In vivo toxicities of nine engineered nano metal oxides to the marine diatom Skeletonema costatum and rotifer Brachionus koreanus

Marine Pollution Bulletin 153 (2020) 110973

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In vivo toxicities of nine engineered nano metal oxides to the marine diatom Skeletonema costatum and rotifer Brachionus koreanus

T

Stella W.Y. Wonga, Guang-Jie Zhoua, , Kevin W.H. Kwoka,b, Aleksandra B. Djurišićc, ⁎⁎ Jeonghoon Hand, Jae-Seong Leed, Kenneth M.Y. Leunga,e, ⁎

a

The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Hong Kong, China c Department of Physics, The University of Hong Kong, Pokfulam, Hong Kong, China d Department of Biological Science, College of Science, Sungkyunkwan University, Suwon, South Korea e State Key Laboratory of Marine Pollution (City University of Hong Kong), Tat Chee Avenue, Kowloon, Hong Kong, China b

ARTICLE INFO

ABSTRACT

Keywords: Toxicity Nanomaterials Diatom Rotifer Biomarker

This study compared in vivo acute toxicities of nine engineered nano metal oxides to the marine diatom Skeletonema costatum and rotifer Brachionus koreanus. The sequence of their toxicities to S. costatum, based on growth inhibition, was: nano zinc oxide (nZnO) > nTiO2 (rutile) > nMgO > Annealed nMgO > nTiO2 (anatase) > γ-nAl2O3 > nIn2O3 > α-nAl2O3 > nSnO2. Similarly, nZnO was also the most toxic to B. koreanus, but the other nano metal oxides were non-lethal. nMgO and nZnO were confirmed to trigger reactive oxygen species (ROS) mediated toxicity to the two marine organisms, while nTiO2 (both anatase and rutile forms) likely induced oxidative stress as shown by their acellular ROS production. nZnO may also cause damage in the endocrine system of B. koreanus, as indicated by the increased transcription of retinoid X receptor. Annealed nMgO reduces its toxicity via removal of O2– and impurities from its surface.

1. Introduction Metal oxides are important in the development of modern technologies as they have great diversity in their physical and chemical properties. Various nano forms of metal oxides have been designed and produced for industrial and commercial uses over the past few years (e.g., Chavali and Nikolova, 2019; Gebre and Sendeku, 2019). Following use, it is possible that they may be released into natural aquatic environments with concomitant exposure to aquatic organisms (Lai et al., 2018). These nano metal oxides can then inhibit the growth and destroy the morphology and photosynthesis of aquatic plants like microalgae, as well as impede the behaviour and development of aquatic animals like crustaceans and fish (see the review Wong et al., 2013 and references cited therein). Although there are several toxicity datasets of nano zinc oxide (nZnO) for marine organisms (e.g. Wong et al., 2010; Wong and Leung, 2014; Yung et al., 2015), the ecotoxicities of other nano metal oxides to aquatic organisms, especially for saltwater species, are still largely unknown (Wong et al., 2013), precluding accurate ecological risk assessments of this class of chemical substances.

The marine diatom Skeletonema costatum is ubiquitously present in coastal waters worldwide, and it is regarded as ecologically important component of marine food chains. Due to its particular vulnerability to exposure to contaminants, S. costatum has been widely used in toxicity tests (e.g., Zhang et al., 2019a, 2019b). As algae constitute the largest and most widely distributed group of photosynthetic organisms in aquatic ecosystems, the toxicity tests on S. costatum can help better understand the impacts of nano metal oxides to ecosystem health through algal growth inhibition testing. The monogonont rotifer Brachionus koreanus is advocated as another ecotoxicological model organism due to its short life-cycle, easy maintenance in the laboratory (i.e., small size, hardiness and dormant resting eggs) and genetic homogeneity (Dahms et al., 2011). For example, a recent study indicated that B. koreanus is the most sensitive species among ten test organisms to the exposure of accidental spill of palm stearin (Zhou et al., 2019). B. koreanus also has the added advantage of being used in revealing molecular mechanisms as its genome has been sequenced (http://rotifer.skku.edu:8080/Bk). Based on this genome database, several biomarker genes were identified and the transcriptional regulation of B. koreanus in response

Corresponding author. Correspondence to: Kenneth M.Y. Leung, State Key Laboratory of Marine Pollution (City University of Hong Kong), Tat Chee Avenue, Kowloon, Hong Kong, China. E-mail addresses: [email protected] (G.-J. Zhou), [email protected] (K.M.Y. Leung). ⁎

⁎⁎

https://doi.org/10.1016/j.marpolbul.2020.110973 Received 16 December 2019; Received in revised form 5 February 2020; Accepted 10 February 2020 Available online 20 February 2020 0025-326X/ © 2020 Elsevier Ltd. All rights reserved.

