Effect and mechanism of TiO2 nanoparticles on the

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Contents lists available at ScienceDirect. Ecotoxicology and ... dicating the impairment on the photosynthesis via damaging the reaction center of PS II. .... our previous papers (Long et al., 2012; Zhang et al., 2017; Gao et al.,. 2018). Briefly ..... accA acetyl-CoA carboxylase alpha subunit. F: TAGTTTGTGCTTCGGGTGG. 202.
Ecotoxicology and Environmental Safety 161 (2018) 497–506

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Effect and mechanism of TiO2 nanoparticles on the photosynthesis of Chlorella pyrenoidosa Ayyaraju Middepogua,1, Jie Houa,1, Xuan Gaoa, Daohui Lina,b, a b

T



Department of Environmental Science, Zhejiang University, Hangzhou 310058, China Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Zhejiang University, Hangzhou 310058, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Green algae Nanomaterials Chloroplast Photosynthetic process Energy metabolism

Titanium dioxide nanoparticles (n-TiO2) have been used in numerous applications, which results in their release into aquatic ecosystems and impact algal populations. A possible toxic mechanism of n-TiO2 on algae is via the disruption of the photosynthetic biochemical pathways, which yet remains to be demonstrated. In this study, Chlorella pyrenoidosa was exposed to different concentrations (0, 0.1, 1, 5, 10, and 20 mg/L) of a type of anatase n-TiO2, and the physiological, biochemical, and molecular responses involved in photosynthesis were investigated. The 96 h half growth inhibition concentration (IC50) of the n-TiO2 to algae was determined to be 9.1 mg/L. A variety of cellular and sub-cellular damages were observed, especially the blurry lamellar structure of thylakoids, indicating the n-TiO2 impaired the photosynthetic function of chloroplasts. Malondialdehyde (MDA) and glutathione disulfide (GSSG) significantly increased while the glutathione (GSH) content decreased. This implies the increased consumption of GSH by the increased intracellular oxidative stress upon n-TiO2 was insufficient to eliminate the lipid peroxidation. The contents of photosynthetic pigments, including chlorophyll a (Chl a) and phycobiliproteins (PBPs) in the exposed algal cells increased along with the up-regulation of genes encoding Chl a and photosystem II (PS II), which could be explained by a compensatory effect to overcome the toxicity induced by the n-TiO2. On the other hand, the photosynthetic activity was significantly inhibited, indicating the impairment on the photosynthesis via damaging the reaction center of PS II. In addition, lower productions of adenosine triphosphate (ATP) and glucose, together with the change of gene expressions suggested that the n-TiO2 disrupted the material and energy metabolisms in the photosynthesis. These findings support a paradigm shift of the toxic mechanism of n-TiO2 from physical and oxidative damages to metabolic disturbances, and emphasize the threat to the photosynthesis of algae in contaminated areas.

1. Introduction Nanomaterials are increasingly used in industrial production as well as in electronic, biological, and medical research (Patra et al., 2012). Titanium dioxide nanoparticles (n-TiO2) are among the most commonly produced nanomaterials with wide applications in a variety of industries including but not limited to solar cells, cosmetics, food, and environmental remediation due to their excellent photocatalytic activity (Chorianopoulos et al., 2011; Tong et al., 2012; Jang et al., 2016; Bendjabeur et al., 2018). As a result, n-TiO2 are increasingly released in aquatic environments, which results in inevitable contamination (Amde et al., 2017). The estimated n-TiO2 concentration in sediments in Switzerland was up to 2.4 mg/kg, which might be further increased with the increasing use of n-TiO2 (Gottschalk et al., 2009). The possible effects of n-TiO2 on aquatic life, especially algal sources of primary



1

production, have become a large concern (Kulacki and Cardinale, 2012). Many studies have investigated the effects of n-TiO2 on algae, and growth inhibition have been extensively observed (Hong and Otaki, 2006; Kulacki and Cardinale, 2012). Very diverse half growth inhibition values (IC50) have been reported, which are related to physical and chemical properties of n-TiO2 and culture media (Ji et al., 2011; Menard et al., 2011; Metzler et al., 2011; Lin et al., 2012; Magdolenova et al., 2014; Gao et al., 2018). Evidence showed that nanoparticles (NPs) on the surface of algal cells might reduce light availability for the photosynthesis, resulting in a shading effect (Schwab et al., 2011; Long et al., 2012; Wang et al., 2016). The cell surface binding and/or internalization of NPs may elevate intracellular reactive oxygen species (ROS) level, which would thus induce consumption of the antioxidants (e.g., glutathione, GSH) and affect the activity of related enzymes (such

Corresponding author at: Department of Environmental Science, Zhejiang University, Hangzhou 310058, China. E-mail address: [email protected] (D. Lin). The two authors contributed equally to this work.

