Size matters – The phototoxicity of TiO2 nanomaterials

Environmental Pollution 208 (2016) 859e867

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Size matters e The phototoxicity of TiO2 nanomaterials €ger a, Alexander Bach a, Bryan Hellack c, Anne J. Wyrwoll a, *, Petra Lautenschla d Agnieszka Dybowska , Thomas A.J. Kuhlbusch c, Henner Hollert b, e, f, g, €ffer a, e, g, Hanna M. Maes a Andreas Scha a

Department of Environmental Biology and Chemodynamics, Institute for Environmental Research, RWTH-Aachen University, Worringerweg 1, 52074 Aachen, Germany b Department of Ecosystem Analysis, Institute for Environmental Research, RWTH-Aachen University, Worringerweg 1, 52074 Aachen, Germany c Institute for Energy and Environmental Technology e.V., Bliersheimer Str. 58-60, 47229 Duisburg, Germany d Natural History Museum London, Cromwell Road, SW7 5BD London, UK e College of Resources and Environmental Science, Chongqing University, 1 Tiansheng Road, Beibei, Chongqing 400715, China f College of Environmental Science and Engineering and State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, 1239 Siping Road, Shanghai, China g State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 May 2015 Received in revised form 10 October 2015 Accepted 21 October 2015 Available online 21 November 2015

Under solar radiation several titanium dioxide nanoparticles (nano-TiO2) are known to be phototoxic for daphnids. We investigated the influence of primary particle size (10, 25, and 220 nm) and ionic strength (IS) of the test medium on the acute phototoxicity of anatase TiO2 particles to Daphnia magna. The intermediate sized particles (25 nm) showed the highest phototoxicity followed by the 10 nm and 220 nm sized particles (median effective concentrations (EC50): 0.53, 1.28, 3.88 mg/L). Photoactivity was specified by differentiating free
OH radicals (therephthalic acid method) and on the other hand surface
adsorbed, as well as free
OH, electron holes, and O2 (electron paramagnetic resonance spectroscopy, EPR). We show that the formation of free
OH radicals increased with a decrease in primary particle size (terephthalic acid method), whereas the total measured ROS content was highest at an intermediate particle size of 25 nm, which consequently revealed the highest photoxicity. The photoactivities of the 10 and 220 nm particles as measured by EPR were comparable. We suggest that phototoxicity depends additionally on the particleedaphnia interaction area, which explains the higher photoxicity of the 10 nm particles compared to the 220 nm particles. Thus, phototoxicity is a function of the generation of different ROS and the particleedaphnia interaction area, both depending on particle size. Phototoxicity of the 10 nm and 25 nm sized nanoparticles decreased as IS of the test medium increased (EC50: 2.9 and 1.1 mg/L). In conformity with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory we suggest that the precipitation of nano-TiO2 was more pronounced in high than in low IS medium, causing a lower phototoxicity. In summary, primary particle size and IS of the medium were identified as factors influencing phototoxicity of anatase nano-TiO2 to D. magna. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Daphnia magna TiO2 nanoparticles Solar radiation Reactive oxygen species Phototoxicity

1. Introduction

* Corresponding author. E-mail addresses: [email protected] (A.J. Wyrwoll), Petra. €ger), Alexander.Bach@[email protected] (P. Lautenschla aachen.de (A. Bach), [email protected] (B. Hellack), [email protected] (A. Dybowska), [email protected] (T.A.J. Kuhlbusch), [email protected]. €ffer), Hanna. de (H. Hollert), [email protected] (A. Scha [email protected] (H.M. Maes). http://dx.doi.org/10.1016/j.envpol.2015.10.035 0269-7491/© 2015 Elsevier Ltd. All rights reserved.

Among nanomaterials, titanium dioxide nanomaterials (nanoTiO2) belong to the most produced nanomaterials worldwide (Piccinno et al., 2012). Since the last decades, they are used in a variety of products, such as plastics, paints for façades, as well as in personal care products (PCPs). Furthermore, the photocatalytic activity of nano-TiO2 is used for self-cleaning surfaces and water treatment (Weir et al., 2012; Wang et al., 2009; Aitken et al., 2006;

