p > 0.5). The size of the aggregates was strongly dependent on the nanoTiO2

concentrations, which was expected, given the increased probability of collisions between the particles at higher concentrations (Stumm and Morgan, 1981). As the concentrations increased from 20 to 200 mg·L^-1, the hydrodynamic diameter of the aggregates increased by two-fold for the three types of nanoTiO2. Zeta potential values for the three nanoTiO2 were negative and decreased with the increase of the nanoTiO2 concentration (p < 0.5, Fig. 1 D-F). It is reported that nanoparticles tend to aggregate as the absolute values of zeta potential decrease (Mandzy et al., 2005). The above observations are consistent with the existing literatures about nanoTiO2

aggregation kinetics in ambient water (Ma et al., 2015; Sendra et al., 2017; Sharma, 2009). For example, rapid formation of micrometer-sized aggregates was found for nanoTiO2 in media with ionic strength of 4.5×10^-3 to 1.65×10^-2 M (French et al., 2009).

Addition of IHg or MeHg to the nanoTiO2 dispersions promoted further nanoTiO2 aggregation.

This effect was more pronounced at higher IHg or MeHg concentrations, as well as greater for IHg than for MeHg addition. For example, in the presence 5×10^-7 M IHg, the hydrodynamic diameters of 20 mg·L^-1 A5, A15 and AR20 were 1.57, 1.72 and 1.63 times higher, respectively, than those found in the absence of mercury. Mono- and bi-modal (A5) distributions of the hydrodynamic size were observed in the dispersions of different nanoTiO2 (Fig. S5). For the higher concentrations of 200 mg·L^-1 nanoTiO2 a shift of the peak towards larger sizes was observed for both A15 and AR20. The above results were further confirmed by TEM (Fig. 1 G-L) showing that the three types of nanoTiO2 formed large aggregates in the algal exposure medium. A5 formed cluster-like aggregates with a size > 3 µm, whereas A15 and A20 showed similar morphologies, and the particles were loosely joined. Similar aggregation behavior of nanoTiO2 has been observed in cell culture medium (Allouni et al., 2009) and environmental water (Brunelli et al., 2013).

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The present findings revealed the tendency of the studied materials to form micrometric aggregates, which can settle down. As a consequence, the nanoTiO2 dispersed in the medium that could interact with green alga C. reinhardtii and associated contaminants could be expected to decrease.

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Figure 1. Z-average hydrodynamic diameter (mean size value, A, B, C) and zeta potential (D, E, F) of different nanoTiO2 materials at 20 and 200 mg·L^-1 suspended in algal exposure medium in the absence and presence of IHg or MeHg. Different letters indicate statistically significant differences between the values as obtained by ANOVA and a Tukey’s post hoc test (p < 0.05), where the letter “a” is assigned to the groups with highest mean values. * denote the data is out of the analytical window of Zetasizer Nano ZS. TEM images of different nanoTiO2 materials: anatase 5 nm (A5), anatase 15 nm (A15) and anatase/rutile 20 nm (AR20) suspended in the exposure medium at concentrations 20 mg·L^-1 (G, H, I) and 200 mg·L^-1(J, K, L).

3.2 Sorption of mercury compounds on nanoTiO2

Both IHg and MeHg significantly adsorbed to the nanoTiO2 materials of interest. The increase of A5 concentrations from 20 to 200 mg·L^-1 resulted in a significant increase of the adsorbed fraction of IHg from roughly 0.70 to 0.95 in the medium containing 10^-9 M IHg. No nanoTiO2

concentration dependence was found for A15 and AR20 adsorbing 10^-9 M IHg or MeHg. In mixtures containing 5×10^-7 M IHg and nanoTiO2, the fraction of adsorbedIHg was much lower (0.35-0.40, Fig. 2C) than that for 10^-9 M IHg, as could be expected by the 1000× higher ratio of IHg to nanoTiO2.

