Nanoparticle TiO2 size and rutile content impact bioconcentration and biomagnification from algae to daphnia

Environmental Pollution 247 (2019) 421e430

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Nanoparticle TiO2 size and rutile content impact bioconcentration and biomagnification from algae to daphnia* Xiangjie Chen a, b, Ya Zhu a, b, Kun Yang a, b, Lizhong Zhu a, b, Daohui Lin a, b, * a b

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

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 October 2018 Received in revised form 7 January 2019 Accepted 8 January 2019 Available online 10 January 2019

Little information is available about effect of particle size and crystal structure of nTiO2 on their trophic transfer. In this study, 5 nm anatase, 10 nm anatase, 100 nm anatase, 20 nm P25 (80% anatase and 20% rutile), and 25 nm rutile nTiO2 were selected to investigate the effects of size and crystal structure on the toxicity, bioconcentration, and trophic transfer of nTiO2 to algae and daphnia. In the exposed daphnids, metabolic pathways affected by nTiO2 and nTiO2-exposed algae (nTiO2-algae) were also explored. The 96 h IC50 values of algae and the 48 h LC50 values of daphnia were 10.3, 18.9, 43.9, 33.6, 65.4 mg/L and 10.5, 13.2, 37.0, 28.4, 60.7 mg/L, respectively, after exposed to nTiO2-5A, nTiO2-10A, nTiO2-100A, nTiO2P25, and nTiO2-25R, respectively. The bioconcentration factors (BCFs) for 0.1, 1, and 10 mg/L nTiO2 in daphnia ranged from 21,220 L/kg to 145,350 L/kg. The nTiO2 biomagnification factors (BMFs) of daphnia fed with 1 and 10 mg/L nTiO2-exposed algae were consistently greater than 1.0 (5.7e122). The results show that the acute toxicity, BCF, and BMF all decreased with increasing size or rutile content of nTiO2. All types of nTiO2 were largely accumulated in the daphnia gut and were not completely depurated within 24 h. At the molecular level, 22 Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways of daphnia were impacted by the nTiO2 and nTiO2-algae treatments, including glutathione metabolism, aminoacyl-tRNA biosynthesis, among others. Six and four KEGG metabolic pathways were significantly disturbed in daphnids exposed to nTiO2 and nTiO2-algae, respectively, indicating the presence of algae partially alleviated the negative impact of nTiO2 on metabolism. These findings increase understanding of the impacts of physicochemical properties of nTiO2 on the food chain from molecular scale to that of the whole organism, and provide new insight into the ecological effect of nanomaterials. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Nanomaterial Algae Water flea Bioaccumulation Trophic transfer

1. Introduction In nature, TiO2 exists in three crystalline forms: anatase (tetragonal), rutile (tetragonal), and brookite (orthorhombic), although the first two are more common (Clemente et al., 2012). Due to the unique physicochemical and optical properties, engineered TiO2 nanoparticles (nTiO2) have been widely used in many applications, including cosmetics, paints, and wastewater treatment, among others (Menard et al., 2011). It is estimated that the global production of nTiO2 will be approximately 2.5 million metric tons by the year 2025 (Robichaud et al., 2009). With widespread

*

This paper has been recommended for acceptance by Baoshan Xing. * Corresponding author. Department of Environmental Science, Zhejiang University, Hangzhou, 310058, China. E-mail address: [email protected] (D. Lin). https://doi.org/10.1016/j.envpol.2019.01.022 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

application, these materials will inevitably discharged into aquatic environments and the estimated concentrations may reach 15 ng/L to 16 mg/L in surface waters and sewage (Mueller and Nowack, 2008; Gottschalk et al., 2013; Gondikas et al., 2014) and 44 mg/kg to 2382 mg/kg in sediments (Gottschalk et al., 2009). It is therefore necessary to investigate the toxicity of nTiO2 to aquatic organisms, including the potential ecological effects resulting from trophic transfer. Daphnia, the common water flea, is widely used as a model aquatic organism in toxicity studies, and much work has been done investigating the toxicity of nTiO2 to this organism. The reported medium lethal concentrations (LC50) of nTiO2 to Daphnia magna range from 29.8 mg/L to more than 250 mg/L within 48 h (Ma et al., 2012; Li et al., 2014; Naddafi et al., 2014; Li et al., 2016b; Wormington et al., 2017), dependent on the size or crystal of the
ment et al. (2013) nTiO2 (Xiong et al., 2013; Zhu et al., 2009a). Cle

