Effects of titanium dioxide nanoparticles on leaf cell structure and viability, and leaf elongation in the seagrass Halophila stipulacea

Science of the Total Environment 719 (2020) 137378

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Effects of titanium dioxide nanoparticles on leaf cell structure and viability, and leaf elongation in the seagrass Halophila stipulacea Zoi Mylona a, Emmanuel Panteris a, Theodoros Kevrekidis b, Paraskevi Malea a,⁎ a b

Department of Botany, School of Biology, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece Laboratory of Environmental Research and Education, Democritus University of Thrace, Nea Hili, GR-68100 Alexandroupolis, Greece

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• TiO2 NPs at environmentally relevant concentrations cause toxic effect on seagrasses. • TiO2 NPs impair cell structure, cell viability and growth in Halophila stipulacea. • Actin filaments, endoplasmic reticulum and growth are early markers of TiO2 NP-stress. • TiO2 NPs may pose a significant potential risk to the coastal environment.

a r t i c l e

i n f o

Article history: Received 15 December 2019 Received in revised form 9 February 2020 Accepted 15 February 2020 Available online 18 February 2020 Editor: Daniel Wunderlin Keywords: Marine plant Stress response Cytotoxicity Growth Biomarker Risk assessment

a b s t r a c t The ecotoxicity of titanium dioxide nanoparticles (TiO2 NPs) is of increasing concern due to their extensive use in a variety of applications. This study aims to achieve a better understanding of TiO2 NP ecotoxicity by assessing for the first time their effects on seagrasses. Changes in leaf cell structure and viability, and leaf elongation in Halophila stipulacea exposed under laboratory conditions to environmentally relevant TiO2 NP concentrations (0.0015–1.5 mg L−1) for 8 days were assessed. Actin filament (AF) disturbance firstly occurred in differentiating cells at 0.0015 mg L−1 on the 8th day, while in meristematic cells at 0.15 mg L−1 on the 6th day, both deteriorating concentration- and time-dependently. Endoplasmic reticulum (ER) appeared aggregated firstly at 0.015 mg L−1 on the 8th day and earlier at the highest concentrations, while microtubules and cell ultrastructure appeared unaffected. Dead cells mainly occurred in older leaves; dead tooth, margin and intercostal epidermal cells exceeded 5% at 0.15–1.5 mg L−1. A significant leaf elongation inhibition occurred at 0.015–1.5 mg L−1 in older leaves and at 1.5 mg L−1 in young apical leaves. AF, ER and leaf elongation impairment in H. stipulacea, being susceptible response parameters, could be used as early warning markers. A risk quotient N1 was calculated, indicating that TiO2 NPs may pose a significant risk to the environment. The data presented underline the need for additional TiO2 NP-seagrasses toxicity information, and could be utilized for the protection of the coastal environment. © 2020 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. E-mail address: [email protected] (P. Malea).

https://doi.org/10.1016/j.scitotenv.2020.137378 0048-9697/© 2020 Elsevier B.V. All rights reserved.

Titanium dioxide nanoparticles (TiO2 NPs) are increasingly developed and used because of their unique physico-chemical properties, such as photocatalytic activity and ultraviolet (UV) radiation absorption

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(EPA, 2009). TiO2 occurs naturally in an amorphous form and in three crystalline forms (rutile, anatase and brookite), among which anatase exhibits the highest photocatalytic activity, while TiO2 NPs display a higher potential for catalytic activity and UV absorption due to their higher relative surface area (EPA, 2009). The extensive use of TiO2 NPs in a variety of applications, including medicine (e.g. anticancer drugs), self-cleaning surface coatings, water treatment agents, light-emitting diodes, solar panels, household products and topical sunscreens (EPA, 2009; Movafeghi et al., 2018) inevitably results in TiO2 NP release into the environment. TiO2 NPs are considered as a generator of reactive oxygen species (ROS), due to their photocatalytic activity; excess ROS production can lead to oxidative stress, resulting in damages in cell structure and function (Vale et al., 2016; Khan et al., 2017). Emerging literature provides information on TiO2 NPs toxicity on higher plants, however, most of the toxicological studies dealing with TiO2 NP-higher plant interactions used high, not environmentally-realistic, TiO2 NP concentrations (see review in Thwala et al., 2016 and Rastogi et al., 2017) to obtain acute toxicity responses. Nevertheless, rather conflicting evidence concerning the effects of TiO2 NPs on terrestrial and freshwater plants has been reported. In particular, toxicological studies focused on terrestrial plants showed both a negative and a positive impact of TiO2 NPs (exposure concentrations: 10–40,000 mg L−1; Rastogi et al., 2017). Concerning freshwater plants, toxicological studies assessed sub-cellular (oxidative stress, activity of antioxidants), photosynthetic and growth effects at exposure concentrations in the range of 0.01–2000 mg L−1; most of these studies indicated no toxic effects or toxic effects at high, not environmentally relevant TiO2 NP concentrations (≥10 mg L−1; see review in Thwala et al., 2016; Okupnik and Pflugmacher, 2016; Dolenc Koce, 2017; Spengler et al., 2017; Movafeghi et al., 2018). On the other hand, information on stress responses of marine angiosperms to TiO2 NPs is currently missing. Marine angiosperms occur widely in shallow coastal waters throughout the world, except along Antarctic shores. Seagrass beds provide habitat for diverse invertebrate and fish populations, protect coastlines from the erosive impact of tides and waves, and play a key role in global carbon and nutrient cycling (Hemminga and Duarte, 2000). Seagrasses are also valued as sensitive indicators of human pollution and perturbations (Kemp, 2000); the use of biomarkers in seagrasses is considered as a valuable tool for the evaluation of environmental quality (Ferrat et al., 2003). However, seagrass diminishing, due to a combination of natural factors and anthropogenic impacts, including phytotoxic chemical inputs, has been documented worldwide (Hemminga and Duarte, 2000). Toxicity information in seagrasses is available mainly for conventional chemical pollutants (e.g. see review in Lewis and Devereux, 2009; Malea et al., 2013a, 2013b; Negri et al., 2015; Carve et al., 2018; Mochida et al., 2019), but responses of seagrass species to emerging pollutants of environmental concern have been also recently assessed (Moustakas et al., 2017; Adamakis et al., 2018; Malea et al., 2019; Mylona et al., 2020a, 2020b). The main goal of the present study is to achieve a better understanding of the ecotoxicity of TiO2 NPs. In particular, this study aims to provide insight on the toxicity effect of environmentally relevant TiO2 NP concentrations on seagrasses. TiO2 NP concentrations lower than 76.1 μg L−1 could be considered as environmentally relevant, since modeled predicted environmental concentrations for TiO2 NPs in sewage treatment plant effluents for the EU in 2014 were estimated to be in the range of 2.77 and 76.1 μg L−1 (Sun et al., 2016). Halophila stipulacea (Forsskål) Ascherson, 1867 was chosen among seagrasses because of its extensive distribution, notable ecological importance, bioindication capacity and suitability for ecotoxicological research (Mylona et al., 2020a). This seagrass is a tropical species, native to the Indian Sea and the Red Sea, which invaded the Mediterranean Sea and the Caribbean Sea due to its ability to acclimate to a wide range of environmental conditions (see Oscar et al., 2018 and references therein). Many H. stipulacea beds occur in the shallows, even at b1 m, but it