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Sun et al., 2014; Pelaez et al., 2012). Nano-TiO2 will end up in waste water treatment plants (WWTPs) when nano-TiO2 containing PCPs are washed down the drain or nano-TiO2 included in façade painting is washed off during strong rain events (Kaegi et al., 2008). From WWTPs, it may enter the aquatic environment via the effluent and might pose a risk for aquatic organisms (Gottschalk et al., 2013, 2009). Nano and macro sized anatase TiO2 are semiconductors that can be excited by wavelengths at or below 384 nm, corresponding to their band gap energy at or above 3.2 eV (Mardare et al., 2000; Tang et al., 1994). As a result electron hole pairs are formed (Carp et al., 2004), which are able to react with H2O, hydroxide ions and oxygen to generate reactive oxygen species (ROS) including hydroxyl
radicals (
OH) (Maness et al., 1999) and superoxide radicals ( O2 ) (Chen and Mao, 2007). ROS may induce oxidative stress in organisms exposed to ROS concentrations exceeding their antioxidant defense system (Luo et al., 2006; Lushchak, 2011; Sun et al., 2006). The UV part of solar radiation can enhance the ecotoxicity of nanoc, 2015). TiO2 by a factor ranging from 0.84 to 16 778 (Jovanovi Phototoxicity of nano-TiO2 was determined with vertebrates such as fish and frogs, as well as bacteria, marine phytoplankton and crustaceans such as Gammarus fossarum and Daphnia magna (Dasari et al., 2013; Li et al., 2014a; Miller et al., 2012; Pathakoti et al., 2013; Tong et al., 2013a, 2013b; Zhang et al., 2012; Feckler  et al., 2014; Clemente et al., 2014; et al., 2015; Kal cíkova Mansfield et al., 2015; Li et al., 2014b). Among these organisms Cladocera, including D. magna were identified as the most sensitive organisms (Jovanovic, 2015). In standard Daphnia tests 48 h median effective concentrations (EC50) of 29.7e500 mg/L were observed for the mainly tested mixed phase TiO2 material P25 (Ma et al., 2012a; Marcone et al., 2012; Kim et al., 2014; Amiano et al., 2012). Daphnia tests with solar radiation revealed a considerably enhanced toxicity of nano-TiO2 (P25) to daphnids (48 h EC50: 29.8 mg/L-7.8 mg/L) (Ma et al., 2012a; Marcone et al., 2012; Kim et al., 2014; Amiano et al., 2012). A large diversity of nano-TiO2 materials exists: differing by several properties such as particle size, surface characteristics, or crystal structure. All these characteristics are known to influence photoactivity and thus may influence phototoxicity of TiO2 nanomaterials (Ohtani et al., 1997; Saadoun et al., 1999). Some studies have already shown that the crystal structure of nano-TiO2 (Clemente et al., 2014; Marcone et al., 2012), irradiation wavelengths (Ma et al., 2012b), and organic matter content influence the phototoxicity of nano-TiO2 to daphnids (Amiano et al., 2012). However, to the best of our knowledge, no study has investigated the influence of particle size by testing different sized TiO2 materials of one crystal phase. In contrast, phototoxicity studies with daphnids were mainly performed with P25 (Jovanovi c, 2015; Clemente et al., 2014; Li et al., 2014b; Ma et al., 2012a; Marcone et al., 2012; Kim et al., 2014; Amiano et al., 2012; Kim et al., 2010). Seitz et al. (2014) found that particle surface normalized EC50 values of anatase nano-TiO2 < 100 nm were significantly different from EC50 values for larger particles of 140 nm (D. magna). They conclude that toxicity of anatase TiO2 particles smaller than 100 nm depends on their reactive surface areas (Seitz et al., 2014). Thus, particle size was already shown to be an important property impacting the toxicity of anatase nano-TiO2 to D. magna in absence of UV irradiation. It remains unclear whether the same holds true for the phototoxicity of anatase TiO2 materials. Therefore, we used two different sized, uncoated anatase nano-TiO2 and a bulk anatase TiO2 material in Daphnia sp. acute immobilisation tests with and without solar radiation (OECD, 2004). The photodegradation of organic pollutants by nano-TiO2 occurs either by free
OH radicals or by a direct photocatalytic oxidation by surface adsorbed
OH radicals and valence band holes (Nosaka