13 0.0

0.2 0.4 0.6 0.8

1.0 A ^a ₁₀^-9 _{M IHg}

b b b

b b

0.0 0.2 0.4 0.6 0.8

1.0 B ₁₀^-9 _{M MeHg}

ab a

abc ab

c bc

0.0 0.2 0.4 0.6 0.8 1.0

[nanoTiO₂](mg·L^-1)

C _5×10^-7 _{M IHg}

Adsorbed fraction

AR20 A5 A15

20 200 20 200 20 200

a a

b

a b

a

0.0 0.2 0.4 0.6 0.8

1.0 D _5×10^-7 _{M MeHg}

AR20 A15

A5

20 200 20 200 20 200

b a

c b

c bc

Figure 2. Fraction of Hg adsorbed on A5, A15 and AR20 after 2 h-exposure to mixtures containing IHg and nanoTiO2 or MeHg and nanoTiO2. Different letters indicate statistically significant differences between the values as obtained by ANOVA and a Tukey’s post hoc test (p < 0.05), where the letter “a” is assigned to the groups with highest mean value.

The increase of nanoTiO2 concentrations resulted in an augmentation of the fraction of adsorbed IHg for A15 and AR20. A particle primary size dependent adsorption of the 10^-9 M MeHg on the nanoTiO2 was observed (Fig. 2 B, D). The fraction of the adsorbed MeHg increased in the order: A5 > A15 > AR20. However, no dependence of IHg adsorption on the primary size of nanoparticles was found. The studied nanoTiO2 materials had higher adsorption capacity to IHg than MeHg. For comparable concentrations, the adsorption of 10^-9 M and 5×10^-

14 7 M IHg to 200 mg·L^-1 A5, A 15 or A20 was about two times higher than that of MeHg. These findings are consistent with the existing literature demonstrating a strong adsorption of different metals including Hg by nanoTiO2 materials. For example, 96% (Ghasemi et al., 2012) and 98.6% (Afshar et al., 2017) of IHg was adsorbed to nanoTiO2; 99% of IHg was adsorbed on nanoTiO2 (P25 and anatase synthesized by sol-gel method), and titanate nanotubes (Lopez- Munoz et al., 2016). However, the medium composition, type of nanoTiO2 and contact time in these studies were different, thus direct comparison is not possible. It was suggested that the adsorption mechanism involves adsorption of hydroxylated mercury species (HgClOH and Hg(OH)2) on the nanoTiO2 materials probably through the interaction with –TiOH and –TiO^– active sites (Lopez-Munoz et al., 2016). The chemical speciation calculations (see SI for further details) showed that under the experimental conditions of this study IHg and MeHg are present mainly as –OH and –Cl complexes. Therefore, a mechanism involving the adsorption of these species to the nanoTiO2 surface site could be plausible.

3.3 Toxicity of mixtures containing nanoTiO2 and IHg or MeHg to C. reinhardtii

The toxicity of mixtures containing nanoTiO2 and IHg or MeHg to C. reinhardtii was characterized by following the changes in the percentage of cells with enhanced cellular ROS and membrane damages. In individual treatment groups, different nanoTiO2 materials induced cellular ROS generation and oxidative stress in less than 15% of cells (Fig. 3A). No nanoTiO2

concentration-dependent ROS generation was found.

15 0

10 20 30 A

cd bc d ab

a a a

nanoTiO₂

0 10 20 B

d

nanoTiO₂+10^-9 M IHg cd bc

a

bc ab c

0 10 20 30 C

Oxidative stress (% cells)

nanoTiO₂+10^-9 M MeHg bc

d c

a c ab

cd

0 20 40 60

d

Da ab nanoTiO₂+5×10^-7 M IHg abc

cd abc

bcd

0 40 80 120 E

[nanoTiO₂](mg·L^-1)

0

AR20 200 20 20 200

A15 20 200

A5

nanoTiO₂+5×10^-7 M MeHg

a d c bc bc c b

Figure 3. Percentage of cells with oxidative stress exposed to mixtures containing nanoTiO2

and IHg or MeHg for 2 h. Cells were stained with CellROX® green. Different letters indicate statistically significant differences between the values as obtained by ANOVA and a Tukey’s post hoc test (p < 0.05), where the letter “a” is assigned to the groups with highest mean value.