https://doi.org/10.1016/j.ecoenv.2018.06.027 Received 1 May 2018; Received in revised form 7 June 2018; Accepted 9 June 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

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Material Technology Co., China, which were characterized in detail in our previous studies (Zhang et al., 2017; Gao et al., 2018). The measured particle size was 12.0 ± 3.5 nm, and the properties and transmission electron microscope (TEM) image are shown in Table S3 and Fig. S1 in the Supporting information, respectively. Different concentrations (0, 0.1, 1, 5, 10 and 20 mg/L) of the n-TiO2 were suspended with algal culture in 250-mL Erlenmeyer flasks. Before the inoculation of algal cells, media were sonicated (600 W, 40 kHz, 25 °C) in a bath for 30 min to fully disperse the NPs.

as SOD, CAT, etc.) (Li et al., 2015). Previous studies have demonstrated that the toxicity of n-TiO2 to algae is related to oxidative stress (Morelli et al., 2018; Liu et al., 2018). Li et al. (2015) reported that algae were damaged by the n-TiO2-induced ROS accumulation and the chloroplast was the site of ROS production. Therefore, the damage caused by nTiO2 in the chloroplast could possibly interfere with the light harvest, electron transfer, and energy metabolism in the photosystem (PS), which however remains to be investigated. Moreover, to the best of our knowledge, no research has investigated the effect of n-TiO2 on the gene expression of algae. Photosynthesis is a complex series of biochemical reactions that plants and algae use to convert solar energy, water, and carbon dioxide into ATP and glucose, which plays an important role in algal growth (Singh and Singh, 2015). The process can be divided into two parts: the light-dependent reactions and the light-independent reactions (also referred as the dark reactions) (Lu et al., 2015). In the light-dependent reactions, the ATP synthesis is catalyzed by F0F1 ATP synthase (Elston et al., 1998). Direct evidence has been found that the proton gradient generated by electron transfer complexes and light-harvesting proteins provides the energy for the synthesis of ATP (Simpson and Knoetzel, 1996), which is used to fix carbon from carbon dioxide into the energy storing carbon compounds in the dark reactions (Lu et al., 2015). In green algae, the photosynthetic process occurs in the thylakoid membrane, which composed of four major protein complexes including PS II, cytochrome b6f, PS I, and ATP synthase (Choquet and Vallon, 2000). The light harvesting pigments of the PS, including Chl a and PBPs, are driven to a higher energy state with the help of light energy, which reflects the photosynthetic efficiency and provides information on the structure and function of PS II (Renger, 2013). In addition, the photosynthetic activity including electron transport rate (ETRmax) and maximum photosynthetic yield (Fv/Fm) have been widely used in detection and evaluation of various stresses on the algal photo-system (Kumar et al., 2014; Chae and An, 2016). C. pyrenoidosa is a green unicellular alga, which has been used as a classical ecotoxicological model for aquatic ecosystems because of its extra-sensitivity to contaminants (Li et al., 2013; Shao et al., 2015). In the present study, the toxic effects of a type of anatase n-TiO2 on the photosynthetic system of C. pyrenoidosa were evaluated from cellular, biochemical, and molecular levels. The deleterious impacts were characterized as growth inhibition, ultrastructure damage, and lipid peroxidation. GSH and glutathione disulfide (GSSG) were measured to evaluate the antioxidative responses. Moreover, contents of photosynthetic pigments (Chl a and PBPs), the photosynthetic activity (ETRmax and Fv/Fm), adenosine triphosphatase (ATPase), ATP, glucose, and the expression of photosynthesis-related genes were investigated to gain a systematical understanding of the toxicity of n-TiO2 on the photosynthetic process in algae. The results will provide insightful and theoretical guidance for the risk assessment and safe use of nanomaterials.

