Reduced toxicity of benzethonium chloride to Escherichia coli through adsorption onto titanium dioxide nanoparticles

by Raven Waldron

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in BioResource Research - Toxicology (Honors Scholar)

Presented May 25, 2018 Commencement June 2018

AN ABSTRACT OF THE THESIS OF

Raven Waldron for the degree of Honors Baccalaureate of Science in BioResource Research - Toxicology presented on May 25, 2018. Title: Reduced toxicity of benzethonium chloride to Escherichia coli through adsorption onto titanium dioxide nanoparticles.

Abstract Approved: ______Stacey Harper

Antimicrobial agents are being increasingly used in consumer soaps and detergents, despite a lack of data demonstrating their efficacy in such products. This study investigated the nature of the interaction between different crystalline structures of titanium dioxide nanoparticles and benzethonium chloride(BTC), and explored which structure, if any, could be best used to mitigate the toxicity of BTC in various water systems. The nanoparticles used in the study were characterized using dynamic light scattering. To examine the toxicity of combinations of BTC and titanium dioxide nanoparticles, the 24-hour IC50 was determined. UV Vis spectrophotometry was used to measure cell growth in LB media containing varying concentrations of titanium dioxide nanoparticles and BTC at the IC50. For all crystalline structures and all concentrations in the supernatant treatment, there was a significant difference between cell growth in the treatment media and cell growth in BTC alone at the IC50. Growth was significantly inhibited when cells were treated with resuspended particles with BTC adsorbed onto the surface, indicating that for combinations of titanium dioxide nanoparticles and BTC found in aquatic ecosystems, special attention must be paid to organisms that spend the most time in sediment or near the bottom of bodies of water, where particles would settle out. There was no demonstrably significant difference in cell growth among different crystalline structures of nanoparticles at any concentration. However, P25, anatase, and rutile titanium dioxide nanoparticles were each capable of mitigating the toxicity of benzethonium chloride, and this finding could lead to novel methods for removing these potentially harmful chemicals from different water sources.

Keywords: nanoparticles, titanium dioxide, benzethonium chloride, antimicrobial, anatase, rutile

Corresponding e-mail address: [email protected]

ãCopyright by Raven Waldron May 25, 2018 All Rights Reserved

Reduced toxicity of benzethonium chloride to Escherichia coli through adsorption onto titanium dioxide nanoparticles

by Raven Waldron

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in BioResource Research - Toxicology (Honors Scholar)

Presented May 25, 2018 Commencement June 2018 Honors Baccalaureate of Science in BioResource Research project of Raven Waldron presented on May 25, 2018.

APPROVED:

Stacey Harper, Mentor, representing Oregon State University Department of Environmental and Molecular Toxicology

Kate Field, Committee Member, representing Oregon State University BioResource Research

Davida Brown, Committee Member, representing George Fox University Department of Chemistry

Toni Doolen, Dean, Oregon State University Honors College

I understand that my project will become part of the permanent collection of Oregon State University Honors College. My signature below authorizes release of my project to any reader upon request.

Raven Waldron, Author ABSTRACT

Antimicrobial agents are being increasingly used in consumer soaps and detergents, despite a lack of data demonstrating their efficacy in such products. This study investigated the nature of the interaction between different crystalline structures of titanium dioxide nanoparticles and BTC, and explore which structure, if any, could be best used to mitigate the toxicity of BTC in various water systems. The nanoparticles used in the study were characterized using dynamic light scattering. To examine the toxicity of combinations of BTC and titanium dioxide nanoparticles, the 24-hour IC50 was determined and UV Vis spectrophotometry was used to measure cell growth in LB media containing varying concentrations of titanium dioxide nanoparticles and BTC at the IC50. For all crystalline structures and all concentrations in the supernatant treatment, there was a significant difference between cell growth in the treatment media and cell growth in BTC alone at the IC50. Growth was significantly inhibited when cells were treated with resuspended particles with BTC adsorbed onto the surface, indicating that for combinations of titanium dioxide nanoparticles and BTC found in aquatic ecosystems, special attention must be paid to organisms that spend the most time in sediment or near the bottom of bodies of water, where particles would settle out. There was no demonstrably significant difference in cell growth among different crystalline structures of nanoparticles at any concentration. However, P25, anatase, and rutile titanium dioxide nanoparticles are each capable of mitigating the toxicity of benzethonium chloride, and this finding could lead to novel methods for removing these potentially harmful chemicals from different water sources.

1 INTRODUCTION

Antimicrobial agents are being increasingly used in consumer soaps and detergents, despite a lack of data demonstrating the efficacy or necessity of antimicrobial agents in such products (Tan et al., 2002). As certain antimicrobial materials have been found to be harmful to organisms or to the environment, they have been taken off the market and replaced with new chemicals designed for the same purpose, rather than removed altogether. Triclosan, one such commonly used antimicrobial additive in many consumer products such as liquid soaps, detergents and hand sanitizers, was banned and removed from commercial use in rinse-off antiseptic wash products by the U.S. Food and Drug Administration in 2016 (FDA 2016). Studies citing ineffectiveness of antibacterial washes, environmental impacts, and the development of bacterial resistance contributed to the decision to remove these products from the market (Tan et al., 2002; Jones et al., 1999). However, in response to comments by industry representatives during the investigation, the FDA proposed three alternatives to triclosan: benzalkonium chloride, benzethonium chloride, and chloroxylenol, and recommended more data be collected on these materials in the coming years. This study examined one of these alternatives, benzethonium chloride

(BTC).