In the absence of nanoTiO2, less than 15% of cells were affected by oxidative stress after 2 h exposure to 10^-9 M IHg or MeHg (Fig. 3B, C). These percentages were significantly higher (59% and 88%) for 5×10^-7 M IHg or MeHg exposure (Fig. 3D, E).

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0 5

10 A nanoTiO₂

0 5

10 B nanoTiO₂+10^-9 M IHg

0 5 10 15

20 C nanoTiO₂+10^-9 M MeHg

Mem b ra n e d a m a g e (% cel ls )

0 10 20 30 40

50 D nanoTiO₂+5×10^-7 M IHg

a

d e bc de b cd

0 20 40 60 80 100

[nanoTiO

](mg·L

)

E nanoTiO₂+5×10^-7 M MeHg

0

AR20 200 200 20

20 A15 200

20 A5 a

ab b a ab a a

Figure 4. Percentage of cells with membrane damage exposed to mixtures containing nanoTiO2 and IHg or MeHg for 2 h. Cells were stained with PI. Different letters indicate statistically significant differences between the values as obtained by ANOVA and a Tukey’s post hoc test (p < 0.05), where the letter “a” is assigned to the groups with highest mean value.

The addition of nanoTiO2 significantly reduced the percentage of cells with oxidative stress for high IHg or MeHg concentrations treatments, suggesting the antagonistic interactions.

Among the tested nanomaterials A5 had stronger ability to reduce Hg species induced oxidative

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stress than A15 and AR20. Moreover, this decrease was more pronounced for IHg than for MeHg exposure, which is consistent with a stronger adsorption of IHg on the nanoTiO2. The membrane damage caused by nanoTiO2 materials alone was below 5% (Fig. 4A). Two- hour exposure to 10^-9 M IHg or MeHg resulted in a very low percentage of cells with membrane damage (Fig. 4B, C), while exposure to 5×10^-7 M IHg or MeHg caused membrane damage in 36% and 63% of the cells, respectively (Fig. 4D, E). Addition of nanoTiO2 lead to a decrease of the percentage of cells with damaged membranes in the mixtures containing 5x10^-7 M IHg or MeHg and nanoTiO2 was lower as compared with those observed for the exposure to IHg or MeHg only. The increase of the concentrations of A5, A15 and AR20 mitigated the effect of IHg to a greater extent than that of MeHg.

Overall, co-exposure to nanoTiO2 materials alleviated the generation of excessive ROS and membrane damage in green alga C. reinhardtii in mixtures with high IHg or MeHg concentrations. Such a mitigation of the Hg species-induced effects could be explained by a decrease of the concentrations of the accumulated IHg or MeHg in the mixtures containing nanoTiO2. To verify this hypothesis, the uptake of IHg and MeHg by C. reinhartdtii was determined under the conditions of the toxicity tests.

3.4 IHg and MeHg uptake by C. reinhardtii in mixtures with nanoTiO2

In individual treatments, cellular Hg concentrations were about 2 to 3 times higher for MeHg exposure than for IHg for comparable exposure concentrations. This finding is in line with our previous results showing higher bioaccumulation of MeHg than IHg (Beauvais-Flück et al., 2017). It is also worth noting that cellular concentrations measured at high IHg or MeHg exposure concentrations could be affected by the altered cellular homeostasis and possible leakage given very high percentage of algal cells experiencing oxidative stress or membrane damage.

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As expected by the significant adsorption of mercury species on nanoTiO2, the presence of nanoTiO2 significantly decreased the amount of the accumulated Hg in C. reinhardtii (Fig. 5).

For instance, the reduction of cellular Hg for 10^-9 M IHg in the presence of 200 mg·L^-1 followed the order A5 > A15 > AR20 showing the importance of the primary size of the nanoparticles.

The decrease in IHg uptake was more important than the one observed for MeHg at higher Hg concentrations. The increase of the nanoTiO2 concentrations from 20 to 200 mg·L^-1 resulted in further reduction of IHg or MeHg uptake by C. reinhardtii (p < 0.05) for all tested materials.