also penetrates much deeper water (Lipkin et al., 2003; Scheibling et al., 2018). H. stipulacea has been considered as a potentially ideal cosmopolitan bioindicator of trace metal pollution (Bonanno and Raccuia, 2018), while biomarkers in H. stipulacea have been regarded as sensitive and reliable indicators of nanoparticle (Ag NP) induced stress (Mylona et al., 2020a). The cytoskeleton, endoplasmic reticulum (ER), cell ultrastructure, cell viability and elongation in H. stipulacea leaves were chosen as response parameters, since most of them have been shown to be prime targets of anthropogenic chemical toxicity in seagrasses (Malea et al., 2013a, 2013b; Adamakis et al., 2018; Mylona et al., 2020a, 2020b). Importantly, we are not aware of any published data regarding toxicity effects of TiO2 NPs on actin filament (AF) cytoskeleton, ER organization and cell ultrastructure in higher plants. We investigated under laboratory conditions potential alterations in (a) AFs, microtubules (MTs) and ER in leaf cells at different developmental stages, (b) leaf cell ultrastructure, (c) the viability of leaf cells of different cell types and leaf ages, and (e) elongation of leaves of different ages in H. stipulacea exposed to TiO2 NP concentrations of 0.0015, 0.015, 0.15 and 1.5 mg L−1 for 8 days. Potential detection of toxic effects would allow to determine the lowest effect concentrations for the examined response parameters, to evaluate the relative susceptibility of these parameters, and to assess the risk posed by TiO2 NPs to the coastal environment. However, (a) according to those mentioned earlier, particularly that most toxicological studies assessing TiO2 NP- freshwater plants interactions showed no toxic effects or toxic effects at high, not environmentally-realistic concentrations and (b) provided that nanoparticle aggregation in seawater is more likely because of the high ionic strength and thus, dispersion is more difficult than in freshwater (Xia et al., 2015), we predict that no marked toxic effects on the examined structural and physiological traits, at least at the lowest concentrations applied, will be observed. 2. Materials and methods 2.1. Seagrass collection Halophila stipulacea was collected from Afissos site, Pagasitikos Gulf, Northern Aegean Sea (39°16′38.33″ N, 23°09′24.64″ E) during March 2016 from 15 m depth by scuba diving. At the sampling site, this seagrass forms a monospecific meadow extending from 10 to 20 m depth. Seagrass plants were rinsed in seawater in the field and carried to the laboratory in plastic containers with seawater. 2.2. Treatments and experimental conditions Erect rhizomes, having at least four nodes and ending in an apical bundle, were placed for 24 h under laboratory conditions in constantly aerated aquaria containing seawater from the sampling site to acclimatize. An apical bundle usually has three pairs of leaves; the leaves of the first and second apical leaf pairs termed young, while those of the leaf pair next to the apical buddle (fourth leaf pair) older. TiO2 NPs were purchased from Alfa Aesar GmbH & Co KG, Karlsruhe, Germany (Titanium (IV) oxide, anatase nanopowder 99.9% metal basis, average particle size 31 nm, surface area 51 m2 g−1; lot no. P 10B008). A stock suspension of 150 mg L−1 of TiO2 NPs was prepared in Milli-Q water and ultrasonicated to a high energy sonication (intensity settings 4) for 3 min with a microtip probe (VibraCell 400 W, Sonics & Materials Inc., USA) (Dalai et al., 2013; Perreault et al., 2012) to increase the dispersion stability; the suspension after sonication became homogenously turbid without visible sedimentation (see also Aruoja et al., 2009). After the sonication, TiO2 NP test suspensions were prepared by diluting the supernatant of the stock suspension (e.g. Wang et al., 2016; Okupnik and Pflugmacher, 2016; Spengler et al., 2017). Plants were incubated completely immersed in acid-washed, glassmade aquaria (35 cm*35 cm*20 cm) containing TiO2 NPs at concentrations 0.0015, 0.015, 0.15 and 1.5 mg L−1 in filtered seawater (0.45 μm