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found that the toxicity of different sized and crystalline forms of nTiO2 to Daphnia magna followed an order of 44 mm anatase < 1 mm rutile < 32 nm anatase < 25 nm anatase < 21 nm P25 (80% anatase and 20% rutile) < 15 nm anatase. The toxicity may result from the production of reactive oxygen species (ROS) enhanced by the presence of nanoparticles (NPs), subsequently inducing cytotoxicity (Vale et al., 2016). Anatase nTiO2 caused greater oxidative damage and induced more intracellular ROS than did P25 nTiO2 (Clemente et al., 2015) and rutile nTiO2 (Zhang et al., 2013). Smaller NPs also induced more ROS and greater overall toxicity (Xiong et al., 2013). However, some studies have reported minimal toxicity from nTiO2 exposure. Lovern et al. (2007) demonstrated that nTiO2 had no significant effect on the activity or survival of daphnia. Even a high dose of 20 g/L nTiO2 caused only 60% D. magna mortality after a 48 h exposure (Heinlaan et al., 2008). Given the conflicting data on nTiO2 toxicity, more specific studies are warranted to address the precise toxicity of nTiO2 with regard to different size and crystalline structure. Once accumulated within biota, nanoparticles may be retained within the tissues. Fan et al. (2016) measured the accumulation of nTiO2 in D. magna and observed higher bioconcentration factors (BCFs) for 30 nm rutile nTiO2 at lower concentrations (0.1 and 1 mg/ L) than that at a higher dose (10 mg/L). Zhu et al. (2009b) also observed the accumulation of nTiO2 in the gut of D. magna; however, larger BCFs (1.18
105 L/kg) were measured at the higher concentration (1 mg/L) of P25 than at the lower concentrations (5.66
104 L/kg) (0.1 mg/L) (Zhu et al., 2010a). Li et al. (2016a) reported that equilibrium concentrations of Ti in daphnia increased from 1.3 to 5.6 mg/g after exposure to nTiO2 for 3 h. Additional investigation is therefore needed to understand the bioconcentration of nTiO2 by daphnia, including the role of particle size and crystal phase in this process. A number of studies have reported the trophic transfer of nanoparticles in aquatic food chains (Tahereh et al., 2014; Chae and An, 2016; Chen et al., 2016; Bhuvaneshwari et al., 2017; Hu et al., 2018). Chen et al. (2015) and Iswarya et al. (2018) reported the biomagnification of nTiO2 in a freshwater chain from algae to daphnia (7.83e28.52). Alternatively, biomagnification was not observed (BMFs ¼ 0.024 and 0.009) for nTiO2 transfer from daphnia to zebrafish (Zhu et al., 2010b). Similarly, in a marine benthic food chain, nTiO2 were trophically transferred from the clamworm to the juvenile turbot without biomagnification (BMFs ¼ 0.30e0.33) (Wang et al., 2016b). As the food chain plays an important role in the fate and effects of nanomaterials, it is necessary to explore various factors that regulate material trophic transfer. Therefore, in this study we investigated the trophic transfer of different sized and crystal structured nTiO2 in a simplified twoorder food chain, including the unicellular green algae Chlorella pyrenoidosa and the water flea D. magna at the molecular and organism levels. To the best of our knowledge, this is the first report assessing the effects of physicochemical properties of nTiO2 on the food chain using endpoints ranging from the molecular to the whole organism; as such, this work broaden our understanding of the fate and ecological effect of nanoparticles in aquatic environments. 2. Materials and methods

was prepared immediately prior to use by dispersing the particles into simplified Elendt M7 culture medium (SM7, the composition is shown in the Supporting Information) with bath sonication for 20 min (100 W, 40 kHz, 25  C). The nTiO2 morphology and particle diameter were characterized by transmission electron microscopy (TEM, JEM-1230, Japan) operated at 80 kV. The crystal structures of nTiO2 were analyzed with an X-ray diffraction (XRD, X'Pett PRO, Poland). The hydrodynamic size and z-potential of nTiO2 suspensions in SM7 were determined by a Zetasizer (Nano ZS90, Malvern Instruments Ltd., UK). 2.2. Test organisms Chlorella pyrenoidosa was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences, China. Daphnia magna was obtained from the Institute of Pesticide and Environmental Toxicology, Zhejiang University, China. The algae were cultured in the medium recommended by the Organization for Economic Cooperation and Development (OECD) 201 (OECD, 2006) in a climate controlled chamber (Boxun, BIC-800, Shanghai, China) at 25 ± 1  C with a 14 h light:10 h dark cycle. The daphnids were cultured in SM7 at a constant temperature (22 ± 1  C) with a light to dark cycle of 14 h:10 h, and were fed with 1
106 cells/mL algae daily. The SM7 culture medium was refreshed once per week. 2.3. Acute toxicity assays An acute toxicity test was carried out for the algae and daphnia according to OECD 201 and OECD 202 (OECD, 2004, 2006), respectively. Chlorella pyrenoidosa in the logarithm growth phase were used for the test, with an initial cell density of approximately 1
105 cells/L. Fifty milliliter of the algal suspension and 50 mL of nTiO2 suspension were mixed together in a 250 mL Erlenmeyer flask. All of the flasks were place on an incubation shaker at 25 ± 1  C with 100 mEm2s1 illumination for 14 h following with 10 h dark. After exposure for 96 h, the final algal cell densities were measured by a hemocytometer with an optical microscope (LM, Olympus, CX21, Japan). The medium inhibitory concentration (IC50) was determined by the relationship between algal growth inhibition rate and exposure concentrations. The daphnids used in the experiment were neonates (6e24 h) from a designated brood and were not fed during the experiment. The sensitivity of daphnids was verified with the potassium dichromate method (Oleszczuk et al., 2015) before the nTiO2 exposure experiment. Ten neonates were placed in a 100 mL glass beaker containing 100 mL of nTiO2 suspension. The nTiO2 suspensions were sonicated (100 W, 40 kHz, 25  C) for 20 min before exposure and were renewed every 3 h to maintain the exposure dose since their sedimentation in the culture medium was demonstrated by a settling experiment (Fig. S1 in Supporting Information). The exposure was conducted in a climatic chamber. The beakers were sealed with plastic wrap to limit contamination and water evaporation. Meanwhile, several holes were opened on the plastic wrap to meet the oxygen demand of daphnids. If lethality to animals in the blank did not exceed 10% at the end of the test, the results were considered valid. After exposure for 48 h, organisms without heartbeats were considered dead and a dose-response curve was constructed.