0.0 0.2 0.4 0.6 0.8 1.0 1.2

b

d d c c d

a 10^-9 M IHg

[THg]cell (mg·kg-1 )

A

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

cd b

de b

e c

Ba 10^-9 M MeHg

0 100 200 300 400 500 600 700 a

c b

d c d c

5×10^-7 M IHg C

0 200 400 600 800 1000 1200

[nanoTiO₂](mg·L^-1)

5×10^-7 M MeHg

d b

de bc

e c Da

0

A15 AR20 A5

20 200 20 200 20 200 0

AR20 A5 A15

20 200 20 200 20 200

Figure 5. Total accumulated cellular mercury (THg = IHg + MeHg)cell in C. reinhardtii during exposure to 10^-9 or 5x10^-7 M IHg or MeHg alone and in mixtures with 20 and 200 mg·L^-1 nanoTiO2. Exposure time: 2 hours. Density gradient centrifugation was applied for separation of the algal cells from the nanoTiO2 aggregates (see the accompanying MethodsX paper). The density gradient centrifugation step was kept short to minimize the risk of releasing Hg from algae during centrifugation and that it was experimentally confirmed that centrifugation did

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not affect the intracellular concentration of Hg. Values are mean ± SD (n = 3). Different letters indicate statistically significant differences between the values as obtained by ANOVA and a Tukey’s post hoc test (p < 0.05). The letter “a” is assigned to the groups with highest mean value.

These results demonstrated that the presence of the nanoTiO2 materials mitigate IHg and MeHg cellular concentrations in C. reinhardtii in a manner dependent on the concentration and primary size of the nanomaterials. In agreement with the adsorption of Hg species on the nanoTiO2 materials, cellular Hg concentration decreased with the amount of IHg or MeHg bound to nanoTiO2 and increased linearly with the concentration of the dissolved IHg or MeHg (not adsorbed to nanoTiO2)(Fig. S6). The findings in this study are in agreement with the existing literature for other metals such as Cd, demonstrating diminution of the Cd bioavailability to C. reinhardtii by nanoTiO2, due to the adsorption of Cd by nanoTiO2 and reduction of the free Cd ions in exposure medium (Vale et al., 2014; Yang et al., 2012a; Yang et al., 2012b; Yang et al., 2014; Yu et al., 2018a) as well as Cr(VI) bioavailability decrease to green alga Scenedesmus obliquus by nanoTiO2 (Dalai et al., 2014). It further supports the findings that metal toxicity to unicellular organisms may decline in the presence of nanoTiO2

if the metal-ENM complexes thus formed cannot penetrate the cell membrane (Dalai et al., 2014; Luo et al., 2018; Yu et al., 2018b).

The study also emphasized that the effects of environmental toxicants are dependent on their interactions with ENMs co-existing in the environmental systems. As the uptake of Hg species by green algae, and more generally phytoplankton, is a prerequisite for the biotic transformations, the results imply that nanoTiO2 has a potential to affect the mercury species biotransformation processes, such as MeHg demethylation, Hg reduction or volatilization, known for phytoplankton.

20

4 Conclusion

The present study examined the interactions and effects of cocktails containing nanoTiO2 and mercury compounds on the microalga, C. reinhardtii. Results showed that A5, A15, and AR20 nanoTiO2 materials strongly aggregated in the exposure media. Nevertheless, they adsorbed significant amounts of IHg and MeHg. Oxidative stress and membrane damage observed at high IHg and MeHg concentrations were significantly mitigated in the presence of nanoTiO2. The alleviation of biological effects caused by nanoTiO2 correlated with the decrease of cellular Hg concentrations, which decreased with the amount of adsorbed Hg or MeHg on nanoTiO2. Overall, the effect of Hg was modulated by ENMs co-occurring in the exposure systems; therefore, it is essential to get a better understanding of their interactions with ENMs.

Appendix A. Supplementary material to this article can be found online at …..

Acknowledgments: MTL and VIS acknowledge the financial support of the China Scholarship Council grant [2016]3100, VIS acknowledges the financial support from the Swiss National Science Foundation (project N 166089).

Conflicts of Interest: The authors declare no conflict of interest.