et al., 2003). We examined whether the observed phototoxicity was linked either to the generation of free
OH radicals (terephthalic acid, TA-method) or to the sum of valence band holes, free
and surface adsorbed
OH, as well as O2 (electron paramagnetic resonance spectroscopy (EPR) coupled with the spin trap 5,5dimethyl-1-pyrroline-N-oxide, DMPO method), by measuring photoactivity of the particles with two different methods upon solar radiation (Nosaka et al., 2003; Lipovsky et al., 2012). In aqueous media, besides the pH and the presence of natural organic matter (NOM), the ionic strength (IS) has an influence on the dispersion stability and thus toxicity of nanoparticles (French et al., 2009; Domingos et al., 2010; Ottofuelling et al., 2011). The € mer et al. (2013) for citrate-stabilized Ag latter was shown by Ro nanoparticles, which were significantly more stable and toxic for daphnids in 10-fold diluted ISO medium (865 mM) than in ISO €mer et al., 2013). For nano-TiO2 it is reported medium (8653 mM) (Ro that its hydrodynamic diameter (HD) increases with IS of the medium (Jiang et al., 2009). Moreover, natural waters with lower ionic strength (430e445 mM) (Jones et al., 1993) than ISO medium exist. Therefore, we decided to perform Daphnia sp. acute immobilisation tests in 10-fold diluted ISO medium (OECD, 2004). For comparison, the nanomaterials were additionally tested in undiluted ISO medium. 2. Material and methods 2.1. Characterization of TiO2 powders and suspensions NM 101 (Hombikat UV 100, primary particle size (PPS): 7e10 nm, 100% anatase, Sachtleben), PC 105 (NM 102, PPS: 15e25 nm, 100% anatase, Cristal Global) and Tiona AT 1 (NM 100, PPS: 200e220 nm, 100% anatase, Cristal Global) were used in the framework of the Sponsorship Programme of the Working Party of Manufactured Nanomaterials (WPMN) of the OECD. All TiO2 materials correspond to the batches of the Joint Research Center (JRC) Nanomaterial Repository NM Series NM 101, NM 102 and NM 100. The specific surface area of the TiO2 powders was measured using a multipoint Brunauer Emmett Teller (BET) method on Micromeritics surface area analyzer (Brunauer et al., 1938). The crystal structure of TiO2 and the potential presence of minor phases were examined by X-ray diffraction (XRD) with an Enraf Nonius PDS 120 X-ray diffractometer, equipped with a Co tube and a primary monochromator-slit system, which confined the X-ray beam to monochromatic radiation (Ka1). The results obtained by BET (NM 101: 280 m2/g > NM 102: 77.6 m2/g > NM 100: 9.4 m2/g) and XRD (all 100% anatase, Fig. 1E) confirmed the data supplied by the manufacturer (Tables Se1). Stock suspensions (1 g/L, n ¼ 8) were prepared by dispersing TiO2 materials in deionized water for 15 min by using an ultrasound probe (200 W, pulsed ultrasound: 0.2 s pulse and 0.8 s pause at 100% power, Sonoplus 200 W, BANDELIN Electronic GmbH & Co. KG). Working suspensions (100 or 10 mg/L, n ¼ 3) were diluted from stock suspensions with deionized water. Hydrodynamic diameters (HD) and zeta potentials (ZP) of the particles in the stock and working suspensions were analyzed with electrophoretic and dynamic light scattering (ELS, DLS, Zetasizer Nano, Malvern Instruments). PPS of the TiO2 materials in suspension (100 mg/L in deionized water) were verified by transmission electron microscopy (TEM) images (CM 20, Philips, operated at 200 kV). For this, 10 ml of the suspensions was transferred to a copper grid (Plano) that was placed on a filter paper and subsequently subjected to TEM analysis. TEM images of the nanomaterials (Fig. 1C and D) confirm the data supplied by the manufacturer (7e10 nm and 20e25 nm, Tables Se1). However, PPS of the bulk material determined by TEM

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861

Fig. 1. TEM images of TiO2 particles sampled from NM 100 (A, B), NM 102 (C), NM 101 (D) suspensions (100 mg/L, deionized water) immediately after preparation. Powder diffractograms (E) of NM 101 (blue), NM 102 (red) and NM 100 (black) compared with the line pattern of TiO2 anatase (green) obtained from ICDD-PDF2 database (entry 1078e2486). Percentage of the nominal TiO2 concentration (1.3 mg/L) at test initiation (0 h) and termination (48 h) in 10-fold diluted ISO medium (F). Error bars represent standard deviations on the mean of three replicates (n ¼ 3). Asterisks indicate significant differences between t0 and t48 for each material (p < 0.05). The significant increase of three percent of the NM 100 concentration at test termination is attributed to sampling errors. Repeated measurements at 3.9 mg NM 100/L showed that concentration stayed constant (Tables Se5). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(0.2e1.0 mm, Fig. 1A and B) deviates from the manufacturer's data (200e220 nm, Tables Se1). In contrast to our finding, the JRC Repository report presents a mean PPS for NM 100 of 40e90 nm and 150 nm, measured by two different laboratories. Furthermore, it states that 27.3% of the particles/agglomerates were below 100 nm (Cotogno et al., 20 14). Currently the EU defines nanomaterials as ‘particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nme100 nm’ (European-Commission Commi, 2011). Consequently, NM 100 is not a nanomaterial and is therefore defined in our study, as well as in the JRC Repository report, as a ‘bulk comparator’ (Cotogno et al., 20 14). 2.2. Sedimentation experiment and TiO2 particle size in the test medium A sedimentation experiment was performed to determine how much TiO2 precipitated from the water phase (10-fold diluted ISO medium) at a nominal TiO2 concentration of 1.3 mg/L at 0 h and 48 h after application (n ¼ 3). Furthermore, the HDs and ZPs of the TiO2 particles were measured in the test medium (1.3 mg/L, n ¼ 3) by means of DLS and ELS after 0, 24, and 48 h of application as described in Section 2.1. The signal to noise ratio for the 1.3 mg/L nanomaterial suspensions was too low for DLS measurements. Therefore, higher nanomaterial concentrations (80 mg/L, n ¼ 3) were additionally prepared and measured with DLS only. Pure test medium, not containing TiO2, served as control for Ti and DLS/ELS analysis. Each test vessel contained five daphnids. The test medium was sampled by gently placing the tip of a glass pipette directly beneath the water column surface. The Ti concentration was