2.2. Algal growth assay The algal cells in each sample were counted at least three times using a counting chamber under a light microscope (LM, Olympus, CX21, Japan), and the density in each treatment group was calculated. The initial algal density was 3.66 × 105 cells/mL and the exposure was maintained for 96 h (Long et al., 2012; Zhang et al., 2017; Gao et al., 2018). The control algae exponentially grew in 96 h (Fig. S2). After the 96 h exposure, the growth inhibition ratio (IR) was calculated as follows: IR (%) = (Xc − Xt)/Xc × 100%, where Xc and Xt are the average cell densities (cells/mL) in the control and treatment groups, respectively. The IC50 values, which represent the concentrations of the test substances leading to 50% reduction in the algal growth compared to the control, were calculated from the dose-response curves with SPSS 20.0 for Windows (SPSS, Inc., Chicago, IL, USA). 2.3. Morphology and ultrastructure observations After the 96 h exposure, the algal cell morphologies were observed using a scanning electron microscope (SEM) (Hitachi S4800, Japan) and a TEM (JEM-1230, JEOL, Japan) following the method described in our previous papers (Long et al., 2012; Zhang et al., 2017; Gao et al., 2018). Briefly, the algal cells were fixed in 2.5% glutaraldehyde, dehydrated in gradient concentrations of ethanol, coated with a layer of gold, and then followed by the SEM observation. For the TEM imaging, the algal cells were fixed in glutaraldehyde overnight followed by staining with osmium tetroxide, dehydration, embedding, and ultrathin section. 2.4. Determination of MDA, GSH, and GSSG In each group, algal cells from 200 mL of culture were collected by centrifugation at 8000 g for 10 min after the 96 h exposure. The samples were homogenized as described by Gao et al. (2018). After the centrifugation at 3000g for 10 min at 4 °C, the supernatant was collected and divided for subsequent experiments. Malondialdehyde (MDA) is an oxidation product of lipid peroxidation and an indicator of cell membrane peroxidation. The level of MDA was determined using the thiobarbituric acid reactive substance (TBARS) assay with the MDA assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with procedures detailed in our previous study (Zhang et al., 2017). GSH and GSSG have been used for evaluating the antioxidative and detoxification states in algae. The contained GSH and GSSG levels were measured using the GSH and GSSG assay kit (Beyotime Institute of Biotechnology, Shanghai, China), which were determined using 5,5-dithiobis-(2-nitrobenzoic)-acid (DTNB) as substrate to measure the absorbance at 412 nm according to Griffith (1980) method. All values were normalized by cell density and were reported in nmol/105 cells.

2. Materials and methods 2.1. Algal cultivation and n-TiO2 exposure C. pyrenoidosa was purchased from the Institute of Wuhan Hydrobiology of Chinese Academy of Sciences, China. The algal cells were cultured in 250-mL Erlenmeyer flasks containing 100 mL of the culture medium recommended by the guideline (No. 201) of Organization for Economic Co-operation and Development (OECD) with or without test materials and were kept in an incubation shaker (120 rpm, 25 °C) with illumination by white incandescent lights (100 ± 5 μE/m2/s, light: dark of 14:10 h) (OECD, 2011). The chemical composition of the culture medium is also given in Table S1 in the Supporting information and the detailed test conditions are summarized in Table S2. The anatase n-TiO2 were purchased from Zhejiang Hongsheng

2.5. Contents of photosynthetic pigments and photosynthetic activity The contents of photosynthetic pigments (Chl a and PBPs) in each culture were analyzed according to the method of Johnson et al. (2014) and Deng et al. (2017). After the 96 h exposure, Chl a and PBPs were extracted from the algae in 90% ethanol in the dark for 24 h, followed by 15 min of centrifugation at 3000g, and diluted to 10 mL with 90% 498

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according to previous studies (Fan et al., 2014; Sun et al., 2014; Zhang et al., 2014). Housekeeping gene 18 s rRNA was stable and chosen as an internal control. The amplification efficiencies of the target genes and 18s rRNA were approximately equal. The relative expression ratio (R) was calculated using the 2-ΔΔCt method (Livak and Schmittgen, 2001). The expression of these target genes was normalized against the control. Three biological replicates and three technical replicates were used in the qPCR analysis.

ethanol. The supernatants were transferred into 96-well plates (Corning Incorporated Life Sciences, USA) and the absorbance values of Chl a and PBPs were measured at 750, 665, 652 nm and 562, 615, 652 nm, respectively, using a multi-mode Microplate Reader (Infinite M200PRO, TECAN, Switzerland). All absorbance values were corrected using 90% HPLC-grade methanol as control. Concentrations of Chl a (μg/L) were calculated based on the absorbance according to an equation provided by Deng et al. (2017). The PBPs content was calculated as the sum of phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE) according to the equations provided by Johnson et al. (2014). Finally, the contents of Chl a and PBPs were normalized by the cell density and expressed as μg/105 cells. Photosynthetic activity was measured with a Phytoplankton-PulseAmplitude-Modulated fluorometer (PHYTO-PAM) (Gademann Instruments GmbH, Würzburg, Germany) fitted with a blue diode light source. In brief, the algal cells were stored in the dark for 30 min before the measurements to allow the PS II reaction center to open (re-oxidize) and the electron transport chain to be fully oxidized. PAM parameters including maximum electron transfer rate (ETRmax) and maximum optical quantum yield (Fv/Fm) were automatically calculated using the PhytoWin software (v2.13; Heinz Walz Gmbh, Effeltrich, Germany) as defined by Ritchie and Bunthawin (2010).