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Whatman GF/C) and filtered seawater with no added TiO2 NPs (control). The seawater (salinity 34.4 psu, pH 7.86, dissolved titanium 0.723 ± 0.049 μg L−1) was collected from the sampling site. The laboratory conditions were 16 h day/8 h night regime, ambient temperature 22 ± 1 °C/18 ± 1 °C day/night, and photon flux density 120 ± 20 μmol m−2 s−1; cool white fluorescent tubes (Phillips, 8 W) emitting light in a visible spectrum with wavelength between 400 and 700 nm (enhanced irradiation between 400 and 500 nm) were used. The aquaria were constantly aerated using pumps. The suspensions in the aquaria were changed every 24 h to maintain nanoparticle exposure during the experiment (see sedimentation analysis). The experiment was run in triplicate. After 2, 4, 6 and 8 days, plant samples were randomly removed for effect detection. 2.3. Nanoparticle characterization Primary particle size and morphology of TiO2 NPs were assessed in ultrasonicated stock suspension (150 mg L−1 in Milli-Q water) by Transmission Electron Microscopy (TEM) using a JEOL JEM 1010 TEM (JEOL Ltd., Tokyo, Japan). The hydrodynamic size of TiO2 NPs in ultrasonicated stock suspension and suspensions at concentrations 0.0015 and 1.5 mg L−1 in seawater was determined by dynamic light scattering (DLS) measurements, and the surface charge of the particles by zeta (ζ) potential measurements in triplicate using a Nano ZS Zetasizer (Malvern Instrument, Worcestershire, UK). The crystalline phase and size identification of NPs was conducted using a Rigaku Ultima+ powder X-ray diffractometer (XRD; 40 kV, 30 mA, Cu Kα radiation), according to the appropriate JCPDS database (Kourtidou et al., 2019). A sedimentation analysis was conducted at 0.0015 and 1.5 mg L−1 TiO2 NP suspensions in seawater to assess the stability of nanoparticle suspensions over time by examining changes in optical absorbance at regular time intervals. Light absorption of the supernatant was measured spectrophotometrically (Camspec M501, Single Beam Scanning, UV/Visible, Shimadzu, Tokyo, Japan) in triplicate at the wavelength of 323 nm (see also Della Torre et al., 2015) every 15 min up to the 6th h, every 1 h from the 6th to 12th h, and every 6 h from the 12th to 36th h. Sedimentation profile was obtained plotting normalized concentration values (C/Co) vs. time; C represents TiO2 NP concentration at the specific time point, and Co the initial concentration at 0 h (Spengler et al., 2017; Morelli et al., 2018). 2.4. Imaging of cytoskeleton, ER and cell ultrastructure AFs, MTs and ER were examined in interphase leaf cells of the meristematic and differentiating zones of the first, youngest apical leaf pair. Leaves from six apical bundles were examined per cell component, concentration and incubation day; razor blade-cut leaf segments (ca. 2 × 2 mm) were obtained, and three segment subsamples were studied in each case. AF staining was performed with TRICT-phalloidin (Sigma) according to the protocol applied by Panteris et al. (2009), modified by Adamakis et al. (2018). MT and ER immunostaining were performed as described by Malea et al. (2013a) and Zachariadis et al. (2001), respectively, with slight modifications. In brief, after fixation leaf segments were incubated in an enzyme mixture consisting of 2.5% cellulase (Onozuka R10, Duchefa), 2% macerozyme (R10, Duchefa), 1% Driselase (Sigma) and 1% β-glucuronidase (Sigma) to digest cell walls; for MT labeling, rat anti-α-tubulin (YOL1/34, AbD Serotec, Kidlington, UK) and FITC-anti-rat (Invitrogen, Carlsbad, CA) were used, both diluted 1:40 in PBS; for ER labeling, leaves were first incubated with mouse anti-HDEL (2E7, Santa Cruz) and afterwards with FITC-anti-mouse (Sigma), both diluted 1:40 in PBS. All the specimens were examined with a Zeiss LSM780 Confocal Laser Scanning Microscope (CLSM) with the appropriate filters and images were acquired with the ZEN2011 Software. Observations were made on about 20–30 cells per image in at least three sample images. The effects on AFs were estimated,