2.1. Preparation and characterization of nTiO2 suspensions 2.4. Bioconcentration of nTiO2 in daphnia Five types of nTiO2 powders with different diameters and crystal phases, including 5 nm anatase (nTiO2-5A), 10 nm anatase (nTiO210A), 100 nm anatase (nTiO2-100A), 20 nm P25 (80% anatase and 20% rutile) (nTiO2-P25), and 25 nm rutile (nTiO2-25R), were obtained from Aladdin (USA). A stock suspension (100 mg/L) of nTiO2

Bioconcentration experiments included a 24 h uptake phase followed a 24 h depuration phase. Juvenile (7e14 d) daphnids were transferred into fresh SM7 to completely empty their guts for 24 h prior to experiment initiation. The daphnids were then exposed to

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0, 0.1, 1, and 10 mg/L nTiO2 suspensions. For each concentration, 80 daphnids were placed in a 100 mL glass beaker containing 100 mL of test suspension. Similar to the toxicity test, the beakers were incubated in a climatic chamber at 22 ± 1  C with a light to dark cycle of 14 h: 10 h, and the medium was refreshed every 3 h. After the exposure for 0, 3, 6, 12, and 24 h (uptake phase), 10 organisms were separated from each beaker with a Pasteur pipette and were rinsed with 0.1 M phosphate buffer (PBS) three times before analysis. After the 24 h exposure, the remaining daphnids were transferred to beakers containing 100 mL fresh SM7 medium for depuration. After 3, 6, 12, and 24 h of depuration, 10 specimens were separated for analyses. In addition, daphnids were randomly selected from the 3 replicates for each exposure concentration and were observed with optical microscopy after the uptake and the 24h depuration period. The daphnids were not fed for the duration of the experiment. Titanium (IV) concentrations in water and biota were measured as described below. The concentration ratio of nTiO2 in daphnia (dry weight) to the culture medium was calculated as the bioconcentration factor or BCF (Bour et al., 2015). The uptake rate constant (ku) and efflux rate constant (ke) were also quantified. The concentrations of nTiO2 in daphnia were linearly fitted with the exposure time. The slope of the linear regression equation was influx rate I (mg/g/h). The ratio of I to the aqueous nTiO2 concentration was defined as ku (L/g/h), and ke (h1) was calculated as the slope of the linear fitting between the natural logarithm of the percentage of nTiO2 remaining in D. magna and the depuration time (Fan et al., 2016). 2.5. Trophic transfer of nTiO2 from algae to daphnia Algae in the logarithm growth phase (1
106 cells/L) were exposed to 0, 1, and 10 mg/L nTiO2 in the OECD culture medium for 24 h as described above for the above algal acute toxicity assay. The algal suspensions with nTiO2 were then transferred into 100 mL glass beakers. Ten juvenile daphnids (7e14 d) were added into each beaker. After a 24 h exposure period, algae and daphnids were collected. The daphnids were separated from the algae with a Pasteur pipette and were rinsed with 0.1 M PBS prior to analysis. A 20-mL algal suspension was dropped onto a counting plate and the algal cell density was measured by optical microscopy (LM, Olympus, CX21, Japan). Separately, 50 mL of the algal suspension was centrifuged at 3000g for 15 min. The precipitated pellet was collected and mixed into 2 mL PBS to obtain a concentrated algal suspension. Algal cells were separated from free and looselyattached nTiO2 using a sucrose gradient centrifugation method (Perreault et al., 2012) as described in the Supporting Information. The nTiO2 concentration in algae and daphnids were quantified as described below. The nTiO2 biomagnification factor or BMF was calculated as the concentration ratio of nTiO2 in the daphnia to that in the algae. 2.6. Microscopic observations The histology and ultrastructure of nTiO2-exposed daphnids was analyzed by transmission electron microscopy (TEM, Hitachi, H7650, Japan) and scanning/transmission electron microscopy with energy disperse X-ray spectroscopy (S/TEM-EDS, Tecnai G2 F20 STWIN, America). After being exposed to nTiO2 for 24 h, 5 animals were transferred into a 2 mL Eppendorf tube containing 2.5% glutaraldehyde. The glutaraldehyde fixation solution was removed after being incubation overnight at 4  C and the daphnids were rinsed with 0.1 M PBS (15 min) three times. The daphnids were then treated with 1% OsO4 for 1 h and washed with the PBS three times. The daphnids were then dehydrated in an ethanol gradient

