Linking the Physicochemical Properties of Calcined Titania Nanoparticles with Their Biocidal Activity

Titanium dioxide nanoparticles (nTiO2) show biocidal activity when exposed to UV illumination. Modification of their physical properties can expand their photoresponse region toward visible light. In this study, such modification was made through a sol-gel synthesis followed by calcination at a range of temperatures (250–900 ◦C), generating a series of nTiO2 particles with different crystal phases, sizes, porosities, zeta potentials, and BET surface areas. The unique properties of nTiO2 were linked to their toxicity to the marine bacterium, Vibrio fischeri. A modified “Flash” high-through put assay was used to test the viability of these marine organisms after short term (15–60 min) exposure under visible light only to the individual groups of nTiO2 (500–2000 μg/mL). Linear regression analysis indicated that across all concentrations and time points, high biocidal activity correlated with the amorphous and anatase crystal phases, high BET surface area, high pore volume and small crystal size. The linkage between physicochemistry and nanotoxicity would be helpful for future design of more efficient and sustainable nTiO2.

Applications using conventional nTiO 2 are restricted by its photosensitivity to UV wavelengths.Various methods (e.g., doping, surface coating, calcination) are used to decrease the band-gap energy of nTiO 2 which increases its photosensitivity to visible/near visible radiation wavelengths [23][24][25][26][27].Although these procedures often create more sustainable and effective materials, their toxic threat to the environment and "at-risk" species must be addressed.

Synthesis and Physicochemical Characterization
The procedures to synthesize and characterize the nTiO 2 in this study have been published previously [41].Briefly, titanium tetraisopropoxide (TTIP, Sigma-Aldrich, St. Louis, MI, USA) as a TiO 2 molecular precursor was added to isopropanol (i-PrOH, Fisher Inc, Waltham, MA, USA) and water.The molar ratio of the H 2 O/i-PrOH/TTIP sol was 100/30/1.After hydrolysis and condensation reactions under vigorous stirring, TiO 2 particles were formed.They were recovered by filtration and purified by refluxing with acetonitrile (Fisher Inc) several times.Finally, they were dried at room temperature for 24 h.The resulting "as synthesized" (as-syn) nTiO 2 particles were calcined at temperatures of 250-900 • C in a furnace (Paragon HT-22-D) to transform them to crystal phase TiO 2 particles.
The crystal size of TiO 2 was determined by using the Scherrer equation from the XRD peak broadening analysis at (101) obtained using a Kristalloflex D500 diffractometer (Siemens, Munich, Germany) with Cu Kα (λ = 1.5406Å) radiation.Its BET surface area and pore volume were measured based on nitrogen adsorption and desorption isotherms using Tristar 3000 analyzer (Micromeritics, Norcross, GA, USA).The zeta potential of nTiO 2 was immediately measured using a ZetaSizer nano ZS (Malvern Inc., Southborough, MA, USA) in 10-20 µg/mL suspensions of photobacterium marine broth (Carolina Biological Supply, Burlington, NC, USA) after ultra-sonication (see below).The data (4 measures) were collected and calculated into zeta potentials using the Smoluchowski equation [42,43].Averages of zeta potentials were analyzed using a one-way ANOVA followed by a Tukey's post-analysis and graphed using Graph Pad Prism 5.An attempt was made to measure particle size distribution using dynamic light scattering (DLS).This technique measures the diffusion of particles moving under Brownian motion, and converts this to size and size distribution using the Stokes-Einstein relationship.However, a stable solution of suspended particles is necessary for valid measurement of Z-averages (d.nm), (i.e., the average diameter of the particles).Because of the high osmolarity of the marine broth, the individual samples were settled out of suspension within a few minutes (below the instruments minimal time for the initial cuvette readings), preventing DLS particle size measurements.