analyzed according to ISO 11885 (ISO 11885, 2009) by inductively coupled plasma optical emission spectrometry (ICP-OES, Ti limit of quantification 5 mg/L) after microwave digestion of the samples at 200  C and 100 bar with nitric acid (65%) and hydrofluoric acid (40%). 2.3. Photoactivity experiment 2.3.1. Terephthalic acid (TA) fluorescence method The formation of free
OH radicals was measured with the terephthalic acid (TA, Sigma Aldrich) fluorescence method described by Hirakawa and Nosaka (2002). TiO2 stock suspensions (1 g/L, n ¼ 3) were either kept under dark conditions or irradiated for 15 min with a mercury-vapor lamp which emitted a spectrum comparable to sunlight (280e800 nm, Bright Sun UV Desert, 70 W, Lucky Reptile). The spectrum (Fig. S-1), as well as the irradiance of UVA (2.36 mW/cm2) and UVB (0.15 mW/cm2) at the surface of the beakers was recorded by using a calibrated spectrometer (AvaSpec, ULS 3648 200e1100 nm, Avantes). UVA and UVB irradiations are comparable to that of a midsummer day (04.07.2000, 13:36, UVA 4.10 mW/cm2 and UVB 0.12 mW/cm2) in Westerland, Germany (Sandmann, 2001). After irradiation, the IS of the TiO2 suspensions was enhanced by adding potassium chloride (143 g/L). Consequently, TiO2 particles precipitated from the suspensions as explained by the DerjaguinLandau-Verwey-Overbeek (DLVO) theory. Thereafter, fluorescence intensities of the reaction product hydroxyterephthalic acid (TAOH) were recorded with a micro well plate reader (Infinite 200 M, Tecan) at 420 nm after excitation with 310 nm. Intensities were converted to TAOH concentrations (mmol/L) by reading the fluorescence intensity from a TAOH (Sigma Aldrich) calibration curve.

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All fluorescence intensities were corrected for that of the negative control (0 mg TiO2/L). Due to a too low signal to noise ratio,
OH generation was not measurable at relevant TiO2 concentrations in the test medium.

control daphnids lower than or equal to 10% (0 mg TiO2/L) (OECD, 2004). Each test series was conducted at least twice (n ¼ 8), except for the LL experiment in undiluted ISO medium with NM 101, which was conducted only once (n ¼ 4).

2.3.2. DMPO (5,5-dimethyl-1-pyrroline N-oxide) method All TiO2 stock suspensions were measured via electron paramagnetic resonance (EPR) spectroscopy (n ¼ 3) to determine the generation of free and surface adsorbed
OH radicals, valance band
holes and O2 after five minutes irradiation with full spectrum light (315e800 nm, Natural Light, Former repti GLO 2.0 compact, 50 W, Exo Terra) according to Lipovsky et al. (2012). The following EPR settings were used for all measurements that were performed at room temperature: magnetic field: 3365 G, sweep width: 100 G, scan time: 30 s, number of scans: 3, modulation amplitude: 2.000 G, receiver gain: 1000. Quantification was carried out on first derivation of the EPR signal of the DMPOeOH quartet as an average of total amplitudes in arbitrary units (AU). For quality control dH2O þ DMPO was checked as blank value. As for the TAOH experiment, measurements at relevant TiO2 concentrations were not reliable.