2.8. Statistical analysis Statistical analysis was carried out using the SPSS 20.0. The IC50 and 95% confidence intervals were calculated by the probit regression. All values were presented as a mean ± standard error (n = 3). The differences between the control and treatments were analyzed by a oneway ANOVA with Tukey's post hoc tests using SPSS 20.0. Normality and variance homogeneity were previously verified. The probability levels of P < 0.05 (*) and P < 0.01 (**) were defined as statistical significance. 3. Results 3.1. Inhibition of algal growth

2.6. Measurements of ATPase activity, ATP, and glucose As shown in Fig. 1, the growth of algae was significantly decreased in a dose-depended manner after the expose to different concentrations of the n-TiO2 for 96 h. The IR increased from 3.62% to 85.3% with increasing n-TiO2 concentration from 0.1 to 20 mg/L. The 96 h IC50 was calculated to be 9.1 mg/L (95% confidence interval = 7.3–11 mg/L).

After the centrifugation, functionally intact chloroplasts were isolated according to the protocol of Hildebrand (2006). The collected chloroplasts were suspended and incubated with 15 mM dithiothreitol in ice for 15 min, and the total ATPase activity was measured according to the instruction of an ATPase assay kit (Nanjing Jiancheng Bioengineering Institute, China) using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher, Waltham, MA, USA) at a wavelength of 660 nm. Achieved values were normalized by the cell density and expressed as U/105 cells. ATP and glucose in the collected supernatant of algae were measured. The ATP content was measured by an Enhanced ATP Assay Kit (Beyotime Institute of Biotechnology, China) using the Microplate Reader based on the reaction of firefly luciferase with ATP which results in a bioluminescent product (Prioli et al., 1985). Glucose is converted by hexokinase in the presence of ATP into glucose-6-phosphate, which then reacts with the oxidized form of nicotinamide adenine dinucleotide (NAD+) via glucose 6-phosphate dehydrogenase (G-6-PDH) to form 6-phosphogluconate and the reduced form of nicotinamideadenine dinucleotid (NADH). Glucose concentration was then determined by NADH optical absorption at 340 nm. A commercial kit for the measurement of glucose was obtained from Sigma Chemical Co. (St Louis, MO, USA). The values of ATP and glucose were normalized based on the cell density and expressed as nmol/105 cells.

3.2. Morphological and ultrastructural damages The morphology and ultrastructure changes of the algal cells after the exposure to n-TiO2 are visualized in the SEM (Fig. 2) and TEM (Fig. 3) images. Algal cells in the control group exhibited a smooth surface and integrated cell wall (Fig. 2A). No morphological damage was observed after the exposure to 0.1 mg/L n-TiO2 (Fig. 2B). However, in the 1, 5, 10, and 20 mg/L groups, adsorptions of n-TiO2 on algae were extensively found (Fig. 2C–F). Dramatic alterations in cell wall including cell surface disruption and shrinkage occurred when the concentration of n-TiO2 increased to 10 mg/L (Fig. 2E). In the 20 mg/L group, extensive surface irregularity was also found, indicating the occurrence of wall rupture and degradation (Fig. 2F). Fig. 3A shows an untreated algal cell with a pyrenoid, lipid droplets, and intact thylakoid membrane structure with clear stroma lamellae. No significant ultrastructure damage was found in the 0.1 mg/L group (Fig. 3B). When the exposed concentration exceeded 1 mg/L, plasmolysis was observed as a common symptom in algae, and the degree of plasmolysis increased with the increase of n-TiO2 concentration (Fig. 3C–F). Membranolysis also occurred when the concentration of nTiO2 increased to 10 mg/L (Fig. 3E). In the 20 mg/L group, distinct cell wall damage along with the cell internalization of n-TiO2 were found in a necrotic algal cell, and the lamellar structure of thylakoids in the chloroplast showed blurry boundaries and lysis (Fig. 3F).

2.7. Gene expression analysis For gene expression analysis, total RNA was extracted from 100 mg of collected algal cells in the 0 (control), 0.1, and 20 mg/L groups using RNAiso Plus (TaKaRa, Dalian, China). Isolation, purification, and quantification of total RNA and quantitative real-time polymerase chain reaction (PCR) were performed. The purity of extracted RNA was assessed by determining the absorbance ratios of 260–280 nm (A260/ A280) and 260–230 nm (A260/A230). Only RNA samples with A260/ A280 values in the range of 1.8–2.1 and A260/A230 values above 2.0 were used in further experiments. One µg of total RNA was processed through reverse transcription in the presence of oligo-dT primers using a PrimeScript RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Quantitative real-time PCR was performed on an iQ5 Multicolor RealTime PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The gene-specific primers of 14 genes (Table 1) related to energy metabolism, photosynthesis, and cell proliferation were designed