Marine Pollution Bulletin 153 (2020) 110973

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to various environmental stressors had been examined (e.g., Kim et al., 2011; Lee et al., 2019). These biomarker genes include heat shock proteins (HSPs) and antioxidant enzymes, and DNA repair processes (Kim et al., 2011). In this study, HSPs (HSP70, HSP90α1, HSP90α2 and HSP90β), Glutathione S-transferases (GST-A, -O, -S, -Z and -Z2), DNAdependent protein kinase (DNA-PK), and retinoid X receptor (RXR) were selected as the candidate genes for the investigation of gene expression and genotoxicity in the rotifer upon exposure to nine different nano metal oxides. HSPs are molecular chaperones which can assist in folding of denatured or incomplete proteins back to their native forms by intervening in their inappropriate interactions with other proteins (Feder and Hofmann, 1999). GSTs are multifunctional antioxidant enzymes with diverse functions, basically divided into four types: (1) phase II metabolism of xenobiotics via catalysing glutathione (GSH) conjugation; (2) facilitating transport of endogenous substrates across membranes; (3) protection against oxidative stress; and (4) assisting the intracellular translocation of molecules as nonenzymatic carrier proteins (or ligandins) such that they do not accumulate excessively at a localized region (Marrs, 1996). DNA-PK plays a key role in the DNA double-strand breaks repair pathway (Woo et al., 1998) and RXR may be a receptor related to lipid homeostasis in B. koreanus (Lee et al., 2019). There were three main objectives in this study. Firstly, this study compared in vivo acute toxicities of nine engineered nano metal oxides including nano α-alumina (α-nAl2O3), nano γ-nAl2O3 (γ-nAl2O3), nano indium oxide (nIn2O3), nano magnesia (nMgO), annealed nMgO, nano tin oxide (nSnO2), nano titanium dioxide (nTiO2 in anatase and rutile forms) and nZnO towards S. costatum and B. koreanus. Secondly, an attempt was made to identify genetic biomarkers that may be utilized for assessing sub-lethal toxic effects of these nano metal oxides to the rotifer, and providing insights into their toxic mechanisms. Lastly, based on the results of the gene expression experiment, the levels of in vivo reactive oxygen species (ROS) generated by three selected nano metal oxides (i.e., nMgO, nSnO2 and nZnO) in the rotifer were further evaluated.

The solution was then transferred to an EPR tube and the tube was illuminated with 365 nm UV for 2 min. The measurement was initiated after the ambient light or UV light illumination. Typical instrumental settings were as follow: microwave power 20 mW, microwave frequency 9.74 GHz, modulation frequency 100 kHz, amplitude 2G, timeconstant 655 ms, and sweep time 84 s.

2. Materials and methods

2.3. Acute toxicity tests on the rotifer

2.1. Chemical characterizations

The rotifer B. koreanus used in this study were maintained at the Department of Molecular and Environmental Bioscience, Hanyang University, Korea. They were cultured in UV light-disinfected, 1-μm FASW (15‰, 25 ± 1 °C, 12 h light:12 h dark), and maintained by feeding with the marine microalga Tetraselmis suecica. However, the rotifers were unfed during the chemical exposure periods. In total, nine sets of standardized acute toxicity tests were conducted for the nine nano metal oxides. For each test, at least six concentration treatments (including a seawater control; Table S1) were applied and the concentration range was selected based on a range finding test. Each of the concentration treatments consisted of three replicates, each being a culture plate well containing 10 Brachionus neonates (< 12 h old) in 1 mL of the test solution. The exposure was static and lasted 24 h, at the end of which any dead rotifers were recorded under a stereomicroscope.

2.2. Acute toxicity tests on the diatom 72-h growth inhibition tests were conducted on S. costatum (strain code: CCMP 1335; CCMP, USA) following the OECD Guidelines for the Testing of Chemicals–Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Test Guideline OECD-201 (2011) with some minor modifications. Algal cells with initial density of 105 cells mL−1 were exposed to different concentrations (Table S1) of the nine test chemicals dispersed in 2 mL of f/2-Si medium in 24-well multidishes (Nunc; Naterville, USA; n = 4 for each concentration treatment; salinity, 30 ± 0.5‰; pH, 8.0 ± 0.1). In order to assess possible shading effects imposed by the insoluble solids of the metal oxides, an experimental design adapted from Hund-Rinke and Simon (2006) was utilized. A transparent multidish was stacked on top of another multidish, and the algae were only incubated in the lower chamber. To test the toxicity effects of chemicals, the test compounds and algae were mixed together in the lower plate wells with only f/2-Si medium in the upper wells, while to assess the shading effects, the test materials and algae were separated in the upper and lower wells, respectively. The walls and undersides of the multidishes were then covered with black paper such that light could only pass through the upper wells to reach the lower dish (Fig. S1). To ensure more uniform dispersal of the metal oxides, which tend to aggregate and settle down over time, the dishes were shaken atop a titer plate shaker for 15 min every 2 h at a mediocre speed of ~100 rpm. The whole set-up was incubated in an environmental chamber with 16 h light: 8 h dark cycle and 25 ± 2 °C for 72 h. Initial and final cell counts were determined using a haemocytometer to calculate the average growth rate during the test period.

All nano metal oxide powders were purchased from Nanostructured & Amorphous Materials Inc. (Los Alamos, NM, USA). nMgO was further annealed in a tube furnace in the presence of oxygen (700 °C, 12 Torr) for 1 h. All glasswares used in the study were acid-washed in 10% HNO3, rinsed with Milli-Q water and autoclaved before use. For the average primary particle size, the nano metal oxide powders were examined under transmission electron microscope (TEM; Tecnai G2 20 S-TWIN at 200 kV, Philips, Eindhoven, The Netherlands) equipped with selected area electron diffraction (SAED). The samples were divided into 3 replicates, with random selection of 20 nanoparticles for measurement within each replicate. The measurements were made on the photomicrographs by the software Image J version 1.44 (NIH, Washington). For aggregate size distribution, the nano metal oxide powders (15 mg each) were dispersed in 150 mL of filtered artificial seawaters (FASW; salinity, 30 ± 0.5‰; pH, 8.0 ± 0.1; sea salt: Tropic Marine, Germany; filtered through 0.45 μm membrane filter), and stirred continuously with a magnetic stirrer (~200 rpm) at room temperature (25 ± 2 °C) for at least 4 d before inspection by laser diffractometry (LD; LS 13 320 Series, Beckman Coulter Inc., Fullerton, USA). Measurement time was 60 s, and each sample was analyzed twice on the LD. Electron-Paramagnetic Resonance (EPR) was employed to detect the acellular reactive oxygen species generation by nanomaterials. EPR spectra were recorded using a Bruker EMX EPR spectrometer at room temperature. The solution for the measurement was prepared by adding 0.02 M 5,5-dimethyl-l-pyrroline-N-oxide (DMPO; free radical spin trap) to 1 mg mL−1 nanoparticles' solutions (30‰ FASW or f/2-Si medium).