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(30%, 50%, 70%, 80%, 90%, 95%, and 100%) for approximately 15e20 min at each step and in 100% acetone for 20 min. The specimens were then placed in a 1:1 mixture of absolute acetone and resin for 1 h at ambient temperature, transferred to 1:3 mixture of absolute acetone and resin for 3 h, and remained in pure resin overnight. Finally, the samples were transferred to 1.5 mL Eppendorf tubes containing 100% resin and were heated at 70  C for approximately 9 h. Seventy to ninety nm sections were obtained by ultra-thin knife (Reichert, America) and were stained by uranyl acetate and alkaline lead citrate for 15 min. The resultant samples were then analyzed by TEM and S/TEM-EDS. 2.7. Metabolite extraction and derivatization After being exposed to 1 mg/L nTiO2 or algal suspensions pretreated with 1 mg/L nTiO2 for 24 h, 80 daphnids were collected and placed in 2 mL Eppendorf tubes, which were then frozen in liquid nitrogen and stored in 80  C prior to metabolite extraction. Daphnia and daphnia fed with fresh algae were used as controls for the nTiO2 and nTiO2-algae treatments, respectively. Extraction and derivatization of metabolites was performed using the methods by Garreta-Lara et al. (2016) and as described in the Supporting Information. The metabolites were analyzed by gas chromatography mass spectrometry (GC-MS, Agilent 5975I, America). The GC-MS was equipped with a DB-5MS column (Phenomenex, 30 m
0.25 mm ID
0.25 mm) and operated in EI mode at 70 eV. The oven temperature program was set at 70  C for 4 min, and then increased (5  C/min) to 300  C and held for 5 min; the delay time was 6 min. Helium was used as the carrier gas with a flow rate of 1.0 mL/min. The injection temperature was 290  C. The source temperature and interface temperature were 230  C and 280  C, respectively. The m/z values were monitored in full scan mode, ranging from 33 to 600 (Wu et al., 2017). A quality control (QC) sample was prepared by mixing aliquots of all samples (a pooled sample). The metabolites were identified by comparison with the NIST 14 library by Mass Hunter Qualitative Analysis software B.07.00 (Agilent, USA). Ribitol was used as an internal standard; additional details are provided in the Supporting Information. Only metabolites with matching scores of >70% were considered reliable. Principal component analysis (PCA) was performed using SIMCA-P 11.5 software. MetaboAnalyst 3.0 (https://www.metaboanalyst.ca/ MetaboAnalyst/) was used to construct heatmaps of metabolites and to identify metabolic pathways in daphnia. Venn diagrams were constructed using http://bioinformatics.psb.ugent.be/webtools/ Venn/. 2.8. Titanium measurement The algal cells or daphnia samples were digested with 2 mL pure HNO3 (AR,  69.0%) at 120  C until the acid had vaporized. The nTiO2 released from the organisms or in aqueous samples were further decomposed into titanium (IV) ions by heating with sulfuric acideammonium sulfate solution (Zhu et al., 2010b). The concentration of titanium (IV) ions was determined with an atomic absorption spectrometer (PerkinElmer, AAS-700, American) equipped with a graphite furnace (the determination conditions of Ti using AAS were in the Supporting Information), which was thereafter used to calculate the nTiO2 concentration. 2.9. Statistical analysis All experiments were conducted in triplicate. The results were presented as mean values with standard deviations. A one-way analysis of variance (ANOVA) with Duncan's multiple comparison test was used to evaluate significant differences between the

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control and treatment groups with the aid of statistical software, IBM® SPSS® Statistics 22.0. A p < 0.05 was used to determine statistical significance.

3. Results and discussion 3.1. nTiO2 characteristics XRD spectra (Fig. S3) show that nTiO2-5A, nTiO2-10A, and nTiO2100A are anatase, nTiO2-25R is rutile, and the ratio of anatase to rutile in nTiO2-P25 is approximately 80:20, all of which is consistent with the material information provided by the manufacturer. The purities of nTiO2-5A, nTiO2-10A, nTiO2-100A, nTiO2-P25, and nTiO225R were determined to be 99.7%, 98.3%, 94.6%, 94.0%, and 99.9%, respectively (Fig. S4). As revealed by TEM (Fig. S5), the nTiO2 were spherical or cylindrical, with particle sizes of 4.6 ± 2.6 nm, 11.2 ± 2.9 nm, 103.1 ± 24.6 nm, 20.3 ± 4.3 nm, and 27.4 ± 5.2 nm. Their hydrodynamic sizes in SM7 were measured to be 929 ± 62 nm, 908 ± 3 nm, 763 ± 12 nm, 953 ± 50 nm, and 908 ± 91 nm (Table 1). Notably, the particles did aggregate significantly in SM7. The near neutral zeta potentials (0.9 ± 0.2, 5.8 ± 0.3, and 5.8 ± 0.1 mV for example, Table 1) likely account for the observed aggregation (Clemente et al., 2012).

3.2. Acute toxicities of nTiO2 to algae All TiO2 types had inhibitory effects on the growth of algae (Fig. 1A). The inhibition rates gradually increased with increasing exposure concentration. Specifically, the calculated 96 h IC50 of nTiO2-5A, nTiO2-10A, nTiO2-100A, nTiO2-P25, and nTiO2-25R to algae were 10.3 ± 0.1, 18.9 ± 0.9, 43.9 ± 2.7, 33.6 ± 2.8, and 65.4 ± 6.6 mg/L, respectively. It is also clear that acute toxicity decreased with increasing particle size. This is expected and consistent with previous reports (Deng et al., 2017; Wang et al.,
ment et al., 2013; Ji et al., 2011). The smaller the parti2016a; Cle cle size, the larger the specific surface area of nTiO2, which increases the probability of reacting with algal cells. nTiO2 aggregates could also trap algal cells and affect growth by blocking light and
ment et al., 2013; Ma et al., 2015; Middepogu material exchange (Cle et al., 2018; Lin et al., 2012). It is also clear from the order of IC50 values that acute toxicity decreased with increasing of rutile content in the nTiO2. This finding is also not surprising given that anatase nTiO2 are known to be more reactive than rutile particles due to differences in the band gap and in surface chemistry (Hirakawa et al., 2007; Luttrell et al., 2014). In addition, the accumulation of ROS induced by nTiO2 could further cause toxicity. Some studies have investigated the relationship between ROS and the structural characteristics of nTiO2. For example, Xiong et al. (2013) showed that smaller particles triggered greater generation of ROS and increased toxicity. Zhu et al. (2009a) also reported on the toxic effects on Chinese hamster ovary (CHO) cells, with toxicity being 10e20 nm anatase > 50e60 nm anatase > 50e60 nm rutile, which was consistent with that of oxidative damage. As such, it is clear that anatase nTiO2 causes more oxidative damage than rutile particles.