3.3. The contents of MDA, GSH, and GSSG As shown in Fig. 4A and B, MDA, GSH, and GSSG contents in the algal cells varied differently after the exposure to n-TiO2. The content of MDA significantly increased with increasing n-TiO2 concentration (P < 0.05 in the 5 and 10 mg/L groups), which was up to more than 3fold of the control (P < 0.01) in the 20 mg/L group (Fig. 4A). By contrast, as the n-TiO2 concentration exceeded 1 mg/L, GSH in the algal cells significantly decreased (P < 0.01) and corresponded to approximately 60% of the control level after the exposure to 5, 10, and 20 mg/L n-TiO2. Meanwhile, GSSG in the algal cells showed an initial increase, 499

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Table 1 Sequences of primers used for the Real-time PCR. Gene

Encoded protein

Primer sequences (from 5’ to 3’)

Length (bp)

accA

acetyl-CoA carboxylase alpha subunit

202

accD

acetyl-CoA carboxylase beta subunit

dgat

acyl CoA: diacylglycerol acyltransferase

me

malate dehydrogenase

pepc

phosphoenolpyruvate carboxylase

cah2

carbonic anhydrase

rbcL

large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

hla3

inorganic carbon transporter

psbA

PS II protein D1

psbB

photosystem II P680 chlorophyll a apoprotein

chlL

light-independent protochlorophyllide reductase subunit

cox2

cytochrome c oxidase subunit II

atpB

ATP synthase CF1 beta subunit

ftsH

cell division protein FtsH

18s rRNA

18S ribosomal RNA

F: TAGTTTGTGCTTCGGGTGG R: ACATCYCACGCAGGTTGAC F: TAGTTTGTGCTTCGGGTGG R: CAATAAGGGCTTTCGGTTCA F: GGCACAAAGAGTTCACCGT R: ACAAACTTGAGGTGGGTG F: CCCTCTCGTTCCCCTTTTATT R: AAATGCTGACGCAAGTGTGA F: GACTATCCCCTTCAGCCACTC R: AAACAGCTCCTCAGCCATCTT F: GACTCCAACATTGCGAAGAT R: GGAAGAGGTCGGTCAGGT F: ATACCGTGAGGAGGACCTTGGA R: CCAGGTGAAGAAAACCAATACA F: TGATGTGCTTCCTCACCCT R: TCCAAAGTGTCCTGGTCCT F: GAAAACGAATCAGCTAACGAAG R: AAGCAGTGAACCAAATACCAAC F: CACCGTCTGATTGAAGAGTTGC R: GATGTTCCTTTCCGTCGTTCTG F: TCGCAGCAAACCGAATTGTT R: GCTGCTGGCACCAGACTT F: GAAGTGGATAATCGTATGGTTG R: CTGCATCACATTTTGCTCCTAA F: GTTTCGTTCAAGCTGGTTCT R: GTTCTTGTAAGCCACCCATT F: ACAAAGTGACCGAAATCCAGAA R: TTACGAATTGGGAGACTAGAAG F: GAGTATGGTCGCAAGGCTGAA R: AACCTGACAAGGCAACCCAC

227 145 158 185 109 194 189 190 187 180 115 105 129 200

3.5. Photosynthetic activity The change in the PS II functional parameters indicated that the nTiO2 treatments altered PS II energy transfer (Fig. 5A and B). ETRmax was very sensitive to the n-TiO2 exposure, which exhibited a significant decrease at the concentration of 0.1 mg/L (P < 0.01) and remained approximately at two-third of the control value with the increase of nTiO2 concentration (P < 0.01) (Fig. 5A). On the other hand, the Fv/Fm exhibited a similar dose-dependent reduction, and significant decreases were observed in the algae exposed to 5, 10, and 20 mg/L n-TiO2 (P < 0.01) (Fig. 5B). 3.6. ATPase activity and ATP and glucose Fig. 1. Inhibition ratios (IR) of the algal growth after exposed to different concentrations of the n-TiO2 for 96 h. The solid line in the figure was fitted with a probit regression model. The dashed lines locate the IC50 value.

The ATPase activity in the chloroplast along with the ATP and glucose contents in the algae are shown in Fig. 5C and D. The ATPase activity was inhibited and significant decreases were found for the algae exposed to 5, 10, and 20 mg/L n-TiO2 (P < 0.01), which was about 60% of the control level (Fig. 5C). Similarly, the contents of ATP and glucose in the algae declined with increasing n-TiO2 concentration. For the ATP content, significant decreases were found in the 0.1, 5, 10, and 20 mg/L treatment groups (P < 0.01), while the content of glucose showed a dose-dependent decline after the exposure to 1 (P < 0.05) and 5, 10, and 20 mg/L n-TiO2 (P < 0.01) (Fig. 5D).

peaking at 5 mg/L, and then gradually dropped back but remaining significantly higher than the control in the 10 and 20 mg/L groups (P < 0.01, Fig. 4B).