2.4. Gene expressions of rotifer exposed to the nine nano metal oxides Transcriptional expressions of HSP70, HSP90α1, HSP90α2, HSP90β, GST-A, GST-O, GST-S, GST-Z, GST-Z2, DNA-PK, and RXR genes were investigated in this study to assess the cellular and DNA damage and repair in the rotifers in response to exposure of each of the nine nano metal oxides. For each treatment, approximately 80,000–100,000 Brachionus neonates (< 12 h old) were exposed to 50 mL of each of the chemicals dispersed in FASW (15‰, 25 ± 1 °C, 12 h light:12 h dark). The exposure was divided into two parts. First, apart from nZnO, 3 concentrations (1, 5, 10 mg L−1) were selected arbitrarily for eight of the NMs (α-nAl2O3, γ-nAl2O3, nIn2O3, nMgO, annealed nMgO, nSnO2, nTiO2 (anatase), nTiO2 (rutile)) due to limited knowledge on their no 2

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observed effect concentrations (NOECs) or solubility, and samples were harvested at 24 h after the exposure. HSP70, HSP90α1, GST-A, GST-O, GST-S and GST-Z were investigated using real-time qRT-PCR subsequently. Secondly, only one concentration (i.e., NOEC: 10 mg L−1) was chosen for nZnO, and sampling took place at 1, 3, 6, 12, 18 and 24 h to investigate the time-dependent expressions of the genes. HSP70, HSP90α1, HSP90α2, HSP90β, GST-A, GST-O, GST-S, GST-Z, GST-Z2, DNA-PK, and RXR expressions were then studied. For each treatment, the rotifers were ground with mortar and pestle, and then homogenized in 3 volumes of TRI reagent (Molecular Research Center, Inc., Cincinnati, OH) with the aid of a Teflon homogenizer to extract total RNA, which was stored at −80 °C until analysis. Total RNA (2 μg) from each sample was reverse transcribed to single-strand cDNA in 20 μL reaction mixture (comprising 1 μL each of SuperScript™ III reverse transcriptase (Invitrogen, Carlsbad, CA), 10 mM dNTPs, 0.1 M diothiothreitol (DTT), RNase inhibitor (RNase OUT, Invitrogen)), 4 μL of 5× PCR reaction buffer and 10 pM of oligo d(T) anchor primer). Polymerase chain reaction (PCR) was then conducted in the following order: 65 °C/5 min, 50 °C/60 min, 70 °C/15 min and 37 °C/20 min. For real-time quantitative PCR (qRT-PCR), 1 μL of cDNA was utilized along with 0.2 μM primer set of each investigated gene and Brachionus 18S rRNA gene (Table S2), and the amplification conditions were: 94 °C/ 4 min, 35 cycles of 94 °C/30 s, 55 °C/30 s, 72 °C/30 s; and 72 °C/ 10 min. Melting curve analysis was performed consecutively at 95 °C/ 1 min, 55 °C/1 min, 80 cycles of 55 °C/10 s (+0.5 °C/cycle) to validate amplification specificity. The specific amplified products were probed for using the DNA-binding dye SYBR® Green (Molecular Probes, Eugene, OR), and fluorescence detection was carried out with CFX96TM Real-Time Detection System (Biorad, Hercules, CA). PCRs were performed in triplicate, and Brachionus 18S rRNA gene was used to normalize the expression levels between samples and to compensate for any difference in reverse transcriptase efficiency. The threshold cycle Ct, which refers to the PCR cycle number where fluorescence is detected above an arbitrary threshold and decreases linearly with increasing input target quality, was tabulated by the CFX Manager Software (Biorad). The fold change of mRNA expression of a gene, expressed relative to the control, was determined by the equation 2−ΔΔCt (Livak and Schmittgen, 2001), where ΔΔCt = (Ct target genesample − Ct 18S rRNAsample) − (Ct target genecontrol − Ct 18S rRNAcontrol).