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compared to the control, investigating z-series projections with Image J Software and expressed by an arbitrary scale of 1 to 3. Ultrastructural observations were conducted by TEM in the fourth leaf pair, derived from the rhizome used for cytoskeleton examination. Ultrathin sections were examined with a JEOL JEM 1011 TEM (JEOL Ltd., Tokyo, Japan) equipped with a Gatan ES500W digital camera (Gatan Anc., Tokyo, Japan), at 80 kV, and micrographs were obtained with Digital Micrograph 3.11.2 Software (Panteris et al., 2018). 2.5. Evans Blue staining Untreated and TiO2 NP- treated leaves of the first and second apical leaf pairs (one leaf per pair) and the fourth leaf pair of three rhizomes per concentration and day were incubated at 0.25% aqueous Evans Blue staining (Malea et al., 2013a). The occurrence of dead cells (stained blue) was examined in the tip, middle part and basal part of leaves for each leaf cell type separately (tooth, margin and intercostal epidermal cells, and mid-rib, lateral intramarginal and cross vein cells; Malea, 1994) under a Zeiss AxioImager Z.2 light microscope equipped with a MRc5 Axiocam. All tooth and margin epidermal cells per leaf part were examined, while the observation for the intercostal epidermal and vein cells was conducted in three randomly selected areas per leaf part (about 700 cells per area). Measurements were expressed as percentages (%); emphasis was placed on the first occurrence of dead cell percentages higher than 5%. The dye of leaves stained with Evans Blue was also extracted, the optical density (OD) of extracts was measured in triplicate with a spectrophotometer (PharmaSpec UV-1700; Shimadzu, Tokyo, Japan), and the OD of TiO2 NP-treated leaves was expressed as a percent increase over control (Mylona et al., 2020a). 2.6. Leaf elongation Erect rhizomes were marked by tagging at the beginning of the experiment (nine rhizomes per concentration). The initial length of leaves of the first and second apical leaf pairs (one leaf per pair) and of the leaves of the fourth leaf pair was measured (including the petiole, in mm). Elongation (in mm) of these leaves was measured after 2, 4, 6 and 8 days, as the length of newly formed leaf segments from the beginning of the experiment. Toxicity index (TI, %), based on leaf elongation, was also calculated according to the following formula: TI = [(LEC − LET) / LEC] ∗ 100, where: LEC = leaf elongation in the control, LET = leaf elongation at NP treatment (Idrees et al., 2015, modified). 2.7. Data analysis Non-parametric tests were used, since preliminary analysis on raw and log-transformed data indicated unequal variances and severe violation of the normality assumption. Kruskal-Wallis Analysis of Variance and Mann-Whitney U test were applied to determine significant differences in response parameters among treatments, and between TiO2 NP treatments and the control, respectively. Spearman's rank correlation coefficient (ρ) was calculated to identify correlations between response parameters (AF disturbance, dead intercostal epidermal cell percentage, OD, TI based on leaf elongation). Statistical analysis was performed using the IBM SPSS®25. 2.8. Risk assessment The risk posed by TiO2 NPs was evaluated considering a direct release of sewage treatment plant (STP) effluents into a coastal area as a worst-case scenario. The risk quotient (RQ) was calculated according to the European approach (ECB, 2003), by dividing the predicted environmental concentration (PEC) by the predicted no effect concentration (PNEC). An initial dilution factor (DF:10) was also taken into account, since discharges to a coastal area are subject to a notable dilution, but

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initial dilution may only occur on calm days in low tidal range areas (ECB, 2003; Mylona et al., 2020a). Thereby, the RQ was calculated following the equation: RQ = (PEC / DF) / PNEC. PECs (mode, and 15% and 85% quantiles) provided by the advanced model of Sun et al. (2016) for TiO2 NPs in STP effluents in the EU in 2014 (13.7, 2.77 and 76.1 μg L−1, respectively) were used. The PNEC can be determined by using the no observed effect concentration (NOEC, Crane and Newman, 2000) divided by a suitable assessment factor based on the quantity and quality of data; in particular, the highest assessment factor (=1000) proposed for deriving PNEC for seawater for long-term studies was applied due to the scarcity of available data (PNEC = NOEC / 1000) (ECB, 2003; ECHA, 2008; Mylona et al., 2020a). An RQ value above 1 indicates that a risk cannot be excluded and thus, a refinement of the risk assessment is recommended (ECB, 2003).

3. Results 3.1. Nanoparticle characterization TEM analysis of TiO2 NPs in stock suspension showed that primary particles had both almost spherical and square shape, a mean (±standard deviation) size of 23.61 (±7.93) nm, which was smaller than the manufacturer's average particle size (31 nm), and a size range of 7.8–67.7 nm (n = 822) (Fig. 1A, B, Table 1). DLS measurements showed that hydrodynamic diameter, zeta potential and polydispersity index of TiO2 NPs in stock suspension had a value of 2385 nm, 2.17 ± 0.14 mV and 0.473, respectively; the corresponding values for TiO2 NP suspensions at concentrations 0.0015 and 1.5 mg L−1 in seawater were 722.1 nm, −3.72 ± 0.91 mV and 0.685, and 1356 nm, 2.37 ± 2.80 mV

Fig. 1. TEM micrograph (scale bar: 100 nm) (A) and size distribution (in nm) (B) of TiO2 NPs in stock suspension (150 mg L−1), X-ray diffraction (XRD) pattern of TiO2 NPs (C), and sedimentation profile of TiO2 NPs at 0.0015 and 1.5 mg L−1 concentrations in seawater (D).

Z. Mylona et al. / Science of the Total Environment 719 (2020) 137378

and 0.694, respectively (Table 1, Appendix A). XRD analysis of TiO2 NPs revealed an average size of about 20 nm, and also a crystalline phase of both anatase and rutile with a percentage of 85.7% and 14.3%, respectively (Table 1); the diffraction peaks are shown in Fig. 1C. Optical absorbance analysis at TiO2 NP suspensions at concentrations 0.0015 and 1.5 mg L−1 in seawater showed a reduction of absorbance during the first 6 h at both concentrations, followed by a steady state at 0.0015 mg L−1 and a slower reduction up to 24 h at 1.5 mg L−1; after 24 h, optical absorbance at 1.5 mg L−1 decreased more rapidly up to 30 h. After 24 h of absorbance analysis, about 70% of TiO2 NPs remained in suspension at both concentrations (Fig. 1D).