3.4. The content of photosynthetic pigments The photosynthetic pigments (Chl a and PBPs) of algae after the 96 h n-TiO2 exposure are presented in Fig. 4C. With increasing n-TiO2 concentration, the content of Chl a increased from 20 nmol/105 cells to 45 nmol/105 cells; the 20 mg/L group exhibited significant higher Chl a than the control (P < 0.01). In contrast, PBPs contents remained steady after the exposure to 0.1, 1, 5, and 10 mg/L n-TiO2; however, a significant increase was found in the 20 mg/L group, which was 328% of the control level.

3.7. Gene expressions The expressions of genes involved in the energy metabolism, photosynthesis, and cell proliferation in algal cells were examined to explore possible toxic mechanisms of the n-TiO2. The result showed that the n-TiO2 induced various alterations in the gene expression in algae cells (Fig. 6). After the exposure to 0.1 mg/L n-TiO2, the relative mRNA level of most genes remained close to the control levels except for the significant down expression of rbcL (P < 0.05). In the 20 mg/L group, 3 genes exhibited significant down-regulations, among which dgat and 500

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Fig. 2. SEM images of the algae after the exposure to (A) 0, (B) 0.1, (C) 1, (D) 5, (E) 10, and (F) 20 mg/L of the n-TiO2 for 96 h. The white arrows point to cell surface attached n-TiO2. The red arrows point to alterations in cell wall, including surface disruption, shrinkage, and irregularity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

obliquus (15.3 mg/L), and K. brevis (10.69 mg/L) (Zhu et al., 2008; Lin et al., 2012; Li et al., 2015). To clarify the action of n-TiO2 on algae, the morphological and ultrastructural damages were examined, which were characterized as cell wall/membrane damage, plasmolysis, and cell internalization of n-TiO2. Similar ultrapathological changes have been identified by other researchers (Schwab et al., 2011; Chen et al., 2012; Gao et al., 2018), implying that these injuries commonly occur in algae exposure to n-TiO2. Meanwhile, the adsorption of n-TiO2 on the algae, which was extensively observed in the treatment groups, could block light from reaching the photosynthesis center and induce the shading effect (Morelli et al., 2013; Wang et al., 2016). Moreover, it is worth noting that the lamellar structure of thylakoids in the chloroplast exhibited blurry boundaries and lysis after the exposure to n-TiO2, which was compelling evidence of the impairment of photosynthetic electron

cah2 dramatically decreased to lower than 0.2-fold and ftsH declined to approximately 0.3-fold of the control. Contrarily, 4 genes showed differential up-regulations. The expression of me, pepc, and chlL were significantly increased to 4.2, 1.7, and 3.5-fold, respectively (P < 0.05), and psbB was significantly up-regulated to above 2-fold of the control (P < 0.01). The expressions of accA, accD, hla3, psbA, cox2, and atpB remained unchanged.

4. Discussion In the present study, the growth inhibition of different concentrations of the anatase n-TiO2 on the green algae exhibited a clear doseresponse relationship; the determined IC50 was 9.1 mg/L, which was comparable with recorded values on C. pyrenoidosa (4.9 mg/L), S. 501

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Fig. 3. TEM images of the algae after the exposure to (A) 0, (B) 0.1, (C) 1, (D) 5, (E) 10, and (F) 20 mg/L of the n-TiO2 for 96 h. Bars represent 500 nm. The panes in A and F locate the thylakoids. The white arrows point to plasmolysis and membranolysis. The red arrow points to the cell internalization of n-TiO2. The yellow star locates the cell wall breakage. Abbreviations: P, pyrenoid; SG, starch grain; LD, lipid droplets. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

transport system. This finding prompted us to explore the in-depth toxic mechanism of n-TiO2 on the photosynthesis of algae. Apart from the shading effect, oxidative stress along with lipid peroxidation has been considered as another dominant factor in the toxic effect of NPs on algae (Chen et al., 2012; Long et al., 2012; Xia et al., 2015). In the present study, the MDA levels in the algae increased after the n-TiO2 exposure, which was in line with previous observations (Chen et al., 2012; Long et al., 2012; Xia et al., 2015). GSH is an important biomarker to estimate oxidative stress because of its important role in eliminating redundant ROS and decreasing the toxic effects of pollutants to algae, along with the production of GSSG (Chen et al., 2012). In the present study, GSH in the algae was significantly decreased in a dose-dependent manner, while the GSSG content showed

the opposite trend, indicating that the consumption of GSH was insufficient to eliminate the lipid peroxidation caused by the exposure to high concentrations of n-TiO2. In this case, excessive intracellular ROS and lipid peroxidation induced by the n-TiO2 could have resulted in subcellular structural damages as evidenced by Liu et al. (2018). In algal cells, chloroplast, as the site of the photosynthesis, has been regarded as the main source of ROS production (Alberts and Lewis, 2002; Gill and Tuteja, 2010). Together with the cell internalization of n-TiO2 and ultrastructural damage of chloroplast in this study, it could be speculated that the combination of physical effect and lipid peroxidation induced by the n-TiO2 might affect the normal function of chloroplast by deactivating the enzyme and pigments in the thylakoid system, which could further result in the disorder of photosynthesis and 502