Storage and Light Exposure of Modified nTiO 2 Samples
After the calcination process, synthesized nTiO 2 samples were stored in the dark upon receipt until used.In preparation for the plate exposures, stock suspensions of each sample were ultra-sonicated using a Daiger Ultrasonic processor (Model GE 750 watt, Sonics ® , Newtown, CT, USA) at a reduced amplitude of 20% for 5-7 min under dim fluorescence lighting.Stock concentrations (10-fold) were prepared and exposed under low fluorescent lighting to the 96 well plates containing the population-he adjusted bacteria plate (see below).The 96 well microplate, containing bacteria exposed to the various samples of modified nTiO 2 was automatically transferred into the luminometer and the chamber door closed from all visible light.The microplate remained in the dark for the remainder of the 60 min exposure.

Microorganisms and Viability Assay
Vibrio fischeri (ATCC 49387) were grown in photobacterium broth (chemical composition listed in reference [44]) on agar slants in test tubes.Their log growth was empirically determined to be 48-72 h after the initial inoculation.Photo-bacterium marine broth (10-20 mL) was added to each inoculated agar tube without agitation to make suspensions of the micro-organism.The micro-organisms were held in complete darkness until the time of their exposure (see below) which was done under a dimmed visible (fluorescence) lighting.Suspensions of bacteria (200 µL) were transferred to a 96 well, white culture plate (Costar Inc., Washington, DC, USA).Their luminescence signal of these untreated suspensions was first adjusted with additional broth to produce a uniform baseline emission of 800-1000 chemiluminescent units.Control readings were taken of wells containing: (broth +/− organisms; broth +/− nTiO 2 samples; broth +/− organisms +/− nTiO 2 samples).None of control treatments generated significant signal and were discounted in the calculations.For the viability testing, aliquots of the individual groups of as-syn-nTiO 2 (500-2000 µg/mL) were administered to wells containing population-corrected Vibrio fischeri at 10-fold concentrations (n = 6 well/concentration).Readings were taken <30 s after exposure and used as the (t = 0) baseline value.Reduction of luminescence was recorded at three post-exposure time intervals (t = 15, 30, and 60 min) using a LMAX II 384 luminescent plate reader (Molecular Device, Sunnyvale, CA, USA) and the results were used to measure loss of viability.Each assay was replicated 4-6 times and representative data presented.Data were collected using Softpro5 (SoftPro, Raleigh, NC, USA) and transferred to Excel software where they were normalized to their individual baseline (t = 0).Linear correlation R 2 values were calculated and plotted against individual physicochemical characteristics using Excel software.

Transmission and Scanning Electron Microscopy
For transmission electron microscopy (TEM) examination of nTiO 2 , samples were sonicated (2510R-DH, Branson Ultrasonics, Danbury, CT, USA) in HPLC grade methanol (Pharmco, Brookfield, CT, USA) for 5 min, transferred to a carbon coated 200 mesh, copper grid (EMS), and photographed using a JEM-2010F (JEOL) high resolution TEM (HR-TEM) with field emission of 200 kV.The diameter of particles was measured using ImageJ software (National Institute of Health, New York, NY, USA).For biological samples, the bacteria were exposed in Eppendorf 5 mL tubes for 1 h, pelleted in marine broth, and washed several times in warm marine broth to remove all non-internalized particles.The pellets were fixed overnight in cold cacodylate-buffered 4% glutaraldehyde (Poly Scientific, Bayside, NY, USA) and processed using standard procedures.For scanning electron microscopy (SEM), samples were exposed to the individual as-syn-nTiO 2 samples, pelleted and fixed for 2 h in cold cacodylate-buffered 4% glutaraldehyde (Poly Scientific, Bayside, NY, USA).They were then transferred to a 0.4 µm nuclear pore filter and sputter-coated with 50 Angstrom gold-palladium before the examination with a JEOL SEM (JSM-5900 LV).

Physicochemical Properties
Table 1 summarizes the characterization of nTiO 2 , as reported elsewhere [41].The as-syn-nTiO 2 -100 sample had the largest BET surface area of 482 m 2 /g and pore volume of 0.659 cm 3 /g.Increasing the calcination temperatures decreased the BET surface area and pore volume as expected [45,46].Increasing the calcination temperature increased the crystal size and shifted amorphous, non-photoactive nTiO 2 to anatase phase and subsequently to rutile phase.The zeta potential values of as-syn-nTiO 2 measured in 500 µg/mL marine broth suspensions were all negatively charged (−24 to −28 mV) at these experimental conditions.The measurement of aggregate sizes by DLS was prevented due to rapid precipitation of each nTiO 2 suspension in the highly osmolar photobacterium, dehydrated marine broth.