Fig. 1. Acute toxicity of nTiO2 to algae (A) and daphnia (B). In each figure, different letters beside the data represent significant differences.

3.3. Acute toxicities of nTiO2 to daphnia The measured 24 h LC50 of potassium dichromate to daphnia was 1.12 mg/L (Fig. S6), confirming that the organisms were suitable for use (Oleszczuk et al., 2015). The five nTiO2 types all had significant acute toxicity on the daphnia and the dose-responsive curves are shown in Fig. 1B. The 48 h LC50 of nTiO2-5A, nTiO210A, nTiO2-100A, nTiO2-P25, and nTiO2-25R to daphnia were 10.5 ± 1.6, 13.2 ± 1.4, 37.0 ± 0.8, 28.4 ± 3.8, and 60.7 ± 3.9 mg/L, respectively. Notably, these values are largely comparable to values previously reported for daphnia. For example, LC50 of 10 nm anatase nTiO2 to Ceriodaphnia dubia and Daphnia pulex were 3.0e15.9 mg/L and 6.5e13.0 mg/L, respectively (Hall et al., 2009); P25 had a EC50 of 29.5 ± 7.22 mg/L to D. magna (Picado et al., 2015). It is also clear that the LC50 values to daphnia were significantly
ment et al. (2013) also lower than for the algae. Similarly, Cle demonstrated that daphnia had a higher acute sensitivity to nTiO2 than did algae, rotifers, and plants. The toxicity of nTiO2 to daphnia also decreased with increasing size and rutile content of the

Table 1 The hydrodynamic size and z-potential of the different nTiO2 types. nTiO2-5A hydrodynamic sizes, nm z-potentials, mV

b

908 ± 3 7.6 ± 0.2e

nTiO2-10A b

929 ± 62 6.7 ± 0.2d

nTiO2-100A a

763 ± 12 5.8 ± 0.3c

nTiO2-P25 b

953 ± 50 0.9 ± 0.2b

Note: The concentration of nTiO2 was 50 mg/L in SM7 medium (pH ¼ 7.0). Within rows, different letters represent significant differences.

nTiO2-25R 908 ± 91b 5.8 ± 0.1a

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particles. It has been reported that daphnia can ingest particles less than 50 mm without by non-selective mechanisms (Hund-Rinke and Simon, 2006). The nTiO2 that enters the intestinal tract may cause damage to intestinal cells, resulting in observable toxicity. The smaller size and anatase nTiO2 are known to be more reactive, causing the higher toxicity (Gaya and Abdullah, 2008).

3.4. Accumulation of nTiO2 in daphnia through aqueous exposure Light microscopic images (Figs. 2 and S7) were obtained to visually analyze the uptake and depuration of nTiO2 by daphnia. It can be seen that nTiO2 were mainly accumulated in the intestinal tract of daphnids, although a small amount of nTiO2 was also found on the body surfaces and thoracic appendages after the 24 h exposure. After the depuration for 24 h, the nTiO2 on the body

Fig. 2. Light microscopic images of daphnia without any treatment (A) and treated with 10 mg/L nTiO2-5A (B: uptake; G: depuration), nTiO2-10A (C: uptake; H: depuration), nTiO2-100A (D: uptake; I: depuration), nTiO2-P25 (E: uptake; G: depuration), and nTiO2-25R (F: uptake; K: depuration). Red arrows point to nanoparticles. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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surfaces and in the thorax was no longer evident; however, there was still a small amount of nTiO2 remaining in the gut. This indicates that it was difficult for nTiO2 to be cleared after ingestion. Pinheiro et al. (2013) observed the distribution of P25 in all tissues (gut, sub-endothelial tissue, and other tissues) of D. magna exposed to a relatively high concentration (11.25 mg/L) of P25, whereas P25 were only detected in the gut at a lower concentration of 1.4 mg/L. Hence, the intestine of daphnia can be the main target organ for nTiO2. Fig. 3 shows TEM images of gut sections from the control and the nTiO2 treated daphnids. Longitudinal (Fig. 3AeD) and transverse (Fig. 3E) sections of the intestinal tract from the control animals exhibits the general structure of enterocytes. It can be seen that the intestine epithelium distributed regularly and was composed of cuboidal to columnar cells with a thick layer of microvilli on the edge, which were separated from the gut muscle by a granular basement membrane (Nogueira et al., 2006; Mendonça et al., 2011; Bacchetta et al., 2018). The intestine was highly stacked with numerous mitochondria and other organelles in the cytoplasm. The epithelial cells can secrete the peritrophic membrane (PTM), protecting the gut from bacteria as well as regulating the exchange of nutrients and enzymes (Heinlaan et al., 2011). PTMs were observable both in the control and the nTiO2 treated groups. However, the microvilli of the gut lumen were damaged after the exposure to 1 mg/L nTiO2 for 24 h, with different degrees of shedding or dissolution being evident. Two phases (absorptive and holocrine phases) of gut epithelial cells are present in Fig. 3H with respect to electron-dense and electron-lucent cells (Heinlaan et al., 2011). As reported by Schultz and Kennedy (1976), once gut epithelial cells differentiate into two different phases, the cells can be disrupted and release cellular contents and digestive enzymes into the gut lumen. Moreover, some cells were notably disorganized or were even without a nucleus (Fig. 3H and I). The digestive cells were separated from smooth muscle with a gap observed between smooth muscle and basal lamina. Empty spaces formed between adjacent digestive cells, and significant quantities of autophagy vacuoles and lamellar bodies were produced in the cytoplasm (Fig. 3FeJ). Similar damage caused by nanoparticles to daphnia have been previously observed (Dalai et al., 2013). Fig. 3K-O show the presence of nanoparticles in the gut and the associated EDS analysis (Fig. S8A) confirming the accumulated particles as TiO2. The ‘water drinking’ behavior of daphnia is known to facilitate the uptake of nanoparticles (Rosenkranz et al., 2009), and nTiO2 aggregates accumulated in the intestine may be too large to be efficiently eliminated (Zhu et al., 2010a). The body burden (BD) values in daphnia dramatically increased with increasing exposure concentration, particle size or rutile content of nTiO2 (Fig. 4). The exception to this was nTiO2-10A, which exhibited the highest accumulation at lower concentrations. As evident in Table 1, all nTiO2 particles aggregated to similar sizes. However, the accumulation of ROS depends on the structural characteristics of nTiO2 noted above, which may influence BD values. Table 2 lists the calculated ku and ke constants. The ku values largely decreased as the size or rutile content of nTiO2 increased; the opposite was observed for the ke values. This indicates that nTiO2 with smaller size or higher anatase content had a higher potential to be retained within the biota. The calculated BCFs of nTiO2 in daphnia (Table 2) ranged from 21,220 L/kg to 145,350 L/kg, dependent on the exposure concentration and physicochemical properties of nTiO2. These BCF values are significant and even higher than those of lipophilic chemicals, such as polycyclic aromatic hydrocarbons (900 ± 12, 910 ± 10, 1376 ± 27, and 1485 ± 15 L/kg for phenanthrene, anthracene, fluoranthene, and pyrene, respectively) (Li et al., 2018). It is notable that the BCFs decreased with increasing exposure concentration. In