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Fig. 4. The contents of MDA (A), GSH and GSSG (B), and Chl a and PBPs (C) in the algal cells exposed to different concentrations of the n-TiO2 for 96 h. * and * * denote significant (P < 0.05) and very significant (P < 0.01) differences from the control, respectively.

Fig. 5. Changes of (A) ETRmax, (B) Fv/Fm, (C) the ATPase activity, and (D) ATP and glucose contents in the algae against the concentration of n-TiO2. * and ** denote significant (P < 0.05) and very significant (P < 0.01) differences from the control, respectively.

503

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Fig. 6. Relative mRNA levels of 14 genes in the algal cells exposed to 0, 0.1, and 20 mg/L n-TiO2 for 96 h. * and ** denote significant (P < 0.05) and very significant (P < 0.01) differences from the control, respectively.

Fig. 7. Possible toxic mechanisms of n-TiO2 on the photosynthesis of Chlorella pyrenoidosa. The downward blue and the upward red arrows stand for down and up regulations, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

expression of dgat (encoding diacylglycerol acyltransferase) was substantially down-regulated, implying the reduction of triacylglycerol (TAG) biosynthesis in the algal cells (Chen and Smith, 2012). Malic enzyme (ME) was considered to be a major supplier of nicotinamide adenine dinucleotide phosphate (NADPH), which is critical to the intracellular fatty acid content (Tang et al., 2010), and phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the competing pathway of lipid accumulation (Chen et al., 1998). Thus, the simultaneously up-regulated expression of me and pepc indicated the accelerated synthesis and transformation of malic acid as well as inhibited lipid accumulation. On the other hand, cah2 and rbcL respectively encodes carbonic anhydrase and RuBisCO small subunit involved in the carbon fixation in the photosynthesis (Fukuzawa et al., 1990; Razzak et al., 2017); their expressions also declined, suggesting the carbohydrate biosynthesis in algae was suppressed by the n-TiO2. Furthermore, the down-regulation of cell division protein ftsH gene could be directly responsible for the observed inhibitory effect on the algal growth (Itoh et al., 1999). These results were consistent with the low ATP and glucose levels in the algal cells as well as with the algal growth inhibition. In particular, the genes involved in the subunit of PS II (psbB) and lightindependent synthesis of chlorophyll (chlL) (Zhang et al., 2007) were significantly up-regulated, which corroborates the increased contents of photosynthetic pigments observed in this study. As summarized in Fig. 7, our findings point to possible toxic mechanisms of n-TiO2 on the algal growth via inhibition of electron transport and photosynthetic processes in the chloroplast, as well as suppressing the lipid and carbohydrate biosynthesis and cell division at the gene expression level. In addition, the stimulated synthesis of photosynthetic pigments in algal cells might be a compensatory effect to maintain vital activities and overcome the toxicity of n-TiO2.

metabolism. Photosynthetic pigments are responsible for the absorption of light in the photosynthetic process, and their contents often changed (decreased in general) in response to NPs exposure (da Costa et al., 2016; Xiao et al., 2016; Deng et al., 2017). However, in the present study, the content of photosynthetic pigments including Chl a and PBPs concurrently increased, which was consistent with the findings using Chlamydomonas reinhardstii exposed to copper oxide nanoparticles (Melegari et al., 2013). There is a debate about the inductive effects of NPs on pigments content of algae; one explanation is that NPs-induced ROS can attack some pigments, which could convert to Chl a under stress and lead to higher Chl a in the algal cells (Chen et al., 2012). Since Chl a and PBPs showed consistent increases in the present study, it is more likely that the adhesion of NPs to the algal surface reduced the availability of light and stimulated the chlorophyll production as a compensation to overcome the shading effect (Kulacki and Cardinale, 2012). On the other hand, the significant decrease in the photosynthetic activity (ETRmax and Fv/Fm) indicated the impairment of electron transport and photosynthesis via damaging the reaction center of PS II. In recent studies, the inhibitory effect of NPs on algal growth was linked to suppressed photosynthesis (Xiao et al., 2016; Deng et al., 2017), while the potential mechanism at biochemical and molecular levels remained unclear. In our study, the suppressed activity of ATPase along with lower productions of ATP and glucose indicated that disruptions of material and energy metabolisms in the photosynthesis could be involved in the algal growth inhibition caused by the n-TiO2, with the molecular mechanisms further discussed below. For the first time, this study revealed that n-TiO2 disrupted material and energy metabolisms in the photosynthesis of algae at the molecular level. Genes such as accA, accD, dgat, me, and pepc play a crucial role in the lipid biosynthesis (Fan et al., 2014). In the present study, the 504