Transmission Electron Microscopy
TEM was used to examine the effects of calcination temperatures on particle size and crystal phase as shown in Figure 1.The crystal size increased when as-syn-nTiO 2 was calcined at higher temperatures.ImageJ software determined the sizes of the particles, calcined at 250, 500 and 700 • C to be 5.7 ± 1.5 nm, 14.0 ± 2.9 nm and 47.5 ± 21.9 nm, respectively.

Physicochemical Properties
Table 1 summarizes the characterization of nTiO2, as reported elsewhere [41].The as-syn-nTiO2-100 sample had the largest BET surface area of 482 m 2 /g and pore volume of 0.659 cm 3 /g.Increasing the calcination temperatures decreased the BET surface area and pore volume as expected [45,46].Increasing the calcination temperature increased the crystal size and shifted amorphous, nonphotoactive nTiO2 to anatase phase and subsequently to rutile phase.The zeta potential values of assyn-nTiO2 measured in 500 μg/mL marine broth suspensions were all negatively charged (−24 to −28 mV) at these experimental conditions.The measurement of aggregate sizes by DLS was prevented due to rapid precipitation of each nTiO2 suspension in the highly osmolar photobacterium, dehydrated marine broth.

Transmission Electron Microscopy
TEM was used to examine the effects of calcination temperatures on particle size and crystal phase as shown in Figure 1.The crystal size increased when as-syn-nTiO2 was calcined at higher temperatures.ImageJ software determined the sizes of the particles, calcined at 250, 500 and 700 °C to be 5.7 ± 1.5 nm, 14.0 ± 2.9 nm and 47.5 ± 21.9 nm, respectively.HR-TEM was also used to examine the effect of calcination temperature on crystal size and crystal phase of the samples as shown in Figure 2. The as-syn-nTiO 2 -100 sample (Figure 2A) exhibited an amorphous structure while the sample calcined at 500 • C (Figure 2B) showed crystals of 15-20 nm sizes and lattice spacing of 0.35 nm which corresponds to the (101) plane of anatase phase.As-syn-nTiO 2 calcined at 700 • C (Figure 2C) showed well-defined rutile crystals with sizes of 40-50 nm.The measured lattice spacing was 0.32 nm, corresponding to the (110) plane of rutile phase.
HR-TEM was also used to examine the effect of calcination temperature on crystal size and crystal phase of the samples as shown in Figure 2. The as-syn-nTiO2-100 sample (Figure 2A) exhibited an amorphous structure while the sample calcined at 500 °C (Figure 2B) showed crystals of 15-20 nm sizes and lattice spacing of 0.35 nm which corresponds to the (101) plane of anatase phase.As-syn-nTiO2 calcined at 700 °C (Figure 2C) showed well-defined rutile crystals with sizes of 40-50 nm.The measured lattice spacing was 0.32 nm, corresponding to the (110) plane of rutile phase.SEM was used to examine the control and treated microorganisms as presented in Figure 3.The control Vibrio fischeri (Figure 3A) appeared spherical and elongated with a thick outer membrane and an occasional single flagellum projecting from one pole.In contrast, nTiO2-treated organisms (Figure 3B) were more variable in appearance, with normal bacteria interspersed with rounded or overtly disrupted organisms.The seemingly normal organisms projected multiple filamentous extensions to each other and to the damaged Vibrio fischeri.Cell walls of the affected bacterium appeared damaged in spots which was followed by a breakdown of the cell membrane, cell rounding and the eventual leakage of their contents.