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Fig. 3. TEM images of the gut from control daphnia (AeE) and daphnia exposed to nTiO2-5A (F and K), nTiO2-10A (G and L), nTiO2-100A (H and M), nTiO2-P25 (I and N), and nTiO225R (J and O) for 24 h. D, K, L, M, N, and O images are magnified from the rectangle located areas in C, F, G, H, I, and J images, respectively. MV: microvilli; m: mitochondrion; N: cell nucleus; n: nucleus; L: gut lumen; l: lysosome; BL: basal lamina; AV: autophagy vacuole; LB: lamellar body; GM: gut muscolaris; NP: nanoparticles; red arrow head: PTM (peritrophic membrane); *: secretory cell. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. The uptake and depuration kinetics of 0.1 mg/L (A), 1 mg/L (B), and 10 mg/L (C) nTiO2 by daphnia.

agreement with the BD values, BCFs decreased with increasing size or rutile content of nTiO2, except for the comparable data between nTiO2-5A and nTiO2-10A. The BCFs of 0.1 and 1 mg/L P25 (21 nm) in daphnia were previously reported to be 56,563 L/kg and 116,281 L/ kg, respectively (Zhu et al., 2010a), which are comparable to our results. 3.5. Food chain transfer of nTiO2 from algae to daphnia through dietary exposure The nTiO2 BMFs of daphnia fed with 1 and 10 mg/L nTiO2exposed algae (thereafter named nTiO2-algae) were consistently greater than 1 (5.7e122, Table 3), indicating biomagnification of

nTiO2 from algae to daphnia. In addition, BMFs decreased with increasing nTiO2 concentration. It is also evident that BMFs largely decreased with increasing particle size and rutile content of the nTiO2 under both exposure concentrations. This was in line with the effects of particle size and crystal structure on the acute toxicity and bioaccumulation of nTiO2 with algae and D. magna. Iswarya et al. (2018) determined the trophic transfer of anatase nTiO2 (<25 nm) and rutile nTiO2 (100 nm) from Chlorella sp. to Ceriodaphnia dubia. The authors reported BMFs of 75, 300, and 1200 mM nTiO2 at 16.88 ± 1.67, 13.80 ± 3.48, and 4.58 ± 1.22 for the anatase and 23.52 ± 1.34, 1.64 ± 0.81, and 2.22 ± 0.31 for the rutile particles, respectively. Chen et al. (2015) measured the transfer of 10 mg/L P25 from Scenedesmus obliquus to Daphnia magna and

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Table 2 Accumulation and depuration of nTiO2 in daphnia. Conc.

Indicators

nTiO2-5A

nTiO2-10A

nTiO2-100A

nTiO2-P25

nTiO2-25R

0.1 mg/L

ku (L/g$h) ke (1/h) Conc. in medium (mg/L) Conc. in daphnia (g/kg) BCF (L/kg)

2.61 ± 0.17a 0.057 ± 0.006b 0.059 ± 0.005ab 6.36 ± 0.54b 107360 ± 2330ab

2.68 ± 0.13a 0.052 ± 0.008b 0.056 ± 0.001b 8.18 ± 0.90a 145350 ± 1242a

2.04 ± 0.16b 0.080 ± 0.005a 0.068 ± 0.007a 6.32 ± 0.55b 92580 ± 2429b

2.26 ± 0.11b 0.071 ± 0.006a 0.062 ± 0.005ab 6.29 ± 0.41b 101080 ± 1872ab

0.86 ± 0.08c 0.075 ± 0.004a 0.040 ± 0.004c 2.25 ± 0.15c 56380 ± 1171c

1.0 mg/L

ku (L/g$h) ke (1/h) Conc. in medium (mg/L) Conc. in daphnia (mg/kg) BCF (L/kg)