2.5. Statistical analysis

2.4. Phototoxicity experiments 2.4.1. Experimental test set up (light sources) A fluorescent tube (Osram Lumilux cool white, HO 49W/840) which was enclosed by a polycarbonate cover was used as light source for the Daphnia sp. acute immobilisation tests with laboratory light (LL). It's spectrum (Fig. S-2) shows two small peaks in the UV range (313 nm and 365 nm). However, these wavelengths are absorbed by the polycarbonate cover, absorbing wavelengths  czyk et al., 2010). smaller than 390 nm (Djenane et al., 2001; Ogon Phototoxicity experiments with simulated solar radiation (SSR) were irradiated with the mercury vapor lamp described in Section 2.3.1. 2.4.2. Test organism One daphnid (Daphnia magna Straus, clone 5) per test beaker was cultured in 100 ml Elendt M4 medium in a climate chamber (20 ± C) with a photoperiod of 16 h light and 8 h dark (OECD, 2004). Daphnids were fed with Desmodesmus subspicatus (0.2 mg C/daphnid) three times a week and the medium was renewed once a week. 2.4.3. Daphnia sp. acute immobilisation test (OECD 202) Acute toxicity tests were performed with <24 h old neonates of Daphnia magna (five per test beaker) according to the OECD guideline 202 (48 h exposure duration) (OECD, 2004). No additional feeding occurred before the experiment. For each TiO2 material parallel test series were run with either LL or SSR under a 16 h light/ 8 h dark regime. Immobility of daphnids was monitored after 24 h and 48 h of exposure. Each test series consisted of five or seven treatment groups with different TiO2 concentrations in diluted ISO medium of 0.08e5 mg/L (SSR) and 0.6e50 mg/L (LL) for the nanomaterials and 0.6e50 mg/L for NM 100 (both SSR and LL). In tests with undiluted ISO medium NM 101 was tested at concentrations of 1.28e50 mg/L (SSR) and 12e100 mg/L (LL). NM 102 concentrations of 0.21e50 mg/L in SSR and LL experiments were tested. One control (0 mg TiO2/L) was tested in each test series. All treatment groups and controls consisted of four replicates. The desired test concentrations were obtained as described in Section 2.1 by using either undiluted (only nanomaterials; ISO medium composition see Tables Se6) (ISO 6341, 2012) or 10-fold diluted ISO medium (all TiO2 materials) as test medium. Tests performed in 10fold diluted ISO medium and undiluted ISO medium were valid according to the OECD guideline 202, showing an immobility of

Ecotoxicity data were statistically analyzed with ToxRat® Professional (version 2.10, ToxRat solutions GmbH). Concentration response functions were fitted to the data using probit analysis using linear maximum likelihood regression. The median effective concentration (EC50) was calculated from this function. Significant differences to the control (p < 0.05) were determined using Fisher's Exact Binominal Test with Bonferroni Correction. Significant differences between the treatment groups of the photoactivity experiments, DLS, ELS and sedimentation experiments were determined using student-t test (two sided, p < 0.05). 3. Results and discussion 3.1. Phototoxicity of TiO2 materials to Daphnia magna Phototoxicity tests revealed that toxicity of all tested TiO2 materials was significantly enhanced when organisms were simultaneously exposed to the TiO2 materials and SSR in comparison to exposures under LL (Fig. 4AeF). Hence, our study gives further evidence that standard Daphnia sp. acute immobilization tests underestimate the environmental hazard associated with nanoand non-nanoscale TiO2 particles (Clemente et al. 2014; Mansfield et al., 2015; Li et al., 2014a,b; Ma et al., 2012a,b; Marcone et al., 2012; Amiano et al., 2012; Kim et al., 2010; Seitz et al., 2014; OECD 2004). Beyond this validation our study adds new knowledge to the area of phototoxicity of TiO2 nanomaterials to D. magna, as we investigated the influence of IS of the test medium and particle size on phototoxicity. Our results reveal that the nanomaterials NM 102 and NM 101 were more phototoxic to D. magna than the bulk material NM 100, based on the determined EC50 values (SSR, Tables Se2): 0.5, 1.3, and 3.9 mg/L (0.43e0.65 mg/L, 0.61e3.66 mg/L and 0.16e40.73 mg/L, lower-upper 95% confidence limits (CL)). Furthermore, we found that phototoxicity did not inversely correlate with the PPS of nanoTiO2. In contrary, the intermediate sized TiO2 material NM 102 (EC50: 0.53 mg/L) was found to have the highest phototoxicty followed by the smaller sized nanomaterial NM 101 (EC50: 1.3 mg/L) and the bulk material NM 100 (EC50: 3.9 mg/L, Tables Se2). Hence, our results indicate that the extent of phototoxicity of the tested TiO2 materials must be additionally triggered by other TiO2 properties. Wang et al. (2010) and Albanese and Chan (2011) found smaller nanomaterial agglomerates to be more bioavailable and toxic than larger ones (Wang et al., 2010; Albanese and Chan, 2011). Our DLS results demonstrate that the most phototoxic particle NM 102 formed the largest agglomerates (HD: 625.0 ± 43.5 nm; polydispersity index (PDI): 0.31 ± 0.06), whereas the least toxic particle NM 100 formed the smallest agglomerates in the stock suspension (HD: 260.9 ± 9.3 nm; PDI: 0.19 ± 0.02, Fig. 2B,C). Furthermore, at test initiation (t0) NM 101 (HD: 752.5 ± 36.8 nm; PDI: 0.33 ± 0.01) formed larger agglomerates in the test medium in comparison to the less toxic NM 100 (HD: 303.1 ± 49.0 nm; PDI: 0.27 ± 0.06, Fig. 2A,B). Agglomeration and subsequent sedimentation of NM 102 in the test medium was so pronounced that we were not able to measure its HD in the test medium. Our DLS results do not confirm the hypothesis mentioned above and show that phototoxicity was not triggered by the degree of agglomeration in the test medium. Bozich et al. (2014) found that positively charged Au