Viability Studies
Reduction of luminescence was used as an index of viability and recorded in a dose-response fashion as early as 15 min post-exposure as shown in Figure 4. Data were normalized to their individual post-exposure values at t = 0. Viability data indicated that the toxicity of the nTiO2 series corresponded to their calcination temperatures and a simple rank order indicated that high biocidal toxicity related inversely to higher calcination temperatures (i.e., small crystals demonstrate high biocidal toxicity) (data not shown).SEM was used to examine the control and treated microorganisms as presented in Figure 3.The control Vibrio fischeri (Figure 3A) appeared spherical and elongated with a thick outer membrane and an occasional single flagellum projecting from one pole.In contrast, nTiO 2 -treated organisms (Figure 3B) were more variable in appearance, with normal bacteria interspersed with rounded or overtly disrupted organisms.The seemingly normal organisms projected multiple filamentous extensions to each other and to the damaged Vibrio fischeri.Cell walls of the affected bacterium appeared damaged in spots which was followed by a breakdown of the cell membrane, cell rounding and the eventual leakage of their contents.HR-TEM was also used to examine the effect of calcination temperature on crystal size and crystal phase of the samples as shown in Figure 2. The as-syn-nTiO2-100 sample (Figure 2A) exhibited an amorphous structure while the sample calcined at 500 °C (Figure 2B) showed crystals of 15-20 nm sizes and lattice spacing of 0.35 nm which corresponds to the (101) plane of anatase phase.As-syn-nTiO2 calcined at 700 °C (Figure 2C) showed well-defined rutile crystals with sizes of 40-50 nm.The measured lattice spacing was 0.32 nm, corresponding to the (110) plane of rutile phase.SEM was used to examine the control and treated microorganisms as presented in Figure 3.The control Vibrio fischeri (Figure 3A) appeared spherical and elongated with a thick outer membrane and an occasional single flagellum projecting from one pole.In contrast, nTiO2-treated organisms (Figure 3B) were more variable in appearance, with normal bacteria interspersed with rounded or overtly disrupted organisms.The seemingly normal organisms projected multiple filamentous extensions to each other and to the damaged Vibrio fischeri.Cell walls of the affected bacterium appeared damaged in spots which was followed by a breakdown of the cell membrane, cell rounding and the eventual leakage of their contents.

Viability Studies
Reduction of luminescence was used as an index of viability and recorded in a dose-response fashion as early as 15 min post-exposure as shown in Figure 4. Data were normalized to their individual post-exposure values at t = 0. Viability data indicated that the toxicity of the nTiO2 series corresponded to their calcination temperatures and a simple rank order indicated that high biocidal toxicity related inversely to higher calcination temperatures (i.e., small crystals demonstrate high biocidal toxicity) (data not shown).

Viability Studies
Reduction of luminescence was used as an index of viability and recorded in a dose-response fashion as early as 15 min post-exposure as shown in Figure 4. Data were normalized to their individual post-exposure values at t = 0. Viability data indicated that the toxicity of the nTiO 2 series corresponded to their calcination temperatures and a simple rank order indicated that high biocidal toxicity related inversely to higher calcination temperatures (i.e., small crystals demonstrate high biocidal toxicity) (data not shown).

Linear Correlation
R 2 values were calculated on the viability studies and plotted using Excel software.Similar ranking based on R 2 values were seen at all time-points and concentrations and indicated that high toxicity was associated with large BET surface area and pore volume, and small crystal size.The highest R 2 values, correlating lethality with pore volume, BET surface area and crystal size appeared highest in the groups exposed to 500 μg/mL for 30 min and are graphically presented in Figure 5.These data indicated that high biocidal activity correlated with large BET surface area (R 2 = 0.93, −slope) and pore volume (R 2 = 0.94, −slope), and small crystal size (R 2 = 0.70, +slope).The values of R 2 for pore volume, BET surface area and crystal size after 30 min exposure to 1000 and 2000 μg/mL were calculated to be (0.84, 0.79, 0.55 and 0.66, 0.64, 0.49), respectively.

Discussion
The primary aim of these experiments was to test a set of closely related nTiO2 for their biocidal activity and relate this toxicity to their specific physicochemical properties.The marine bacterium Vibrio fischeri was chosen as the target micro-organism since nTiO2 affects a range of organisms in marine ecosystems [9,[47][48][49][50][51][52].