2.23 ± 0.12b 0.098 ± 0.003c 0.67 ± 0.06a 64.5 ± 0.42b 96200 ± 6522b

2.48 ± 0.11a 0.087 ± 0.006d 0.58 ± 0.04b 81.2 ± 0.93a 139410 ± 1211a

1.55 ± 0.14c 0.136 ± 0.007a 0.62 ± 0.05ab 43.5 ± 3.76d 69770 ± 1375c

1.55 ± 0.18c 0.101 ± 0.004c 0.56 ± 0.03b 50.1 ± 7.24c 88730 ± 1976b

1.16 ± 0.10d 0.122 ± 0.006b 0.69 ± 0.03a 38.3 ± 1.50e 55150 ± 1242d

10 mg/L

ku (L/g$h) ke (1/h) Conc. in medium (mg/L) Conc. in daphnia (mg/kg) BCF (L/kg)

0.46 ± 0.03a 0.082 ± 0.005cd 3.66 ± 0.06d 165 ± 6.73a 45150 ± 2885a

0.48 ± 0.06a 0.077 ± 0.006d 3.53 ± 0.06e 147 ± 5.01ab 41640 ± 416ab

0.333 ± 0.03b 0.090 ± 0.004bc 4.06 ± 0.08b 142 ± 30.4b 34990 ± 1115c

0.43 ± 0.07a 0.094 ± 0.005b 3.86 ± 0.05c 150 ± 2.83ab 38790 ± 1443bc

0.22 ± 0.02c 0.112 ± 0.003a 5.26 ± 0.04a 112 ± 10.5c 21220 ± 2223d

Note: Within rows, different letters beside the data represent significant differences.

Table 3 BMFs of nTiO2 with different concentrations transferred from algae to daphnia. Conc. (mg/L)

nTiO2

Conc. in algae (mg/kg)

Conc. in daphnia (mg/kg)

BMF

1

nTiO2-5A nTiO2-10A nTiO2-100A nTiO2-P25 nTiO2-25R

24.3 ± 4.0a 23.2 ± 4.8a 26.7 ± 5.0a 28.2 ± 5.2a 22.4 ± 1.1a

2423 ± 122c 2839 ± 45.9a 1411 ± 109d 2653 ± 50.7b 848 ± 95.8e

99.7 ± 21.7a 122 ± 35.4a 52.8 ± 14.6b 94.2 ± 15.2a 37.9 ± 2.4b

10

nTiO2-5A nTiO2-10A nTiO2-100A nTiO2-P25 nTiO2-25R

444 ± 21.8c 329 ± 17.9d 868 ± 36.1a 831 ± 11.5a 675 ± 8.8b

6127 ± 85.1b 5268 ± 127c 7738 ± 173a 5378 ± 134c 3846 ± 156d

13.8 ± 3.6ab 16.0 ± 2.4a 8.9 ± 1.1b 6.5 ± 0.7b 5.7 ± 1.3b

Note: Within rows, different letters beside the data represent significant differences.

observed a BMF of 7.83. It has been reported that anatase nTiO2 will become heavily bound to algal cells, whereas rutile nTiO2 were adsorbed loosely on the algae (Gao et al., 2018). Thus, a fraction of rutile nTiO2 may readily desorb from algae before being ingested by daphnia. In addition, smaller nTiO2 (nTiO2-5A and nTiO2-10A) could attach to a greater extent on the algal cells compared with the larger particles (nTiO2-100A) given their higher specific surface area. As a result, daphnia ingested more smaller size nTiO2 or higher anatase content by dietary exposure. Although the initial hydrodynamic sizes of nTiO2 are similar, particle size is a dynamic process, particularly in a biological system. 3.6. Effect of nTiO2 on metabolic profile of daphnia Since a small amount of nTiO2 remained in the body of daphnia through the aqueous exposure and trophic transfer, these materials may cause damage to intestinal cells and subsequently affect metabolism. Fig. 5A and B show heatmaps generated by a hierarchical cluster analysis of 46 metabolites extracted from daphnids exposed to nTiO2 and nTiO2-algae, respectively. Systemic and dendric graphs based on the hierarchical cluster analysis reflect the relationship among the 18 sets of data. Clear separation of six groups represent the different metabolic flux across the different treatments and the metabolites that were regulated differently through different exposure pathways. To better display the significance between metabolites, a principal component analysis (PCA) was conducted. The results of PCA show that both the nTiO2 and the nTiO2-algae treatments (Fig. 5C and D, respectively) significantly