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S1

NanoTiO

materials mitigate mercury uptake and effects on green alga Chlamydomonas reinhardtii in mixture exposure

Mengting Li, Wei Liu and Vera I. Slaveykova*

Environmental Biogeochemistry and Ecotoxicology, Department F.-A. Forel for Environmental and Aquatic Sciences, School of Earth and Environmental Sciences, Faculty of Science, and Institute for Environmental Sciences, University of Geneva, Uni Carl Vogt,

66, boulevard Carl-Vogt, CH-1211 Genève 4, Switzerland

Corresponding author: vera.slaveykova@unige.ch

Suplementary Material

S2 Powdered nanoscale TiO2 particles with different compositions and sizes were purchased from Nanostructured & Amorphous Materials, Inc, USA.

Table S1. NanoTiO2 properties as dry powder as provided by the manufacturer Composite Diameter

(nm)

Purity Specific Surface Area (m²/g)

Bulk density (g/cm³)

pH TEM image

A5 anatase 5 99.8% 150-300 0.25-0.3 2.0-5.0

A15 anatase 15 99% ~240 0.04 -

0.06

5.5-7.0

AR20 anatase/rutile 20 99+% ≥ 5 0.4-0.5 5.5-7.0

S3 Table S2. Chemical composition of algal culture medium (TAP x 4)

Component Concentration (M) Phosphate

KH2PO4 1.01x10^-4

K2HPO4 1.49x10^-4

Ammonium

NH4NO3 1.68x10^-3

CaCl2 2 H2O 8.50x10^-5 MgSO4 7 H2O 1.02x10^-4

Metals

H3BO3 4.75 x10^-5

ZnSO4 7 H2O 1.90 x10^-5 MnCl2 4 H2O 6.25 x10^-5 FeSO4 7 H2O 4.50x10^-6

CoCl2 6 H2O 1.67x10^-6 CuSO4 5 H2O 1.57x10^-6 (NH4)6Mo7O24 4 H2O 2.23x10^-6 Na2EDTA 2 H2O 3.36x10^-5

TRIS acetate

CH3COOH 4.30x10^-3

TRIS 5.00x10^-3

S4 Table S3. Summary of the different treatments used in bioassays with C. reinhardtii

Treatments Concentrations

IHg (M) MeHg (M) A5 (mg L^-1) A15 (mg L^-1) AR20 (mg L^-1)

1 10^-9 0 0 0 0

2 10^-9 0 20 0 0

3 10^-9 0 0 20 0

4 10^-9 0 0 0 20

5 10^-9 0 200 0 0

6 10^-9 0 0 200 0

7 10^-9 0 0 0 200

8 5x10^-7 0 0 0 0

9 5x10^-7 0 20 0 0

10 5x10^-7 0 0 20 0

11 5x10^-7 0 0 0 20

12 5x10^-7 0 200 0 0

13 5x10^-7 0 0 200 0

14 5x10^-7 0 0 0 200

15 0 10^-9 0 0 0

16 0 10^-9 20 0 0

17 0 10^-9 0 20 0

18 0 10^-9 0 0 20

19 0 10^-9 200 0 0

20 0 10^-9 0 200 0

21 0 10^-9 0 0 200

22 0 5x10^-7 0 0 0

23 0 5x10^-7 20 0 0

24 0 5x10^-7 0 20 0

25 0 5x10^-7 0 0 20

26 0 5x10^-7 200 0 0

27 0 5x10^-7 0 200 0

28 0 5x10^-7 0 0 200

S5 The tested combinations of nanoTiO2 and IHg or MeHg (Table S3) correspond to (i) negligible or no effects concentrations (20 mgL^-1 nanoTiO2; 10^-9M IHg or MeHg) and (ii) concentrations at which according to the literature more than 50% of the cells are affected (200 mgL^-1 nanoTiO2; 10^-9M IHg or MeHg). For example, 200 mgL^-1nanoTiO2 were chosen as corresponding to lowest value inducing 50% effect to the most sensitive organism in a multispecies test battery (Kahru and Ivask, 2013). IHg/MeHg were based on our previous work EC50 (48h) for IHg 6x10^-7M and EC50 (48h) for MeHg (2x10^-8M). This approach of testing combinations covering the two “extremes” was chosen given that the study aims to explore the interactions and effects in mixtures, which usually could occur at high dose levels of the mixture components.