proteins present in the sample, and presented as a percentage of DCF fluorescence. The amount of total proteins in each sample was determined using the Bradford method (Bradford, 1976). 2.6. Statistical analysis Data analysis was performed with Graphpad Prism version 5.00 (Graphpad Software, San Diego), and SPSS version 17 (SPSS Inc., Chicago). To determine the EC50 (median effective concentration) and LC50 (concentration causing lethality to 50% of rotifers), data were fitted to a sigmoidal log(dose)-response curve (variable slope) with a four-parameter logistic equation. Statistical difference was implied if the 95% confidence intervals (C.I.) of the EC50 or LC50 values did not overlap. The mean expression levels of each of the studied genes (i.e. HSP70, HSP90α1, HSP90α2, HSP90β, GST-A, GST-O, GST-S, GST-Z, GST-Z2, DNA-PK, and RXR) were compared between the treatments (concentration or time) using one-way Analysis of Variance (ANOVA) with Tukey's post-hoc comparisons. Spearman rank correlation analysis was used to study relationships between transcriptional levels of different genes. Two-way ANOVA using time and exposure concentration of nano metal oxides as fixed factors was used to test for equality of the means of in vivo ROS levels. 3. Results and discussion 3.1. Crystalline shapes, average primary particle and aggregate sizes TEM photos and SAED patterns were used to affirm the crystalline structures of the nanoparticles (Fig. S2). Annealing of nMgO had the effect of homogenizing the particle shapes (Fig. S2 d–e), as the crystalline surface became refined. Furthermore, impurities might become segregated from the particles during the process of annealing (Robach et al., 1998), although this phenomenon was not tested in this study. According to measurements taken with the TEM images, nTiO2 (anatase) had the smallest particle size while α-nAl2O3 had the largest particle size (Table 1). However, the values mostly differed from manufacturer's information except for nIn2O3, nSnO2, nTiO2 and nZnO, thus emphasizing the importance of conducting chemical characterizations in parallel with toxicity tests. Providing accurate information about the physicochemical properties of NMs is the basis for identifying their key characteristics which may influence their biological consequences (Jeevanandam et al., 2018). Annealed nMgO formed the smallest aggregate in seawater while nSnO2 and nZnO agglomerated to larger sizes (Table 1).

2.5. Measurement of in vivo ROS level The method for quantifying ROS level described in Kim et al. (2011) was employed. Approximately 80,000–100,000 Brachionus neonates (< 12 h old) were exposed to each treatment (n = 3) of nMgO (5, 10, 20 mg L−1), nSnO2 (5, 10, 20 mg L−1) and nZnO (1, 5, 10 mg L−1) for 3 and 24 h, separately from those used for mortality or real-time qRT-PCR analysis. After exposure they were homogenized in a buffer (0.32 mM sucrose, 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 1 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride PMSF (pH 7.4)). The homogenate was centrifuged at 10,000 ×g for 20 min at 4 °C, and the supernatant was collected for measurement. This step also helped to settle down any residual nanoparticles which might interfere with the absorbance measurements. 10 μL of the supernatant was then transferred to a well of 96-well black microplates. 170 μL of phosphate-buffered saline (PBS; containing 0.4 M Na2HPO4, 0.1 M NaH2PO4, 1.0 M NaCl, pH 7.4) buffer and 20 μL of 2′7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes) were added to the supernatant for reaction of 40 min at 37 °C. The fluorescence of dichlorofluorescein (DCF), which is an oxidation production of H2DCFDA, was assessed with Thermo™ Varioskan Flash reader (Thermo Fisher Scientific, Inc., Waltham, MA) at an excitation wavelength of 485 nm and emission wavelength of 520 nm. Solutions containing nanomaterials were found not to have interference on the fluorescence measurements. The data were normalized by the amount of total

3.2. ROS formation in seawater with or without UV radiation DMPO is a spin probe which traps the hydroxyl (%OH) and superoxide (O2−) radicals to form DMPO-OH and DMPO-OOH spin adducts respectively (Finkelstein et al., 1980). However, the latter is unstable in aqueous solution, generally decomposing to give DMPO-OH, which forms a quartet 1: 2: 2: 1 EPR signal (Finkelstein et al., 1980). None of the test nano metal oxides generated %OH or O2– in seawater under ambient light (data not shown), but after UV radiation, EPR signals were produced in the presence of nMgO, nTiO2 (anatase), nTiO2 (rutile) and nZnO (Fig. S3a). The 1:2:2:1 peaks can be differentiated clearly from the background noise generated from blank reference (seawater). nZnO gave the highest intensity signal, while nMgO gave the smallest signal. MgO, TiO2 and ZnO are known to produce O2−, %OH and H2O2 (a prerequisite for %OH formation) correspondingly (Sawai et al., 1996; Horikoshi et al., 2003). Thermal annealing has the effect of evacuating oxygen groups (O2– in this case) and impurities that may generate ROS from lattices (Tan et al., 2007), and therefore no EPR signal was detected for annealed nMgO under all conditions. In f/2-Si medium, nTiO2 (anatase) and nZnO were able to generate the radicals in both presence and absence of UV light (Fig. S3b & c), while nTiO2 (rutile) was only 3

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Table 1 General characteristics of the nine nano metal oxides. Chemical

Average primary particle size (nm)a

Average primary particle size (95% C.I.; nm)b

Morphology

Average aggregate size (95% C.I.; μm)c

Specific surface area (m2 g−1)a

True density (g cm−3)a

α-nAl2O3

80

172.0 (112.9–231.2)

1.43 (1.33–1.52)

74

N.A.

γ-nAl2O3 nIn2O3 nMgO Annealed nMgO nSnO2 nTiO2 (anatase) nTiO2 (rutile) nZnO

40–80 30–50 20 N.A. 55 15 40 20

N.A. 24.9 (13.9–35.9) 37.9 (23.5–52.4) 36.0 (21.5–50.5) 71.7 (42.5–100.9) 5.3 (3.0–7.6) 59.7 (27.2–92.2) 20.6 (13.8–27.3)

Sphericala; Pillshapedb Sphericala Faceted/roda,b Polyhedrala,b Facetedb Faceteda,b Sphericala,b Needle-likea,b Nearly Sphericala,b