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Fig. 2. Hydrodynamic diameters (HD) and zeta potentials (ZP) of NM 101 (A), NM 100 (B) and NM 102 (C-1, C-2) in the stock (S-SP 1 g/L, polydispersity index (PDI) nano-TiO2 and bulk: 0.31e0.39 and 0.15e0.19, n ¼ 8), working (W-SP 100 and 10 mg/L, n ¼ 3) and test suspensions (10 fold diluted ISO medium, n ¼ 3) at different time points (0, 24, 48 h). Measurements of NM 102 in the test suspension were not valid because of strong sedimentation of the particles (data not shown). Error bars represent standard deviations on the mean of at least three independent replicates. Asterisks indicate significant differences to the S-SP and circles indicate differences compared to the time point t0 of the specific test suspension (p < 0.05).

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pH of the test medium (6.14e7.84) was higher than the IEP of both nanomaterials, explaining why we determined a negative ZP for both particles in the test medium (Fig. 2A, Tables Se3). The different IEP may be caused by the fact that the percentage of surface atoms/molecule increases significantly, when particle size decreases. Except for NM 101 at high concentrations (EC50: 79.5 mg/L, Tables Se2), effects on the mobility of D. magna only occurred when particles generated ROS, measured either as free
OH radicals (measured by the TA method), or the sum of specific ROS
(measured by the DMPO method), including O2 , free and surface adsorbed
OH radicals and valance band holes. This was for all TiO2 materials only the case under SSR (Fig. 3AB). Thus, we confirm that radical formation is the main factor impacting phototoxicity of the tested nano and macro sized TiO2 materials to D. magna (Kim et al.,

N M

nanoparticles were more toxic to D. magna than negatively charged ones and assumes that this was caused by a higher affinity of the positive charged particles to negatively charged cell surfaces of D. magna (Bozich et al., 2014). For NM 102 in contrast to the other two particles, a positive ZP in the stock suspension was measured (12.2 ± 1.1 mV, Fig. 2 C-2). However, negative ZP of all particles in the test medium were documented (Fig. 2A,B, Tables Se3). Thus, differences in toxicity cannot be explained by an enhanced adsorption of NM 102 to D. magna caused by a positive ZP. NM 102 was found to have a positive ZP in its stock suspension, because the pH of the stock suspension (5.78) was lower than the reported isoelectric point (IEP) of NM 102 (6.00) (Cotogno et al., 20 14). In contrast, the pH of the NM 101 suspension (6.19) was higher than its reported IEP of 5.3e5.7, resulting in a negative ZP (Cotogno et al., 20 14; von der Kammer et al., 2010; Ramakrishnan et al., 2012). The

Fig. 3.
OH radical formation (TA method) and the sum of the measured ROS (DMPO method) in TiO2 suspensions (1 g/L) of NM 101, NM 102 and NM 100 measured with the TA fluorescence method (A) and DMPO method (B) after irradiation with a full spectrum lamp. Error bars represent standard deviations on the mean of the replicates of one experiment (n ¼ 3). Asterisks/circles indicate significant differences between the non-irradiated/irradiated treatment groups and the non-irradiated/irradiated control (p < 0.05). Crosses mark significant differences between the irradiated NM 102 and NM 101 as well as NM 100 treatment groups (p < 0.05).

C NM 100 SSR 140 24 h * * * ° ° ° 120 48 h 100 80 60 40 control 20 0 0.1 1 10 100 -20 NM 100 concentration (mg/L)

Immobility (%)

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E NM 102 LL 140 24 h 120 48 h 100 80 60 40 control 20 0 0.1 1 10 100 -20 NM 102 concentration (mg/L)

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Immobility (%)

864

F NM 100 LL 140 24 h 120 48 h 100 80 60 40 control 20 0 0.1 1 10 100 -20 NM 100 concentration (mg/L)

Fig. 4. Immobility (%) of Daphnia magna exposed to NM 101, NM 102, and NM 100 with either simulated solar radiation (SSR; A, B, C) or with laboratory light (LL; D, E, F) in 10-fold diluted ISO medium. Error bars represent standard deviations on the mean of the replicates from at least two independently conducted experiments. Circles (24 h of exposure) and asterisks (48 h of exposure) indicate significant differences from the control (p < 0.05).