altered the metabolic profile of daphnia. The distribution of principal components of the anatase nTiO2 treatment groups were largely consistent, but were clearly separate from the rutile nTiO2 treatment groups. This indicates a phase-specific response to nTiO2. The distributions and possible relationships of significantly different metabolites induced by different exposure pathways are shown by Venn diagrams (Fig. 5E and F). Six significantly altered metabolites (glycine, maltose, cadaverine, putrescine, palmitic acid, and lysine) were detected in daphnia treated with each of the five nTiO2. Cholesterol, 1-monomyristin, ribonic acid, and benzoic acid exhibited significant differences only under nTiO2-25R, nTiO2-5A, and nTiO2-10A exposures, respectively. In the nTiO2-algae treated daphnids, 5 significantly different metabolites (glycine, cadaverine, putrescine, palmitic acid, and lysine) were detected. The exposures to algae pre-treated with nTiO2-5A, nTiO2-10A, nTiO2-100A, and nTiO2-25R exclusively altered three (d-glucose, d-glucopyranoside, and 1-monomyristin), two (ribonic acid and benzoic acid), one (lactic acid), and three (stearic acid, L-5-oxoproline, and maltose) metabolites of daphnia, respectively. The disrupted Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways were analyzed based on the metabolites of daphnids from all treatment groups (Tables S2 and S3). In summary, 22 KEGG metabolic pathways of daphnia were impacted by the nTiO2 and nTiO2-algae treatments, among which 15 pathway alterations were detected for the two treatments, including glutathione metabolism, aminoacyl-tRNA biosynthesis, fatty acid biosynthesis, among others. The significantly disturbed metabolic pathways through the different exposures are summarized in Table 4. For the nTiO2 treatment, 6 KEGG metabolic pathways were differentially impacted, including glutathione metabolism, biotin metabolism, cyanoamino acid metabolism, galactose metabolism, biosynthesis of unsaturated fatty acids, and starch and sucrose metabolism. For the nTiO2-algae treatment, only 4 KEGG metabolic pathways (glutathione metabolism, biotin metabolism, cyanoamino acid metabolism, and fatty acid biosynthesis) were significantly disturbed, indicating the presence of algae partially alleviated the impact of nTiO2 on metabolism. This was particularly evident for the metabolism of galactose, starch, and sucrose in the exposed daphnia. To the best of our knowledge, this is the first study analyzing the effect of nTiO2 on the metabolic profile of daphnia, though similar studies have been reported for other contaminants. Li et al. (2014) explained the toxicity of nano-Ag by the released Agþ induced disturbance in oxidative stress and energy metabolism. Poynton et al. (2011) determined the top five affected KEGG metabolic

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Fig. 5. Heatmaps generated by hierarchical cluster analysis of daphnia metabolites exposed to 1 mg/L nTiO2 (A) and 1 mg/L nTiO2 exposed algae (B). Different position of color pieces represents corresponding relative contents of metabolites. Red represents metabolite levels up-regulated, blue represents levels down-regulated. The principal component analysis (PCA) of daphnia metabolites exposed to 1 mg/L nTiO2 (C) and 1 mg/L nTiO2 exposed algae (D). The samples are clustered according to the value distributions of components 1 and 2. Venn diagrams of significantly different daphnia metabolites exposed to 1 mg/L nTiO2 (E) and 1 mg/L nTiO2 exposed algae (F). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Table 4 Pathway analysis of metabolites extracted from daphnia exposed to nTiO2 and nTiO2-algae. NPs

Pathway name

P values nTiO2-5A

nTiO2-10A

nTiO2-100A

nTiO2-P25

nTiO2-25R

nTiO2

Glutathione metabolism Biotin metabolism Cyanoamino acid metabolism Galactose metabolism Biosynthesis of unsaturated fatty acids Starch and sucrose metabolism

0.0007** 0.0368* 0.0441* 0.1786 0.0369* 0.1532

0.0010** 0.0405* 0.0484* 0.1947 0.0442* 0.0130*

0.0010** 0.0405* 0.0484* 0.0179* 0.0442* 0.0130*

0.0007** 0.0368* 0.0441* 0.1786 0.0369* 0.0107*

0.0010** 0.0405* 0.0484* 0.0179* 0.2966 0.0130*

nTiO2 -algae

Glutathione metabolism Biotin metabolism Cyanoamino acid metabolism Fatty acid biosynthesis

0.0003** 0.0296* 0.0354* 0.0197*

0.0007** 0.0368* 0.0441* 0.2509

0.0005** 0.0332* 0.0397* 0.0249*

0.0003** 0.0296* 0.0354* 0.2062

0.00005** 0.0441* 0.0527 0.0433*

Note: * and ** represent the significant difference at p < 0.05 and p < 0.01 levels, respectively. Other detected pathways without significant difference are shown in Tables S2 and S3 in the Supporting Information.

pathways of daphnia caused by Cd exposure, including fatty acid biosynthesis, aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, lysine degradation and phenylalanine, and tyrosine and tryptophan biosynthesis. Garreta-Lara et al. (2016) examined the metabolites of daphnia exposed to higher salinity, greater temperature, and hypoxia, and demonstrated that all of the three treatments affected the pyruvate metabolism and Citrate or Krebs cycle in daphnia. 4. Conclusion This work systematically investigated the toxicity and trophic transfer of different sizes and crystal forms of nTiO2 from algae to daphnia. Once internalized by daphnia, nTiO2 mainly accumulated in the intestine, and once there, clearance was incomplete. This exposure to nTiO2 could damage the microvilli and epithelial cells of the gut and induce significant toxicity, including lethality, to the exposed daphnia. The acute toxicity, BCF, and BMF all decreased with increasing exposure concentration, particle size, or rutile content of nTiO2. At the molecular level, 22 KEGG metabolic pathways of daphnia were impacted by exposure to nTiO2 and nTiO2algae, among which 6 and 4 pathways were significantly disturbed by the nTiO2 and nTiO2-algae exposures, respectively. The presence of algae alleviated the impact of nTiO2 on the metabolism of daphnia. These results will be useful to evaluate the ecological effect of nTiO2 and provide important information necessary to adequately assess exposure and risk of engineered nanomaterials in aquatic environments. Conflicts of interest There are no conflicts to declare. Acknowledgements This work was supported by the National Natural Science Foundation of China (21525728 and 21621005) and the National Key Research and Development Program of China (2017YFA0207003). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.01.022. References Bacchetta, R., Santo, N., Valenti, I., Maggioni, D., Longhi, M., Tremolada, P., 2018. Comparative toxicity of three differently shaped carbon nanomaterials on

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