1.90 2.13 1.93 0.74 2.45 1.14 1.55 2.22

100–200 15 50 N.A. 30 240 160 50

3.700 7.180 3.580 N.A. 6.950 3.900 4.230 5.606

(1.82–1.98) (1.83–2.43) (1.30–2.56) (0.72–0.76) (2.13–2.76) (0.92–1.36) (0.99–2.11) (2.11–2.32)

N.A. - Data not available. a Data provided by company. b Data supported by TEM image. c Data supported by LD.

stimulated after being irradiated by UV light (Fig. S3c). The results suggested that the medium in which NMs are dispersed in, as well as the environmental conditions they are exposed to (i.e., with or without UV radiation), have impacts on their ROS induction.

microalgae. For example, the 72-h EC50 values for the freshwater microalgae Scenedesmus sp. and Chlorella sp. exposed to γ-nAl2O3 were 39.5 and 45.4 mg L−1 respectively (Sadiq et al., 2011), while in the present experiment the toxicity on S. costatum was much lower (72-h EC50 = 577 mg L−1). A recent study also reported that the 72-h EC50 values of nAl2O3, nIn2O3 and nSnO2 on the green microalga Pseudokirchneriella subcapitata were 9.4, 110 and 2.1 mg L−1 respectively (Sousa et al., 2019), and these values are once again much lower than those obtained for S. costatum in this study (577.0–2101.0, 739.8 and 5200.0 mg L−1 respectively). A possible reason could be due to the difference of methods used to disperse the chemicals before the toxicity tests. For the two freshwater studies (Sadiq et al., 2011; Sousa et al., 2019), the NMs were dispersed in culture mediums after sonication of the stocks for 15–30 min, while in this study the NMs were only stirred for an extended period of time before dosing, hence time and method of dispersion possibly accounted for the increased aggregate size besides the ionic strength. Comparison between Aruoja et al. (2009) and Hartmann et al. (2010), both of which exposed P. subcapitata to nTiO2 and obtained different 72-h EC50 values of 9.72 mg L−1 (for the former) and 71.1 mg L−1 (for the later), illustrates once again the importance of chemical preparation and storage. Aruoja et al. (2009) prepared the stock solution in algal medium immediately before experiment and then ultrasonicated for 30 min before use, while Hartmann et al. (2010) stored the stock solution in dark after preparation and performed sonication for 10 min before exposure. For the rotifer B. koreanus, no 24-h NOEC or LC50 values could be obtained for eight of the NMs (α-nAl2O3, γ-nAl2O3, nIn2O3, nMgO, annealed nMgO, nSnO2, nTiO2 (anatase), nTiO2 (rutile)) as they could survive well even at very high concentrations (500–5000 mg L−1). The 24-h NOEC of nZnO was estimated as 10 mg L−1 while its 24-h LC50 value was determined as 18.66 mg L−1 (95% C.I.: 16.89–20.62 mg L−1). Clearly, nZnO was thus the most toxic nano metal oxide in this study towards both S. costatum and B. koreanus. It was also reported that the bacterium, alga and protozoan are usually more sensitive to the exposure of nZnO than other nanoparticles (Aruoja et al., 2015). The high potency of nZnO may be due to its release of bioavailable Zn2+ ions, which is shown by other studies (Aruoja et al., 2009; Wong et al., 2010). There have been reports of Al2+ and Sn2+ being more toxic than Zn2+ towards algae (Magdaleno et al., 1997) and crustaceans (Martin and Holdich, 1986). But when aluminum and tin occur as oxides, they became less toxic than ZnO; it is because ion dissolution from aluminum and tin oxides is very limited and hence their toxicities are significantly reduced (Yamamoto et al., 2004). Nonetheless, it is believed that not all toxicity effects of nano metal oxides can be solely explained by their metal ions, and nanoparticulate-specific effects such as cellular abrasion should not be ruled out (Wang et al., 2009; Wong et al., 2010).

3.3. Acute toxicity tests on the diatom and rotifer Concentration-response relationships for the diatoms exposure to the nine different nano metal oxides are shown in Fig. 1. Their toxicity towards S. costatum was ranked in the following order (Table 2): nZnO > nTiO2 (rutile) > nMgO > Annealed nMgO > nTiO2 (anatase) > γnAl2O3 > nIn2O3 > α-nAl2O3 > nSnO2. Similar results were obtained by Wang et al. (2009), which reported the 24 h toxicity towards the nematode Caenorhabditis elegans as nZnO (LC50: 2.3 mg L−1) > nTiO2 (anatase; LC50: 79.9 mg L−1) > nAl2O3 (unreported phase; LC50: 81.6 mg L−1). However, some of the current results were also somewhat contradicting to theoretical expectations. For instance, nTiO2 (anatase) is predicted to be more toxic than its rutile form, because the former is intrinsically more photoreactive than the later in f/2-Si medium (Fig. S3 b–c). nTiO2 (anatase) also has smaller average primary particle and agglomerate sizes, which together with a greater specific area (Table 1) should facilitate easier entry into the cells and promote reactivity. However, since nZnO agglomerated to greater extent than most of the other nano metal oxides and it was found to be the most toxic NMs in this study, it is necessary to find other explanations. The results presented here, though intriguing, should be supported by chemical uptake and in vivo ROS measurements in S. costatum, which were unfortunately unavailable in this study. Extracellular ROS detection by DMPO does not appear to be a good indicator of toxicity in this case as nMgO turned out to be one of the most toxic chemicals towards S. costatum, even though neither %OH nor O2– were induced in f/2-Si medium with or without UV light. Aside from inducing oxidative stress, nMgO has been demonstrated to desiccate cells and caused abrasive mechanical damages to cell membranes (Stoimenov et al., 2002). nAl2O3 (no phase given) also disrupts cell membrane through depolarization (Lin et al., 2008), although its toxic effect on S. costatum is not as great as nMgO. γ-nAl2O3 has been implicated in oxidative stress through increasing manganese superoxide dismutase and in vivo ROS level in mouse skin epithelial cells (Dey et al., 2008), and hence it is possible that it may generate other types of ROS (other than %OH and O2−) that cannot be captured by DMPO. In2O3 and SnO2 individually are usually harmless, though sintering them together can create a new article (indium-tin-oxide) that is far more potent and can generate acellular ROS (Lison et al., 2009). The results obtained from this study also differed from those obtained for other studies on the toxicities of nano metal oxides on 4