2014; Ma et al., 2012b). The photoactivity pattern measured by the TA method (NM 101 > NM 102 > NM 100, Fig. 3A) did not explain the phototoxicity pattern observed in the present study (NM 102 > NM 101 > NM 100, Fig. 4AeC, Tables Se2). Thus, our study shows that phototoxicity was not only caused by the amount of free
OH radicals. With the DMPO method we determined a different photoactivity pattern: NM 102 > NM 101 ¼ NM 100 (Fig. 3B). The different induction patterns observed with the TA and DMPO method in our study may reflect that TA can only react with free
OH radicals and DMPO can additionally react with surface attached

OH radicals, valence band holes and O2 generated by TiO2 (Nosaka et al., 2003). Thus, in our experiments it appears that the formation of free
OH radicals increased with a decrease in primary particle size (TA experiment), whereas the sum of the measured ROS was highest at an intermediate particle size of 20e25 nm (NM 102; DMPO experiment). Besides studies demonstrating that the TiO2 photoactivity increases as particle size decreases (Grela and Colussi, 1996; Jang et al., 2001; Xu et al., 1999), studies exist indicating that photoactivity of TiO2 materials was highest for intermediate nano-sized anatase TiO2 particles (Allen et al., 2008; Almquist and Biswas, 2002; Wang et al., 1997). Almquist and Biswas (2002) compared the photoactivity of anatase TiO2 particles with a size range between 5 and 165 nm with each other and observed an optimal

anatase crystal particle size of around 25 nm. They suggest that for particles smaller than 25 nm, photoactivity depends more on optical and electronic properties including light absorption and scattering efficiencies and charge-carrier dynamics, which strongly depend on particle size. In contrast, for particles greater than 25 nm, photoactivity depends more on the surface area available for redox reactions. The induction pattern observed with the DMPO method in the present study reflects the findings described by Almquist and Biswas (2002). The photoactivity pattern which was determined by means of the DMPO method (NM 102 > NM 101 ¼ NM 102, Fig. 3B) helps to explain the observed phototoxicity pattern: Phototoxicity as well as photoactivity were highest for the intermediate sized TiO2 material NM 102. Although NM 101 and NM 100 exhibited a comparable photoactivity per mass, NM 101 was more phototoxic than NM 100. Considering that nano-TiO2 was shown to adsorb to daphnids and that NM 101 has a much larger surface area than NM 100 (Tables Se1), the daphniaeparticle interaction area might have been much larger than for NM 100, resulting in a more pronounced phototoxicity (Dabrunz et al., 2011). This hypothesis has to be validated by measuring the reactive surface areas of the TiO2 materials in the test medium by means of nanoparticle tracking analysis, as shown by Seitz et al. (2014). The importance of a close contact between daphnids and TiO2 materials becomes obvious when it is considered that
OH radicals have very short lifetimes

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, 1997) enabling them only to diffuse around (about 10 ms) (Hoigne 2e6 nm from their site of origin (Roots and Okada, 1975; Sanner and Pihl, 1969). Here they can cause toxic effects, by oxidizing components of the carapace, e.g., polyunsaturated phospholipids. Such molecules are known to be susceptible to ROS resulting in lipid peroxidation (Maness et al., 1999), which causes oxidative stress (Luo et al., 2006). Ma et al. (2012a,b) also suggested that ROS related phototoxicity of nano-TiO2 to D. magna occurred externally, most probably on the surface of D. magna. Likewise, several studies with Escherichia coli demonstrate that the extent of phototoxicity of nano-TiO2 is closely linked to its adsorption to their cell membranes (Maness et al., 1999; Cho et al., 2005; Gogniat et al., 2006). Moreover, Cho et al. (2005) proved that adsorbed
OH radicals and valence band holes mainly impact the phototoxicity of nano-TiO2 to E. coli. We suggest that the daphniaeparticle interaction area is an important factor influencing the phototoxicity of TiO2 materials. The same was found for anatase TiO2 particles smaller than 100 nm in absence of UV irradiation. (Seitz et al., 2014). Thus, we propose that phototoxicity is a function of the daphniaeparticle interaction area and the anatase TiO2 particle's potential to generate a combination of different ROS, which both depend on particle size. Recently, in the EU and the U.S. there is an ongoing debate whether the bulk and different nano forms of one substance have to be registered and tested separately or not (Aitken et al., 2006; Sun et al., 2014). Our study gives evidence that it is important to consider physicochemical properties such as primary particle size in the environmental hazard assessment of TiO2 materials. Furthermore, we suggest that phototoxicity of the TiO2 materials is not only caused by the generated free
OH radicals, but also by
valence band holes, surface adsorbed
OH radicals and O2 measured by EPR. Thus, the potential of the materials to generate specific ROS, as was measured by EPR, can be used as an indicator for the phototoxicity of TiO2 materials to D. magna. Further studies are necessary to validate our hypothesis. We further examined whether the ionic strength (IS) of the test medium has an influence on the phototoxicity of TiO2 nanomaterials to daphnids by testing them in 10-fold diluted (865 mM) and in undiluted ISO medium (8653 mM). The SSR induced toxicity of NM 101 and NM 102 was less pronounced in ISO (EC50 48 h: 2.9 mg/L and 1.1 mg/L, Fig. S-3A and B; Tables S-4) than in diluted ISO medium after 48 h of exposure (EC50 48 h: 1.28 and 0.53 mg/L, Fig. 4A and B; Tables Se2). We suggest that, in accordance with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the higher ionic strength of the ISO medium induced an enhanced agglomeration, as well as sedimentation and thus a lower toxicity of the nanoparticles compared to those in diluted ISO medium (Derjaguin and Landau, 1941; Verwey and Overbeek, 1948). Our results confirm that the IS of the medium is a further factor influencing the phototoxicity of TiO2 nanomaterials to D. magna. However, it has to be considered that further research is necessary to understand stress effects when using diluted ISO medium. In conclusion, our study demonstrates that physico-chemical properties such as the primary particle size, and water chemical parameters such as IS of the test medium influence the phototoxicity of TiO2 nanomaterials to Daphnia magna. 3.2. Environmental relevance of TiO2 phototoxicity We measured the exposure concentration and investigated whether the sedimentation behavior differed between the tested TiO2 particles. This was done by quantifying the TiO2 concentration at one nominal TiO2 concentration (1.3 mg/L) for each particle in the upper water phase of the test medium during the course of the experiment (0 h and 48 h) by inductively coupled plasma optical emission spectrometry. These results revealed that sedimentation