Linear Correlation
R 2 values were calculated on the viability studies and plotted using Excel software.Similar ranking based on R 2 values were seen at all time-points and concentrations and indicated that high toxicity was associated with large BET surface area and pore volume, and small crystal size.The highest R 2 values, correlating lethality with pore volume, BET surface area and crystal size appeared highest in the groups exposed to 500 µg/mL for 30 min and are graphically presented in Figure 5.These data indicated that high biocidal activity correlated with large BET surface area (R 2 = 0.93, −slope) and pore volume (R 2 = 0.94, −slope), and small crystal size (R 2 = 0.70, +slope).The values of R 2 for pore volume, BET surface area and crystal size after 30 min exposure to 1000 and 2000 µg/mL were calculated to be (0.84, 0.79, 0.55 and 0.66, 0.64, 0.49), respectively.

Linear Correlation
R 2 values were calculated on the viability studies and plotted using Excel software.Similar ranking based on R 2 values were seen at all time-points and concentrations and indicated that high toxicity was associated with large BET surface area and pore volume, and small crystal size.The highest R 2 values, correlating lethality with pore volume, BET surface area and crystal size appeared highest in the groups exposed to 500 μg/mL for 30 min and are graphically presented in Figure 5.These data indicated that high biocidal activity correlated with large BET surface area (R 2 = 0.93, −slope) and pore volume (R 2 = 0.94, −slope), and small crystal size (R 2 = 0.70, +slope).The values of R 2 for pore volume, BET surface area and crystal size after 30 min exposure to 1000 and 2000 μg/mL were calculated to be (0.84, 0.79, 0.55 and 0.66, 0.64, 0.49), respectively.

Discussion
The primary aim of these experiments was to test a set of closely related nTiO2 for their biocidal activity and relate this toxicity to their specific physicochemical properties.The marine bacterium Vibrio fischeri was chosen as the target micro-organism since nTiO2 affects a range of organisms in marine ecosystems [9,[47][48][49][50][51][52].

Discussion
The primary aim of these experiments was to test a set of closely related nTiO 2 for their biocidal activity and relate this toxicity to their specific physicochemical properties.The marine bacterium Vibrio fischeri was chosen as the target micro-organism since nTiO 2 affects a range of organisms in marine ecosystems [9,[47][48][49][50][51][52].

Physicochemical Effects of Calcination Temperatures
The different calcination temperatures (250-900 • C) used in the synthesis produced a series of modified materials with subtle differences in their physicochemical properties.An overview of these properties (Table 1) indicated that low calcination temperatures related to large BET surface area, large pore volume and small crystal size.HR-TEM showed that increasing the calcination temperature from 500 to 700 • C shifted the crystal phase of nTiO 2 from anatase towards rutile.Data from related studies [53][54][55] also showed that altering the calcination temperature changed the crystallographic properties of as-syn-nTiO 2 .Increasing the calcination temperatures also increased the crystal size due to sintering and growth of particles.The crystal sizes measured from the HR-TEM micrographs using ImageJ software and those given in Table 1 which were calculated from XRD data [41], differed slightly.However, both calculations indicated that larger nTiO 2 crystals were formed at higher calcination temperatures.

Biocidal Effects
The SEM micrograph shown in Figure 3B was representative of the exposed Vibrio fischeri.It suggests membrane damage and rounding of the micro-organism as its membranous network broke-down.Other studies have used TEM and biochemical analysis to describe similar effects of UV and visible light activated TiO 2 and have documented a leakage of ions and decreases in membrane fluidity in Escherichia coli (E.coli) and Staphylococcus aureus [56,57].The explanation for the differences in vulnerability of micro-organisms to nTiO 2 is unclear but in part reflects the different thickness of the cell walls of gram + /gram − bacteria [58,59].
A number of research studies have reported on the toxicity of nanoTiO 2 in various micro-organisms (e.g., E. coli, Staphylococcus aureus, Enterococcus faecalis, coliform) and fungi (Candida albicans, Aspergillus niger).These studies have primarily used UV-or visible ("doped") light-activated materials and a variety of endpoints and exposure time-points, making a comparison among studies difficult.In general, bulk and even nanoTiO 2 in most forms, show low-toxicity to organisms and even in the case of UV-activated TiO 2 , concentrations of 800 µg/mL are required to kill 100% of the organism (coliform) in polluted waters [4].This value might be compared with the biocidal effects (Figure 5A) associated with exposures to 500 µg/mL calcined TiO 2 samples.
One study [63] examined the relative phenol degradation activity in a series of nTiO 2 samples generated after calcination at 350-750 • C. Similar to our findings, they found that the materials calcined at lower temperatures showed the highest phenol degradation activity and were most sensitive to visible light.Given this, the present study represents a unique contribution to the field as it examines the biocidal activity of nine molecularly related VLA nano-TiO 2 materials.