Chemical speciation calculations for IHg and MeHg in exposure medium

The speciation of dissolved IHg or MeHg was calculation by using the Windermere Humic Aqueous Model (WHAM) model VII (Tipping, 2007). Its default database was updated for OH^-, Cl^-, SO42- and CO32- binding constants (Powell et al., 2005). The experimentally determined concentrations of initial IHg or MeHg were used as input, together with concentration of different exposure medium components.

Results and discussion

Exposure time (min)

0 20 40 60 80 100 120

Intracellular Hg (x103 mg kg-1 ) 0 2 4 6

IHg MeHg

Figure S1. Intracellular Hg concentration versus exposure time for C. reinhardtii exposed to 6x10^-8 M IHg (black dots) and MeHg (open dots).

S6

0 20 40 60 80 100 120

0.00 0.01 0.02 0.03

0 20 40 60 80 100 120

0.000 0.001 0.002 0.003 0.004 0.005 A

Amount of adsorbed Hg (mg·kg-1 )

20 mg·L^-1 A5 20 mg·L^-1 A15 20 mg·L^-1 AR20

200 mg·L^-1 A5 200 mg·L^-1 A15 200 mg·L^-1 AR20

Contact time (min)

B

Figure S2. Adsorption of IHg on nanoTiO2 under different exposure times. (A) 20 mgL^-1; (B) 200 mgL^-1; the initial IHg concentration was fixed at 5x10^-7 M. The data were fitted with a pseudo-second-order equation.

S7 Figure S3. Schematic representation of the FCM data analysis procedure to determine the gate for algae and nanoTiO2. As first step a FSC-H/FSC-A dot-plot (A) was used to remove eventual instrument background, and then signals present in the gated region were plotted in a count/

versus red fluorescence plot (B) and in a SSC /FSC dot-plot (C) to verify cellular characteristics of size, granularity and chlorophyll autofluorescence. (D), (E) and (F) are the examples of alga, nanoTiO2 and mixture of alga and nanoTiO2.The log FSC-H versus log FSC-A dot-plot was used first to remove cell doublets or artefacts. Then two different plots (log SSC-A versus log FSC-A plot and count versus log red fluorescence plot) were used to discriminate between algal cells and nanoTiO2 on the base of the different size and higher chlorophyll autofluorescence of the alga in comparison with nanoTiO2.

S8 Figure S4. Log-log dot plots of CellROX®green versus chlorophyll fluorescence of C.

reinhardtii: (A) negative control, not exposed to toxicants; (B) positive control, exposed to H2O2. Log-log dot plots of PI fluorescence versus chlorophyll fluorescence of C. reinhardtii (C) negative control, not exposed to toxicants; (D) positive control, heat treated cells. Exposure conditions: 5×10^-3 M H2O2 and 30 min for CellROX®green stains; 90°C and 15 minutes for PI stains. Staining conditions: 5μM CellROX®green and 7μM PI, time 30 min. PI binds to DNA and attaches to RNA following intracellular penetration through impaired cell membranes, but it is excluded from the healthy cells. CellROX® Green Reagent is a probe for measuring oxidative stress in live cells. The cell-permeant dye is weakly fluorescent while in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA.

S9 Figure S5. Size distribution of nanoTiO2 (20, 200 mg L^-1) in the absence and presence of IHg and MeHg in algal exposure medium at 2 h.

S10

-10 -9 -8 -7 -6

-2 -1 0 1 2 3 4

IHg

Log[Hg]_med (M)

Log[THg]cell (mg·kg-1 ) ^A

MeHg

Figure S6. Log-log plot between accumulated mercury in alga [THg]cell and the concentration of dissolved Hg (not adsorbed on nanoTiO2) in exposure medium. Total Hg contents in algal cells display linear relationship with dissolved Hg concentration (non-adsorbed to nanoTiO2) in exposure medium, with slope of 0.47 ± 0.01 (R² = 0.99) and 0.60 ± 0.42 (R² = 0.93) for IHg and MeHg, respectively.

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