3.2. Effects on structural cell components In both interphase meristematic and differentiating cells of untreated young leaves, numerous AFs, uniformly oriented in each cell, were prevalent (Fig. 2A1, B1, B2). Under TiO2 NP exposure, the effects on AFs appeared in time- and concentration-dependent way (Table 2). In meristematic cells, the first response of AFs was detected at 0.15 mg L−1 on the 6th day, where signs of disorientation and initial bundling were observed (Fig. 2A2), while on the 8th day the bundling was extensive (Table 2). At 1.5 mg L−1, the effects on AFs were detected earlier (2nd and 4th day), including alterations similar to those observed at the above concentration (Fig. 2A3); on the 6th day, AFs were branched and further bundled, while on the 8th day an overall diminishing of AFs was observed (Fig. 2A4, Table 2). In differentiating cells, the first effect appeared at 0.0015 mg L−1 on the 8th day, where AFs appeared disoriented and branched (Fig. 2B3). At 0.015 mg L−1, AF effects initiated earlier (4th day), exhibiting signs of disorientation, diminishing and modest bundling (Fig. 2B4, Table 2). On the 6th day F-actin bundling and branching was observed (Fig. 2B5); on the 8th day AFs appeared extensively branched, while the finest filaments disappeared (Fig. 2B6). At the two highest concentrations applied, diminishing, orientation loss and initial bundling appeared even from the 2nd day (Fig. 2B7, B10, Table 2). Afterwards, F-actin bundling and branching were extensive until the 8th day, exhibiting a more or less chaotic pattern of AF bundles (Fig. 2B8, B9, B11, B12). Mean AF disturbance over the incubation period, expressed by the arbitrary scale of 1 to 3, significantly varied with increasing exposure concentration in both differentiating and meristematic cells of young leaves (Kruskal-Wallis Chi-squared; χ2: 13.04, n: 20, df: 4, pb0.05, and χ2: 13.66, n: 20, df: 4, pb0.01, respectively); in particular, a significant difference between the control and the 0.015, 0.15 and 1.5 mg L−1 treatments was found regarding differentiating cells (Mann-Whitney U test, pb0.05), and between the control and the 1.5 mg L−1 treatment regarding meristematic cells (pb0.05). Thereby, the LOEC and NOEC values based on AF disturbance were 0.015 and 0.0015 mg L−1, respectively for differentiating cells, and 1.5 and 0.15 mg L−1, respectively for meristematic cells.

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In untreated young leaves, ER appeared evenly distributed with discrete aggregation spots (Fig. 3A). After TiO2 NP treatment, ER aggregations appeared intensified and/or unevenly distributed, which was observed among adjacent cells or within each cell (Fig. 3B–F). This effect was firstly recorded on the 8th day at 0.015 mg L−1, while at 0.15 and 1.5 mg L−1, earlier. In both meristematic and differentiating young leaf cells, no effects were observed on cortical MTs at any of the concentrations applied; a representative image is given at 1.5 mg L−1 on 8th day, where MTs were abundant and dividing cells (preprophase bands) were present (Appendix B1). In treated older leaves, the cell wall, the chloroplasts and the mitochondria of the epidermal leaf cells were typically organized, even at 1.5 mg L−1 on the 8th day (Appendix B2). 3.3. Cell death The death of some leaf cells due to TiO2 NP treatment was established by Evans Blue staining, whereas no positive Evans Blue staining was noticed in the control. In leaves of the fourth leaf pair (older leaves), the percentage of dead tooth cells first exceeded 5% on the 2nd incubation day (5.6%) and was 13.2% on the 4th day at 0.15 mg L−1. The percentages of dead margin epidermal cells and dead intercostal epidermal cells first exceeded 5% on the 6th day at 0.15 mg L−1 (22.0 and 7.6%, respectively), while no dead vein cells were observed (Fig. 4A1–A4). Comparing the control with the 0.15 mg L−1 treatment as for the percentage of the alive tooth cells on the 4th incubation day, and the percentages of the alive margin and intercostal epidermal cells on the 6th day, a significant difference was found (Mann-Whitney U test, pb0.05). In leaves of the apical bundle (young leaves), a percentage of dead cells higher than 5% was only observed on the 8th incubation day at 1.5 mg L−1 in the intercostal epidermal cells (7.2%) (Fig. 4A5). Comparing the control with the 1.5 mg L−1 treatment as for the percentage of the alive intercostal epidermal cells on the 8th incubation day, a significant difference was found (Mann-Whitney U test, pb0.01). Thereby, the LOEC for epidermal leaf cell death was 0.15 mg L−1 regarding the older leaves and 1.5 mg L−1 regarding the young leaves; the respective NOECs were 0.015 and 0.15 mg L−1. Mean OD values of extracts from older leaves stained with Evans Blue over the incubation period increased with increasing concentration (over 6.4–19.6% to the control's OD). As for extracts from young leaves, mean OD over the incubation period was relatively low (over 3.4–5.4% to the control's OD) at the lower exposure concentrations and comparatively high (19.1%) at 1.5 mg L−1 (Fig. 4B). 3.4. Leaf elongation inhibition The initial length (0 day, mean ± standard error) of young and older leaves was 15.14 (±1.00) mm and 40.34 (± 0.94) mm, respectively.

Table 1 Characterization of TiO2 NPs in stock suspension, seawater suspensions (0.0015 and 1.5 mg L−1) or manufacturer's powder. TiO2NP characterization Primary particle size (nm) Polydispersity index (PDI)

Hydrodynamic diameter (nm)

Surface charge (mV)

Crystal structure (%) Average size (nm) Mean ± standard deviation of three replicates. a n = 822 particles.

Medium

Technique

Mean ± SD

Stock suspension Stock suspension 0.0015 mg L−1 1.5 mg L−1 Stock suspension 0.0015 mg L−1 1.5 mg L−1 Stock suspension 0.0015 mg L−1 1.5 mg L−1 powder powder

TEM DLS

23.61 ± 7.93a 0.473 0.685 0.694 2385 722.1 1356 2.170 ± 0.137 −3.723 ± 0.912 2.368 ± 2.789

DLS

Zeta potential

XRD XRD

Anatase/rutile structure

85.7/14.3 20

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Fig. 2. Cortical CLSM sections of TiO2 NP-treated Halophila stipulacea young leaf cells (1st apical leaf pair), compared to the control. In meristematic cells: A1. Control AF organization. A2. At 0.15 mg L−1, F-actin is disoriented and bundled on the 6th day. A3, A4. At 1.5 mg L−1, AFs exhibit disorientation and initial bundling on the 4th day (A3), while on the 8th day they appear very diminished (A4). In differentiating cells: B1, B2. Control AF organization. B3. At 0.0015 mg L−1, AF disorientation and branching is observed on the 8th day. B4–B6. At 0.015 mg L−1, AF disorientation and slight bundling occurred on the 4th day (B4), in addition to further bundling on the 6th day (B5), followed by extensive branching and fine filament disappearance on the 8th day (B6). B7–B12. At 0.15 and 1.5 mg L−1, AFs are disoriented and bundled on the 2nd day (B7, B10, respectively), demonstrating considerable bundling and branching from the 6th and 4th day, respectively (B8, B11), resulting in a chaotic pattern on the 8th day (B9, B12). Scale bar: 10 μm.