Physicochemical Properties Influence Toxicity
The viability data demonstrated a linkage between the viability of Vibrio fischeri and those modified nTiO 2 groups with large BET surface area and pore volume, small crystal size, and amorphous-anatase crystal phases.The physicochemical properties of particles, especially nanoparticles, are fundamental to their toxicity.Among the properties thought to influence a particle's biological activation include the size, shape or topography, surface area, crystal structure, surface charge, porosity and the tendency to aggregate [64][65][66][67][68][69].Although many studies associate surface area and crystalline size of nTiO 2 with its toxicity, others relate toxicity more to the crystal structure and particle shape/topography.Numerous studies have linked small particle size with higher nTiO 2 toxicity in rodents, eco-species, cell culture, and microorganisms [13,18,64,[70][71][72][73][74][75].Relationships between particle toxicity, and the particle's size and BET surface area have been repeatedly demonstrated in inhalation toxicology studies [70,75,76].It has also been reported that nanosize nTiO 2 can damage human bronchial epithelial cells (BEAS-2B) in the absence of photoactivation [77].On a cellular lever, an ultrafine particle size is thought to facilitate movement through cellular barriers and give the particle closer proximity to vulnerable subcellular systems (e.g., nucleic acids, enzymes) [65,[78][79][80].
The crystal phase of nTiO 2 also seems to influence toxicity with anatase being more toxic to a variety of species [15,64,81,82] while other studies associate a higher toxicity and nucleic acid damage with a mixture of rutile/anatase phases [77,83].
Aggregation is also considered to be a factor in nTiO 2 toxicity since it affects particle shape, size and bioavailability [20,74,76,84].Aggregation is affected by pH, ionic strength, and ionic identity (inorganic and organic) of aqueous suspensions.The relatively low toxicity associated with rutile phase particles has been attributed to their tendency to form larger aggregates in physiological exposure vehicles [15].However, data from our study and others [76] failed to support a role for particle aggregation in nTiO 2 toxicity.In the current study, exposure of our particles in the highly osmolar marine broth resulted in an immediate agglomeration and sedimentation.Yet, the biocidal activity of these materials showed a high correlation with the particle's size and surface properties as measured on dry particles or in distilled water.One explanation for this could be that the nTiO 2 particles, although aggregated, still showed a sufficiently high fractal surface for the formation of ROS and resulting toxicity.It is possible that studying the response to aggregated nanomaterials could be as relevant as that of highly dispersed material if the aggregates retain the characteristics of material (e.g., a large specific surface area, fractal surface, and nanosize dimensions) after agglomeration.In all likelihood the biological activity of nanoparticles relates to physicochemical parameters yet to be rigorously tested in current toxicity screening studies such as redox activity, surface affinity or combinations of more conventional physicochemical properties.
A (primary) mechanism underlying the toxicity of both UV and VLA nTiO 2 centers on ROS formation [9,10].Studies have demonstrated a linear correlation between the amount of ROS generated by TiO 2 and biocidal activity.The larger surface areas of those modified TiO 2 calcined at lower temperatures might provide more absorption sites for UV and visible light and result in higher ROS formation.The larger surface area leads to high reactivity [85,86].It is reasonable to assume that the higher surface area produced by the lower temperature modified TiO 2 in our study would allow for a greater ROS generation and higher toxicity of these damaging free radicals.ROS formation is influenced by both the BET area and particle size [9,23,51,[87][88][89][90].Although this study did not directly address the formation of ROS, an association with ROS and the loss of viability of the Vibrio fischeri is suggested.At no time in the study were the bacteria nor the nTiO 2 materials exposed to UV lighting.The modified particles were exposed to dim fluorescent lighting only during their sonication, as were the Vibrio fischeri during their dosing.We are confident that the biocidal activity recorded in these micro-organisms resulted from their exposure to VLA nTiO 2 and did not result from exposure to UV light.A number of studies have addressed this phenomenon and indicate that visible light is able to photo-activate nTiO 2 and generate ROS [23,88,89,91].