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Fig. 1. Concentration-response relationships for Skeletonema costatum after exposure to each of the nine nano metal oxides for 72 h (n = 4; mean ± 2 s.d.).

fold difference from the control (Cappellen et al., 2002), as they may serve as possible biomarkers for detecting sub-lethal effects and/or exposures of these NMs in the rotifer. All the test genes (i.e., HSP70, HSP90α1, GST-O, GST-S and GST-Z) except for GST-A (which did not meet the threshold criteria) were consistently down-regulated by nTiO2 (anatase) in the rotifer B. koreanus at 24 h (Fig. 2a–f). nMgO also had similar effects on these genes, although significant changes were not always observed at the lower concentrations of 1 and 5 mg L−1 (Fig. 2a, c–d). In contrast, only nSnO2 caused significant up-regulation of HSP70, HSP90α1, GST-S and GST-Z (Fig. 2a–b, e–f), and GST-A was only down-regulated by annealed nMgO and nTiO2 (rutile) (Fig. 2c). Up-regulation of GST-A and GST-O was observed only for α-nAl2O3 (Fig. 2c-d), while its transitional form γ-nAl2O3 only down-regulated HSP90α1 (Fig. 2b). nIn2O3 did not uniformly up-regulate or down-regulate any of the genes, but it had the distinctiveness of increasing gene expressions at the lower concentrations followed by a return to basal levels or a down-regulation at the highest test concentration (Fig. 2a–f). Therefore, suitable reference biomarker genes for the future studies of six of these nano metal oxides are: HSP70, HSP90α1, GST-O, GST-S and GST-Z for nTiO2 (anatase); GST-A for nTiO2 (rutile); HSP70, HSP90α1, GST-S and GST-Z for nMgO; GST-A for annealed nMgO; GST-A and GST-O for α-nAl2O3; and HSP90α1 for γ-nAl2O3. No suitable biomarker was identified for nIn2O3 in this study. The low concentrations of chemicals employed in this study might have prevented development of clear dose-dependent responses.

Table 2 72-h EC50 values (95% confidence interval) of various nano metal oxides to Skeletonema costatum. Values with same letter denote overlapping of 95% C.I. Chemical

72-h EC50 (mg L−1)

α-nAl2O3 γ-nAl2O3 nIn2O3 nMgO annealed nMgO nSnO2 nTiO2 (anatase) nTiO2 (rutile) nZnO

2101.0 (1671.0–2642.0)f 577.0 (494.0–674.0)e 739.8 (580.0–943.7)e 140.4 (132.5–148.8)b 184.0 (156.2–216.7)c 5200.0 (1389.0–19,472.0)f 353.3 (322.8–386.8)d 117.0 (99.6–137.4)b 1.7 (1.6–1.8)a

3.4. Gene expressions of rotifer exposed to nine nano metal oxides Since no NOEC value and meagre solubility data could be obtained for eight of the nano metal oxides (i.e., α-nAl2O3, γ-nAl2O3, nIn2O3, nMgO, annealed nMgO, nSnO2, nTiO2 (anatase), nTiO2 (rutile)), arbitrary concentrations of 1, 5 and 10 mg L−1 were selected to compare their toxicities towards the rotifer. Irregular gene expression patterns were observed for most of the test chemicals (Fig. 2), probably due to the low concentrations employed. The discussion hereafter mostly concentrates on the genes which were consistently changed in one direction across all deployed concentrations and expressed at least two5

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Fig. 2. The mean relative expression levels of HSP70 (a), HSP90α1 (b), GST-A (c), GST-O (d), GST-S (e), GST-Z (f), in the rotifer Brachionus koreanus after exposure to eight nano metal oxides (i.e. α-nAl2O3, γ-nAl2O3, nIn2O3, nMgO, annealed nMgO, nSnO2, nTiO2 (anatase), nTiO2 (rutile)) for 24 h (n = 3, ± 2 s.d.). Dashed line indicates the control level. Error bars with the same letter denote statistical indifference between concentrations (Tukey's post-hoc test, p > 0.05).