Leaves were elongated during the incubation period, both in the control and at TiO2 NP exposures (Fig. 5). Mean elongation of the older leaves over the incubation period significantly decreased with increasing concentration (Kruskal-Wallis Chi-squared; χ2: 19.10, n: 279, df: 4, pb0.01), whereas that of the young leaves did not significantly vary (χ2: 7.9, n: 294, df: 4, pN0.05) (Fig. 5A, B). Comparing the control and TiO2 NP treatments as for

mean elongation of the older leaves over the incubation period, a significant inhibition was detected at 0.015, 0.15 and 1.5 mg L−1 (MannWhitney U test, pb0.01 or pb0.05) (Fig. 5B). As for the young leaves, a significant difference in mean elongation over the incubation period between the highest exposure concentration and the control was observed (Mann-Whitney U test, pb0.01); in particular, a significant inhibition on the 2nd and 4th day was found (Mann-Whitney U test,

Z. Mylona et al. / Science of the Total Environment 719 (2020) 137378 Table 2 Time course of TiO2 NP effects on actin filaments (AFs) in interphase meristematic (m) and differentiating (d) cells of young apical leaves of Halophila stipulacea. TiO2 NP concentrations (mg L−1)

Control 0.0015 0.015 0.15 1.5

2 day

4 day

6 day

8 day

m

d

m

d

m

d

m

d

1 1 1 1 2ac

1 1 1 2ac 2ac

1 1 1 1 2ac

1 1 2ac 2bc 3

1 1 1 2ac 2bc

1 1 2bc 3 3

1 1 1 3 3

1 2bc 3 3 3

Effects on AFs are expressed by an arbitrary scale of 1 to 3; 1: unaffected AFs; 2a: AF diminishing/disorientation; 2b: AF branching; 2c: initial AF bundling; 3: extensive AF bundling/fine filament disappearance.

pb0.05 and pb0.01, respectively) (Fig. 5A). Thereby, the LOEC based on older leaf elongation was 0.015 mg L−1 and on young leaf elongation 1.5 mg L−1, while the respective NOECs were 0.0015 and 0.15 mg L−1. As for the older leaves, mean toxicity index (TI, %) over the incubation period significantly varied with exposure concentration (KruskalWallis, Chi-squared; χ2: 11.54, df: 3, n: 16, pb0.05), being the lowest (21.9%) at 0.0015 mg L−1 and the highest (55.5%) at 1.5 mg L−1 (Fig. 5C). As for the young leaves, mean TI values over the incubation period were lower, compared to those in the older leaves; these TI values (18.2–34.3%) showed a gradual, but not significant increase with increasing TiO2 NP concentration (χ2: 2.10, df: 3, n: 16, pN 0.05) (Fig. 5C). 3.5. Relationship between response parameters In young leaves, AF disruption in both differentiating and meristematic cells, expressed by the arbitrary scale of 1 to 3, showed a significant and positive correlation with the percentage of dead intercostal epidermal cells (ρ = 0.659 and 0.574, respectively; n: 20, pb 0.01). TI based on leaf elongation showed a significant (positive) correlation

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with OD values of extracts from leaves stained with Evans Blue (ρ = 0.644, pb 0.01, n = 20). With regard to older leaves, TI based on leaf elongation significantly and positively correlated with the percentage of dead intercostal epidermal cells and OD values (ρ = 0.537 and 0.555, respectively; n = 20, pb 0.05). 3.6. Risk assessment The NOEC for AF cytoskeleton organization in differentiating cells of young leaves, and for older leaf elongation was found to be 0.0015 mg L−1. This NOEC value was used to determine the PNEC (= 0.0015 mg L−1 / 1000 = 0.0015 μg L−1). Based on this PNEC and on PECs (mode, and 15% and 85% quantiles) for STP effluents (13.7, 2.77 and 76.1 μg L−1, respectively), a RQ of 913, 185 and 5073, respectively was calculated, indicating that a risk cannot be excluded. 4. Discussion Anatase TiO2 NPs were observed to exert toxic effects on H. stipulacea leaves at the cellular and physiological level. As TiO2 NP compounds are poorly soluble (e.g. Thwala et al., 2016), the observed toxic effects may have been mainly driven by nano forms. TiO2 NPs showed a high propensity for aggregation, as indicated by zeta potential and hydrodynamic diameter measurements. Nevertheless, a fraction of TiO2 NPs may have been adsorbed to the cell surface and internalized by cells; in particular, small nanoparticles and nanoparticle aggregates may have passed through cell wall pores, and larger ones through pores formed by nanoparticle-cell interactions (Navarro et al., 2008). The occurrence of both smaller and larger TiO2 NP aggregates in the culture medium is indicated by the high polydispersity index value. The above are corroborated by sedimentation analysis results, which generally indicated a slow sedimentation due to gravitation up to culture medium renewal. Notably, anatase TiO2 NPs have been reported to be

Fig. 3. CLSM sections at maximum intensity projections, depicting ER distribution in cells of untreated and TiO2 NP-treated Halophila stipulacea young leaves (1st apical leaf pair): A. Evenly scattered ER spots in untreated cells. B. Enlargement of ER aggregations after 0.015 mg L−1 TiO2 NPs on the 8th day. C, D. At 0.15 mg L−1, ER aggregations appear unevenly distributed on the 6th day (C), escalating on the 8th day (D). E, F. At 1.5 mg L−1, effects are apparently time-dependent, revealing more aggregations at the end (F) than at the beginning of the experiment (E). Scale bar: 10 μm.