Conclusions
Data generated in this study show an association between the physicochemical properties of nTiO 2 , calcined at a range of temperatures, and its biocidal activity to marine bacteria.Findings indicate that lower calcination temperatures produced larger BET surface areas and smaller crystal sizes which increased the toxicity of nTiO 2 and suggests that calcination temperature should be considered an important parameter on the modification of nTiO 2 biocidal toxicity.The commercially available, inexpensive, rapid "screen" assay used in this study can be useful to test groups of physicochemically related nanomaterials for their potential ecotoxicity risk.Altering a nanomaterials' toxicity with a relatively cost-effective technique promises to reduce its environmental burden and to develop a more sustainable nanomaterial for environmental remediation.
, A = anatase phase, R = rutile; a Calculated as the apparent size along the anatase (101) using the Scherrer equation (D = 0.9 λ/B•cosθ) where D is the average crystalline size, λ is the wavelength of X-ray radiation (0.154 nm), B is the full width at half maxima (FWHM) of the main peak of each phase, and θ is the diffraction angle; b Calculated as the ratio of anatase and rutile using X (%) = (1/[1 + 1.26 (I R /I A )]) × 100 (I A : Integrated intensity of anatase (101) and I R : Integrated intensity of rutile (110)).
, A = anatase phase, R = rutile; a Calculated as the apparent size along the anatase (101) using the Scherrer equation (D = 0.9 λ/B•cosθ) where D is the average crystalline size, λ is the wavelength of X-ray radiation (0.154 nm), B is the full width at half maxima (FWHM) of the main peak of each phase, and θ is the diffraction angle; b Calculated as the ratio of anatase and rutile using X (%) = (1/[1 + 1.26 (IR/IA)]) × 100 (IA: Integrated intensity of anatase (101) and IR: Integrated intensity of rutile (110)).

Figure 4 .
Figure 4. Reduction of luminescence was measured at 15-60 min in response to the series of as-syn-nTiO2 at 500-2000 μg/mL exposures.Higher toxicity corresponded to those nTiO2 materials dried/calcined at the lower temperatures.(A) Toxicity of nTiO2 with 500 μg/mL exposure, (B) Toxicity of nTiO2 with 1000 μg/mL exposure, and (C) Toxicity of nTiO2 with 2000 μg/mL exposure.

Figure 5 .
Figure 5. R 2 values, calculated from the 500 μg/mL, 30 min exposure, are shown.These values are representative of treatments at other times and exposure concentrations.The numbers next to "assyn" sample represent its individual calcination temperature (°C).(A) Viability for pore volumes, (B) Viability for BET surface areas, and (C) Viability for crystal sizes.

Figure 4 .
Figure 4. Reduction of luminescence was measured at 15-60 min in response to the series of as-syn-nTiO2 at 500-2000 μg/mL exposures.Higher toxicity corresponded to those nTiO2 materials dried/calcined at the lower temperatures.(A) Toxicity of nTiO2 with 500 μg/mL exposure, (B) Toxicity of nTiO2 with 1000 μg/mL exposure, and (C) Toxicity of nTiO2 with 2000 μg/mL exposure.

Figure 5 .
Figure 5. R 2 values, calculated from the 500 μg/mL, 30 min exposure, are shown.These values are representative of treatments at other times and exposure concentrations.The numbers next to "assyn" sample represent its individual calcination temperature (°C).(A) Viability for pore volumes, (B) Viability for BET surface areas, and (C) Viability for crystal sizes.

Figure 5 .
Figure 5. R 2 values, calculated from the 500 µg/mL, 30 min exposure, are shown.These values are representative of treatments at other times and exposure concentrations.The numbers next to "as-syn" sample represent its individual calcination temperature ( • C). (A) Viability for pore volumes, (B) Viability for BET surface areas, and (C) Viability for crystal sizes.