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5. Conclusion

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Our study demonstrated that the anatase n-TiO2 could inhibit the algal growth via the impairment of the lamellar structure of thylakoids while influencing the photosynthetic function of the chloroplast. The consumption of GSH in algal cells was insufficient to eliminate the lipid peroxidation caused by the stress from the n-TiO2, and the synthesis of photosynthetic pigments increased as a compensatory effect. Declined photosynthetic activity indicated the impairment of photosynthesis by the n-TiO2 via damaging the reaction center of PS II. The reduced production of ATP and glucose along with altered gene expressions suggested the disruption of material and energy metabolisms in the photosynthesis as a toxic mechanism of the n-TiO2 on the algae. Our findings support a paradigm shift of n-TiO2 toxic mechanism from the shading effect and oxidative stress towards metabolic disturbances, and emphasize the threat of NPs to the photosynthetic process of algae in contaminated water bodies. Acknowledgements This work was supported by the National Natural Science Foundation of China (21477107, 21525728, 21621005 and 21337004) and the National Key Research and Development Program of China (2017YFA0207003). Conflict of interest The authors declare that there are no conflicts of interest in the present experiment. Appendix A. Supporting information Composition of the OECD medium (Table S1); test conditions of the performed experiment (Table S2); properties of the selected n-TiO2 (Table S3); TEM image of n-TiO2 (Fig. S1); exponential growth curve of the control algae under the experimental condition (Fig. S2). Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2018.06.027. References Alberts, B.J.A., Lewis, J., 2002. Molecular Biology of the Cell, 4th edition. Garland Publishing Inc., New York. Amde, M., Liu, J.F., Tan, Z.Q., Bekana, D., 2017. Transformation and bioavailability of metal oxide nanoparticles in aquatic and terrestrial environments. A review. Environ. Pollut. 230, 250–267. Bendjabeur, S., Zouaghi, R., Zouchoune, B., Sehili, T., 2018. DFT and TD-DFT insights, photolysis and photocatalysis investigation of three dyes with similar structure under UV irradiation with and without TiO2 as a catalyst: effect of adsorption, pH and light intensity. Spectrochim. Acta A Mol. Biomol. Spectrosc. 190, 494–505. Chae, Y., An, Y.J., 2016. Toxicity and transfer of polyvinylpyrrolidone-coated silver nanowires in an aquatic food chain consisting of algae, water fleas, and zebrafish. Aquat. Toxicol. 173, 94–104. Chen, J.E., Smith, A.G., 2012. A look at diacylglycerol acyltransferases (dgats) in algae. J. Biotechnol. 162, 28–39. Chen, J., Lang, C., Hu, Z., Liu, Z., Huang, R., 1998. Antisense PEP gene regulates to ratio of protein and lipid content in Brassica napus. seeds J. Agric. Biotechnol. 7, 316–320. Chen, L.Z., Zhou, L.N., Liu, Y.D., Deng, S.Q., Wu, H., Wang, G.H., 2012. Toxicological effects of nanometer titanium dioxide (nano-TiO2) on Chlamydomonas reinhardtii. Ecotoxicol. Environ. Saf. 84, 155–162. Choquet, Y., Vallon, O., 2000. Synthesis, assembly and degradation of thylakoid membrane proteins. Biochimie 82, 615–634. Chorianopoulos, N.G., Tsoukleris, D.S., Panagou, E.Z., Falaras, P., Nychas, G.J.E., 2011. Use of titanium dioxide (TiO2) photocatalysts as alternative means for Listeria monocytogenes biofilm disinfection in food processing. Food Microbiol. 28, 164–170. da Costa, C.H., Perreault, F., Oukarroum, A., Melegari, S.P., Popovic, R., Matias, W.G., 2016. Effect of chromium oxide (III) nanoparticles on the production of reactive oxygen species and photosystem II activity in the green alga Chlamydomonas reinhardtii. Sci. Total Environ. 565, 951–960. Deng, X.Y., Cheng, J., Hu, X.L., Wang, L., Li, D., Gao, K., 2017. Biological effects of TiO2 and CeO2 nanoparticles on the growth, photosynthetic activity, and cellular components of a marine diatom Phaeodactylum tricornutum. Sci. Total Environ. 575, 87–96.

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