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Fig. 4. A. Images of Halophila stipulacea leaf cells: A1. Typical control cells. A2. Dead tooth cells (blue stained cells) of older leaves (4th leaf pair, DOT) after exposure to 0.15 mg L−1 TiO2 NP concentration on the 4th day. A3. Dead margin epidermal (DOM) and dead tooth (DOT) cells of older leaves at 0.15 mg L−1 on the 6th day. A4. Dead intercostal epidermal cells of older leaves (DOI) at 0.15 mg L−1 on the 6th day. A5. Dead intercostal epidermal cells of young leaves (1st-2nd apical leaf pairs, DYI) at 1.5 mg L−1 on the 8th day. Scale bars: 50 μm. A2–A5 images regard the lowest TiO2 NP concentration and the shortest incubation time at which the percentage of dead cells per leaf cell type and leaf age exceeded 5%. B. Optical density (OD, percent increase over control) of extracts from TiO2 NP-treated H. stipulacea young and older leaves stained with Evans Blue; mean values over the incubation period. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

effectively taken up by plants (Movafeghi et al., 2018). H. stipulacea leaf cells may have directly interacted with suspended nanoparticles, since this seagrass leaves consist of only two cell layers and, thus, all leaf cells are in contact with seawater. TiO2 NP toxicity is mainly associated with ROS generation (Vale et al., 2016). Due to their photocatalytic activity, TiO2 NPs can generate radical species upon ultraviolet (UV) and / or natural light exposure; moreover, TiO2 NPs can produce ROS also in the dark (Fenoglio et al., 2009; Wang et al., 2011; Vale et al., 2016). Importantly, Xia et al. (2015), who examined TiO2 NP interaction with a marine microalga found that extracellular ROS generation induced by TiO2 NPs under visible light was negligible, and that TiO2 NP toxicity was related to elevated ROS levels caused by internalization of TiO2 NPs. ROS overproduction was also observed in leaves of the seagrass Cymodocea

nodosa exposed under experimental conditions similar to those of the present study to TiO2 NPs at concentrations of 0.0015 to 0.15 mg L−1; in particular, elevated levels of internal cell H2O2 were observed even at the beginning of the experiment at the lowest concentration tested, and this H2O2 overproduction was related to structural and physiological disturbances (Mylona et al., unpublished data). Accordingly, the observed AF disruption may be due to an overproduction of ROS. AFs have been reported to be very sensitive to ROS; in particular, oxidative stress can oxidatively modify actin and modulate the activity of actin-binding proteins (Schmidt et al., 2016). Considering that NPs can also exert toxic effects on plant cells by reacting with cell components (Navarro et al., 2008), a direct binding of TiO2 NPs to AFs may have also contributed to the observed AF damages. The above explanations are also in accordance to the findings of Wang et al. (2011),

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Fig. 5. Time course of elongation (in mm) of Halophila stipulacea young leaves (1st-2nd apical leaf pair) (A) and older leaves (4th leaf pair) (B) after exposure to various TiO2 NP concentrations and in the control; mean ± SE from about 15 leaves. C. Toxicity index (%) based on leaf elongation at the TiO2 NP treatments; mean ± SE over the incubation period.

who observed that anatase TiO2 NPs cause reorganization and elimination of MTs in Arabidopsis thaliana, and suggested that TiO2 NPs may induce MT damage indirectly by generating ROS or directly by interacting with tubulin. In addition, considering that actin-binding proteins are sensitive to cytosolic calcium imbalance (Wan and Zhang, 2012), the observed AF disruption may have been mediated, at least in part, by a mechanism involving calcium-NP interaction. Remarkably, a perturbation of calcium homeostasis has been considered as a potential mechanism, by which trace metal exposure leads to a disorganization of AF cytoskeleton (Wan and Zhang, 2012).

TiO2 NPs can induce leaf cell death in H. stipulacea, as indicated by our data. The observed occurrence of dead cells may be mainly related to ROS overproduction induced by exposure to increasing TiO2 NP concentrations; in particular, the potential production of excess amounts of ROS may have caused cytotoxicity and genotoxicity, resulting in leaf cell death (see also Yan and Chen, 2019). A high correlation between cell viability reduction and ROS overproduction under nanoparticle (Ag NP) stress has been previously reported for the freshwater plant Lemna gibba (Oukarroum et al., 2013). Considering that F-actin arrays participate in critical functions, such as cytoplasming streaming (Volkmann and Baluška, 1999), the