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of particles started within the first 5e10 min after application, resulting in measured concentrations of the aqueous phase of only around 10e20% of the nominal concentration. Furthermore, the initial sedimentation was more pronounced the larger the PPS was (Fig. 1F). This finding suggests that EC50 values based on the measured TiO2 concentrations in the upper water phase would be around 80e90% lower than those based on nominal concentrations. Thus, phototoxicity of TiO2 materials might have been even higher when effects would have been based on measured than on nominal concentrations. However, daphnids do not stay only in the upper water phase, they show a vertical migration through which they can regularly dip into the nano-TiO2 sediment which obviously formed on the bottom of the beakers (Dodson et al., 1997). To evaluate the environmental risk of the tested TiO2 materials, further research is necessary to clarify whether the particles in the upper water phase or those at the bottom of the test vessel were mainly contributing to the observed phototoxicity. For this, phototoxicity experiments should be additionally performed in a flow through system, as described by Seitz et al. (2013). Furthermore, phototoxicity of nano-TiO2 was shown to depend on the UV dosage and the amount of natural organic matter in natural waters (Li et al., 2014a; Ma et al., 2012b; Tong et al., 2013a; Morris et al., 1995). Therefore, it is necessary to additionally consider the environmental conditions to obtain a complete risk assessment of TiO2 materials (Li et al., 2014a). Acknowledgment The study was funded by the Federal Environment Agency Germany within the framework of the UFOPlan 2010 of the Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety Germany (FKZ 3710 65 413, authors are responsible for the content of the manuscript). Access to nanoparticles characterization (XRD and BET) was provided at the Natural History Museum London within the QualityNano scheme funded by the European Commission under FP7 Capacities Programme Grant Agreement No: 262163. XRD was performed with the help of Dr. Jens Najorka. TEM analyses were carried out at the Ernst RuskaCentre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich with the help of Dr. Marc Heggen. Spectrometer measurements were carried out at the Federal Institute for Materials Research and Testing (BAM) in Berlin with the help of the PhD student Anne-Kathrin Barthel. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2015.10.035. References Aitken, R.J., Chaudhry, M.Q., Boxall, A.B.A., Hull, M., 2006. Manufacture and use of nanomaterials: current status in the UK and global trends. Occup. Med-Oxford. 56 (5), 300e306. Albanese, A., Chan, W.C.W., 2011. Effect of gold nanoparticle aggregation on cell uptake and toxicity. ACS Nano. 5 (7), 5478e5489. Allen, N.S., Edge, M., Verran, J., Stratton, J., Maltby, J., Bygott, C., 2008. Photocatalytic titania based surfaces: environmental benefits. Polym. Degrad. Stab. 93 (9), 1632e1646. Almquist, C.B., Biswas, P., 2002. Role of synthesis method and particle size of nanostructured TiO2 on its photoactivity. J. Catal. 212 (2), 145e156. Amiano, I., Olabarrieta, J., Vitorica, J., Zorita, S., 2012. Acute toxicity of nanosized TiO2 to Daphnia magna under UVA irradiation. Environ. Toxicol. Chem. 31 (11), 2564e2566. Bozich, J.S., Lohse, S.E., Torelli, M.D., Murphy, C.J., Hamers, R.J., Klaper, R.D., 2014. Surface chemistry, charge and ligand type impact the toxicity of gold nanoparticles to. Daphnia magna. Environ. Sci. Nano. 1 (3), 260e270. Brunauer, S., Emmett, P.H., Teller, E., 1938. Adsorption of gases in multimolecular

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