The higher number of GST genes modulated by nTiO2 (anatase) is within expectation, as this chemical has been shown to induce oxidative stress in a number of organisms such as Alexandrium tamarense (Li et al., 2019) and Escherichia coli (Kumar et al., 2011). Although nTiO2 (rutile) generally did not modulate as many genes as its anatase counterpart, another study using a higher concentration (e.g. 100 mg L−1) demonstrated that nTiO2 (rutile) can actually upregulate oxidative stress-related genes in the human hepatoma cell (Petković et al., 2011). Indeed at 10 mg L−1, transcription levels for many of the genes in the rotifer were similar between the two forms as shown in the present study (Fig. 2a–f), and thus rutile might overshadow anatase in terms of the impacts at higher concentrations. Ekstrand-Hammarström et al. (2012) has also shown that rutile polymorphs of TiO2 can have equivalent or even higher ROS induction than its anatase counterparts within normal human bronchial epithelial cells and alveolar cells after 2 h of in vitro exposure. The genotoxic potentials of anatase and rutile forms of TiO2 on bottlenose dolphin leukocytes were also similar (Bernardeschi et al., 2010), which was speculatively attributed to their similar aggregate sizes in solution, and as their uptake and bioavailability became limited, so did their ROS induction capacities. Although γ-nAl2O3 did not regulate any of the GST genes, it might still serve as a potential source of oxidative stress; it is because it can elevate the expression level of manganese superoxide dismutase (MnSOD) in mouse epithelial cells (Dey et al., 2008). Besides, structural changes were found in tissues of the Tilapia Oreochromis mossambicus due to stress developed after exposure to nAl2O3 (Murali et al., 2018). Conversely, alterations of GST genes by nMgO may not always lead to adverse impacts; e.g. Gerloff et al. (2009), exposing human intestinal cells (Caco-2) to various metal oxides, found that nMgO did not cause cytotoxicity even though it decreased the total glutathione content. Future studies require investigation of time-dependent expression patterns of the genes exposed to these NMs, as many genes are often found to be initially upregulated by the chemicals, followed by return to control level and further suppression (Petković et al., 2011). nZnO at the concentration of 10 mg L−1 induced highest HSPs and RXR gene expressions at 3 h, followed by down-regulation at 24 h (Fig. 3a–d, k). HSP90α2 had the maximum gene expression among all test genes, increasing up to 70–80 folds (Fig. 3c). This seems to contradict the claim made by Picard (2002), stating that as HSP90 protein is found plentifully in organisms under normal conditions, its induction is usually not great in face of stress. Besides, it was reported that HSP 70 in the fish Pangasius hypophthalmus was reduced after exposure to nZnO (Kumar et al., 2018), while the HSP gene in THP-1 human monocytic cells was strongly upregulated by nZnO (Safar et al., 2019). Therefore, there should be perhaps a re-evaluation of the status of HSP as a sensitive biomarker. Although the centrifuge steps in our methods are assumed to have removed most of the NMs from the supernatants, this study did not check for the interference of NMs in the results of measurement, and artefacts might have arisen; nonetheless the time-dependent patterns displayed by all studied genes showed that NMs definitely had an effect on the rotifer. The up-regulation of RXR by nZnO possibly indicated the onset of endocrine disruption, as it has been proven that increased RXR mRNA level occurred spontaneously with growth of reproductive organs in both sexes of mud snail (Sternberg et al., 2008). Improper timing of retinoid signalling induced by organotins (which are high-affinity ligands for RXR) may induce imposex in female neogastropods (Nishikawa et al., 2004). It is, therefore, likely that nZnO may act through a similar toxicity pathway on the reproductive system of the rotifer. The GSTs were induced in a similar manner (Fig. 3e–i), although the induction timeline differed among the isoforms. GST-Z2 was the earliest

to be induced at 3 h, followed by peak expressions of GST-A and GST-Z at 6 h, GST-S at 12 h and finally GST-O at 18 h. Therefore, GST-O remained the only up-regulated GST gene by 24 h, although it was starting to display the common trend of decline (Fig. 3f). Caution has to be taken when interpreting the results though, as Shi et al. (2012) showed that upregulation of GST in the mitrochondria of human cell line (MCF-7) did not protect against the cytotoxicity of nZnO, and dissolution of Zn2+ ions over time could have contributed to the inhibition of mitochondrial GST or complexation of GSH. The loss in GST thereafter might have led to increased ROS production and decreased antioxidant capability, eventually resulting in cell death (Zhu et al., 2009; Seiffert et al., 2012). Moreover, it was suggested that the cytotoxicity of nZnO may be viable through a number of pathways besides lipid peroxidation, including damages to the lysosome and mitochondria, apoptosis and increasing intracellular calcium concentrations (Shi et al., 2012). DNA-PK was the only gene to remain down-regulated throughout the whole exposure period (Fig. 3j). Post-translational inactivation of DNA-PK activity has been associated with an auto-regulatory negative feedback loop after initiation of appropriate DNA repair mechanism, or preventing depletion of ATP reserve by inhibiting the repair of DNA-PK which has already been destroyed by apoptosis (Smith and Jackson, 1999). Hence, the observed pattern of repressed DNA-PK gene expression in the rotifer may possibly be due to similar reasons, as DNA damage has been demonstrated to occur as a result of oxidative stress incurred by nZnO in E. coli (Kumar et al., 2011). The correlation between the investigated genes was quite high (Table S3), with HSP70, HSP90α1, GST-S, GST-Z and RXR flagging significant correlations with nine out of ten other genes, while DNA-PK, being significantly correlated with only four out of the ten genes, appeared to be least involved with the other genes. The results suggested that a highly-coordinated and interlinked defence and repair mechanism should have been well-established in the rotifer for it to overcome and combat pollutant-mediated stresses. 3.5. In vivo ROS level in the rotifer Based on the results of the gene expression study, nMgO, nSnO2, and nZnO were selected to investigate their in vivo induction of ROS. In vivo ROS was observed for nMgO (Fig. 4a) and nZnO (Fig. 4c) but was not shown in response to nSnO2 (Fig. 4b). For nMgO, there was no interactional effect between concentration and time on the ROS level, and only concentration was a significant factor (Two-way ANOVA; Concentration*Time: F3,16 = 0.609, p > 0.05; Concentration: F3,16 = 53,417, p < 0.001; Time: F1,16 = 4.038, p > 0.05; Fig. 4a). Thus, nMgO mediated ROS generation in the rotifer is concentration dependent. For nZnO, concentration and time interacted significantly on the ROS generation in the rotifer, in which the concentration dependency of nZnO mediated ROS generation was more pronounced in 3 h exposure (i.e. with a steeper slope) than in 24 h exposure (Twoway ANOVA; Concentration*Time: F3,16 = 5.790, p < 0.01; Concentration: F3,16 = 37.351, p < 0.001; Time: F1,16 = 0.040, p > 0.05; Fig. 4c). nSnO2 apparently provoked toxicity responses in the rotifer B. koreanus through other channels than ROS production, as HSP70, HSP90α1, GST-A, GST-S and GST-Z gene expressions were significantly altered at 10 mg L−1 and at the same time, no ROS was detected in both in vivo and acellular compartments. nSnO2 is usually non-toxic towards cells or organisms (Lison et al., 2009), and the reason behind its incitation of these genes merits further investigations. Moreover, the observed GST induction in the rotifer due to nSnO2 may not be restricted to oxidative stress, as there are many types (or isoforms) of 7