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observed AF disruption in young leaf cells may have impacted cellular functions and ultimately contributed to a loss of cell viability. This assumption is corroborated by the positive correlation found among AF disturbance and the occurrence of dead intercostal epidermal cells. On the other hand, to our knowledge, no data on cell death upon exposure to TiO2 NPs are available for higher freshwater plants (see also Thwala et al., 2016); in particular, no necrotic lesions in fronds of L. minor exposed to 0.0080 to 80 mg L−1 concentrations of TiO2 NPs (anatase, b25 nm) were observed (Dolenc Koce, 2017). Leaf cell death in H. stipulacea after exposure to the highest TiO2 NP concentrations applied appeared to be leaf age- and leaf cell typedependent, in accordance to previous findings concerning the interaction between H. stipulacea and Ag NPs (Mylona et al., 2020a). The metabolically more active young leaves of the apical shoot showed lower levels of cell death, compared to older leaves, as they grow protected by the next leaf pair and potentially display a higher detoxifying capacity (Richir et al., 2013). The observation that dead epidermal leaf cells were observed, whereas no dead vein cells occurred could be explained by the assumption that H. stipulacea epidermal leaf cells directly interact with suspended nanoparticles (see also Mylona et al., 2020a). Titanium dioxide NPs can inhibit the growth of H. stipulacea, as indicated by the observed dose- and leaf age-dependent impairment of seagrass leaf elongation. Inhibition in leaf elongation by increasing TiO2 NP concentration, along with the observed reduction in cell viability indicates an increasing deterioration of the global state of H. stipulacea with increasing nanoparticle concentration. Leaf elongation impairment could be due to direct or indirect damaging effects of nanoparticles on cell component organization and cell functions, resulting even in cell death (see also Yan and Chen, 2019). That a marked impairment of young leaf elongation was observed only at the highest exposure concentration applied is in accordance with cell death observations, both indicating that the young apical leaves of H. stipulacea are less susceptible to TiO2 NP toxicity than older leaves. In accordance to our findings, a reduction in growth parameters (relative frond number, frond average length and dry weight) of the freshwater plant Spirodela polyrhiza with increasing concentration (0.05 or 0.1 to 10 mg L−1) of TiO2 NPs (anatase, 8 nm) during 20 days of experimentation has been also reported (Movafeghi et al., 2018). On the other hand, the remaining few toxicological studies focused on TiO2 NPfreshwater plant interactions (e.g. Kim et al., 2011; Song et al., 2012; Li et al., 2013; Dolenc Koce, 2017) showed plant growth inhibition only at very high nanoparticle concentrations (N200–500 mg L−1). Overall, our results indicate that TiO2 NPs can induce adverse effects on H. stipulacea at environmentally relevant concentrations, thus substantially not supporting our initial hypothesis. The lowest effect concentrations observed are lower, compared to the lowest concentration at which toxic effects of TiO2 NPs on freshwater higher plants under laboratory conditions have been detected (0.05 mg L−1; Movafeghi et al., 2018), despite nanoparticles display a higher propensity for aggregation in seawater. In general, as mentioned earlier, in most toxicity tests for TiO2 NPs and freshwater plants, no adverse effects or adverse effects at high, not environmentally- realistic TiO2 NP concentrations were observed (≥10 mg L−1; see review in Thwala et al., 2016; Okupnik and Pflugmacher, 2016; Dolenc Koce, 2017; Spengler et al., 2017). This variation in effect levels may be due to differences in plant species morphology and physiology, response parameters sensitivity, nanoparticle physico-chemical properties, exposure concentrations, culture media characteristics and experimental techniques (see also Yan and Chen, 2019). In this context, it should be mentioned that the results of the present study, considering the experimental conditions and the fact that H. stipulacea is able to acclimate to a wide range of environmental conditions and to occur at the shallows even at b1 m, can be regarded as environmentally relevant. AF disruption mainly in differentiating cells of H. stipulacea young leaves, being observed even at the lowest tested concentration and at early time at the remaining concentrations applied, appears to be an

early warning indicator of risk posed by TiO2 NPs to this seagrass species. Inhibition of leaf elongation, particularly in older leaves and ER impairment in young leaf cells also appear to be reliable and susceptible indicators of TiO2 NP stress. These findings are generally consistent with those of recent studies assessing Ag NP toxic effects on several structural, biochemical and physiological traits of H. stipulacea and Cymodocea nodosa (Mylona et al., 2020a, 2020b); in particular, the present and previously reported data mainly highlight the relevance of AF disturbance in seagrass leaf cells as a biomarker of stress induced by nanoparticles. Considering a direct release of STP effluents into a coastal area as a worst-case scenario, our data indicated that TiO2 NPs may pose a significant potential risk to H. stipulacea and thus, to this seagrass meadows functioning. This finding is further supported by the fact that the lowest effect concentrations observed for the most sensitive response parameters are comparable with measured TiO2 NP concentrations in waste water treatment plant effluents, as well as that the modeled results used for risk assessment are in consensus with those of analytical studies (Gottschalk et al., 2013 and references cited therein; Sun et al., 2016). In this context, it should be also taken into account that the increasing use of TiO2 NPs will result in the release of larger nanoparticle amounts into the environment in the near future, thus augmenting the threat to coastal ecosystems. 5. Conclusions We present here the first attempt to assess the toxicity effect of TiO2 NPs on seagrasses. TiO2 NPs proved to be able to impair structural and physiological traits of H. stipulacea at environmentally relevant concentrations, and to pose a significant potential risk to the coastal environment; AF and ER impairment in leaf cells and leaf elongation inhibition appeared to be reliable and early markers of TiO2 NPinduced stress. The present study underlines the need for additional toxicity data for TiO2 NPs and seagrasses, and for a refinement of the environmental risk assessment of TiO2 NPs. Future research should also focus on the mechanisms underlying the toxicity of TiO2 NPs on seagrasses. Our findings contribute to a better understanding of TiO2 NP ecotoxicity and of anthropogenic chemicals impact on seagrass ecosystems, and could be utilized in biomonitoring programs for the protection of the coastal environment. Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2020.137378. CRediT authorship contribution statement Zoi Mylona: Methodology, Formal analysis, Investigation, Writing original draft, Visualization. Emmanuel Panteris: Conceptualization, Methodology, Investigation, Resources, Writing - review & editing. Theodoros Kevrekidis: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. Paraskevi Malea: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments Authors are grateful to Assoc. Prof. D. Fatouros (School of Pharmacy, AUTH, Greece), and Prof. T. Kechagias and Assoc. Prof. G. Vourlias (School of Physics, AUTH, Greece) for their support with TiO2 NP

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