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Fig. 2. (continued) 8

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GSTs, and each of them carries different physiological functions (as seen in plants; Marrs, 1996). In vivo ROS measurement is a better method for predicting oxidative stress than extracellular ROS detection, as the spin trap DMPO only allows detection of ·OH and O2−, which means that a whole range of different spin probes for more ROS species need to be employed for inclusion of all existent ROS generated by the nano metal oxides, and the current DMPO approach probably leads to underestimation.

4. Conclusion Among the nine nano metal oxides, nZnO was the most toxic one towards the diatom S. costatum while nSnO2 was the least toxic, according to the 72-h EC50 values. nZnO was also the most toxic towards the rotifer B. koreanus, while the other nano metal oxides were found to be non-lethal. As nZnO is commonly used in commercial products like sunscreens and showed to be relatively highly toxic to the diatom and 9

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Fig. 4. The mean relative expression levels of in vivo ROS in the rotifer Brachionus koreanus after exposure to nMgO (a), nSnO2 (b) and nZnO (c) for 3 and 24 h (n = 3, ± 2 s.d.). Dashed line indicates the control level. Bars with the same letter denote statistical indifference between concentrations (Tukey's post-hoc test, p > 0.05).

rotifer, further research should be conducted to determine its ecological impact on the coastal marine environment. nMgO and nZnO were confirmed to bring on ROS-mediated toxicity to the two marine organisms, while nTiO2 (anatase and rutile) might induce oxidative stress

also as shown by their acellular ROS production in seawater. nZnO may also wreak havoc in the endocrine system of organisms, as indicated by the increased RXR transcription. Annealed nMgO can help to reduce its toxicity via removal of O2– and impurities from its surface. For some 10

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NMs which do not appear to induce toxicity via oxidative stress (e.g. nSnO2), gene expressions need to be probed for better understanding of their toxicity mechanisms such as cellular abrasion, inflammation and endocrine disruption. Further chemical characterizations (e.g. in vivo ROS measurement) and gene expression studies should also be carried out to elucidate the results for S. costatum. There are various limitations for this study, as the metal ion release from the metal oxides were not investigated nor were metal salt controls included, and metal ions may play a part in contributing towards their overall toxicity. This study provides a useful dataset for the construction of an aquatic toxicity database for NMs. Such a database can provide information for scientists, manufacturers and regulators across the world such that they can develop environmentally friendly products for commercialization, and reducing the ecological impacts of NMs by imposing restriction on their releases.

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CRediT authorship contribution statement Stella W.Y. Wong: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Guang-Jie Zhou: Formal analysis, Writing - original draft, Writing - review & editing. Kevin W.H. Kwok: Investigation, Writing - review & editing. Aleksandra B. Djurišić: Formal analysis, Methodology, Writing - review & editing. Jeonghoon Han: Investigation, Methodology. Jae-Seong Lee: Formal analysis, Writing - original draft, Writing - review & editing, Methodology. Kenneth M.Y. Leung: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing. 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. Acknowledgements This work was supported by the Research Grants Council of the Hong Kong SAR Government via the General Research Fund (Project no.: 17305715). GJ Zhou was supported by the Hong Kong Scholars Program (Project No. XJ2012050) and RGC via Collaborative Research Fund (project no. C7044-14G) and Theme-based Research Scheme (project no. T21-711/16-R). The authors thank Ms. Helen Leung and Ms. Cecily Law for technical support, and the anonymous reviewers for their valuable comments on this manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2020.110973. References Aruoja, V., Dubourguier, H., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 407, 1461–1468. Aruoja, V., Pokhrel, S., Sihtmäe, M., Mortimer, M., Mädler, L., Kahru, A., 2015. Toxicity of 12 metal-based nanoparticles to algae, bacteria and protozoa. Environ. Sci. Nano 2, 630–644. Bernardeschi, M., Guidi, P., Scarcelli, V., Frenzilli, G., Nigro, M., 2010. Genotoxic potential of TiO2 on bottlenose dolphin leukocytes. Anal. Bioanal. Chem. 396, 619–623. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cappellen, D., Luong-Nguyen, N.H., Bongiovanni, S., Grenet, O., Wanke, C., Šuša, M., 2002. Transcriptional program of mouse osteoclast differentiation governed by the macrophage colony – stimulating factor and the ligand for the receptor activator of NFκB. J. Biol. Chem. 277, 21971–21982.

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