Titanium dioxide nanoparticles have also been widely used in products such as sunscreens, toothpastes, and white pigments in paints and coatings, and are frequently washed down drains and off skin into water sources (Suttiponpamit et al., 2011; Chen et al., 2007). Due to their variety of practical uses, they are increasingly produced and

2 therefore increasingly present in the environment and wastewater (Guzman et al., 2006;

Mueller and Nowack, 2008; Jomini et al., 2015). Due to their wide use, titanium dioxide nanoparticles are likely to be introduced as a co-contaminant with BTC in wastewater, drinking water, and aquatic ecosystems. Because of concerns surrounding the impact of antimicrobial agents in household products on naturally occurring microbes in aquatic ecosystems, it is important to understand how titanium dioxide nanoparticles present in the same water could affect the toxicity and behavior of BTC in the environment.

Titanium dioxide nanoparticles have also been explored as a means of remediating, removing or breaking down contaminants and pollutants in water systems, including antimicrobial alternatives to triclosan (Savage et al. 2005; Adesina 2004;

Chitose et al. 2003). Nanotechnology is increasingly investigated as a method of remediation due to the unique properties of materials at the nanoscale (Kung and Kung,

2004). Previous studies have confirmed that triclosan can be photocatalytically oxidized and remediated by titanium dioxide nanoparticles (Yu et al., 2006). Adsorption also plays a role in the complete neutralization of triclosan, specifically targeting the intermediates produced by photocatalysis (Yu et al., 2006). The two main mechanisms by which titanium dioxide nanoparticles could interact with antimicrobial agents are hypothesized to be: (1) photocatalysis in the presence of UV light and (2) physical adsorption onto the surface of the particles. The latter is the focus of this study.

BTC can adsorb onto the surface of titanium dioxide particles with a primary particle size of 0.29 ± 0.04 µm (Yaremeko & Petryshyn 2013). However, the specific interaction between BTC and titanium dioxide nanoparticles has yet to be investigated.

3 The increased relative surface area of nanoparticles compared to their bulk counterparts could correlate to an increased ability to adsorb materials onto their surface. In addition, special attention must be paid to the media in which particles are incubated, as different solutions can potentially affect the agglomeration and therefore total surface area of the particles in solution (Jiang et al., 2009).

Crystalline structure of titanium dioxide nanoparticles can also contribute to differences in physiochemical properties. Titanium dioxide nanoparticles can be synthesized in a variety of different structures, the most common of which are anatase, rutile, and brookite (Reyes-Coronado et al., 2008). Anatase is usually considered to be the most phocatalytically active (Diebold 2002). The most commonly used titanium dioxide nanomaterial in toxicity testing is Degussa P-25 (P25), an 80:20 mixture of anatase and rutile polymorphs. Each type of titanium dioxide nanoparticle has its own unique characteristics, which contribute to the overall behavior of particles in solution and in mixtures with other materials. The observed differences in sedimentation and aggregation kinetics for different crystalline structures could have profound impacts on the fate and transport of nanomaterials in aquatic ecosystems (Liu et al., 2011). The anatase to rutile ratio of titanium dioxide nanoparticles affects photoreactivity, agglomeration, and dispersion properties. This demonstrates that crystalline structure can indeed affect the interaction between the particles and other materials in solutions and mixtures (Suttiponpamit et al., 2011; Lui et al., 2008; Lui et al., 2011). Additionally, lead adsorption is stronger to anatase titanium dioxide nanoparticles than to anatase- rutile mixtures (Giammar et al., 2006). However, the difference in adsorption of

4 antimicrobial agents across different crystalline structures of titanium dioxide nanoparticles has yet to be examined.

Today, over 85,000 chemicals are marketed for use in industrial and commercial applications (EPA 2018). For the majority of these chemicals that do not fall under specific regulations governing the production of pesticides, cosmetics, and food, manufacturers are not required to generate data about their toxicity and impact before using them in massive quantities. Because of the vast breadth of materials entering our ecosystems, the science of toxicology must concern itself not only with the impacts of individual materials, but also the complex mixtures that are actually present in our waste water, drinking water, and the environment. Examining the way different materials interact with each other to enhance or mitigate both on and off-target effects is crucial to understanding the relevant toxicity of materials being investigated.

This study aimed to investigate the nature of the interaction between different crystalline structures of titanium dioxide nanoparticles and BTC, and explore which structure, if any, could be best used to mitigate the toxicity of BTC in water systems such as wastewater and drinking water. It also sought a better understanding of the behavior of BTC in aquatic ecosystems, where it is likely to be present as a co-contaminant with titanium dioxide nanoparticles. Such information could be beneficial in determining the safety of continuing to use BTC in consumer products.

5 METHODS

Test organisms

For the evaluation of the toxicity of antimicrobial alternatives to triclosan, a sample of Escherichia coli K-12 was purchased from Carolina Biological Supply Company

(MicroKwik culture, Burlington, NC, USA) and cultured in lysogeny broth (LB) media (10 mg/L tryptone, 5 mg/L yeast extract, and 10 mg/L sodium chloride). E. coli was plated on

LB nutrient agar for storage, then cultured and maintained in 250 mL Erlenmeyer flasks with 150 mL of culture media. Organisms were housed at 37 °C in a MaxQ 4450 incubated shaker (Thermo Fisher Scientific, Waltham, MA, USA) at low speed until they reached 24 hours of growth.

Determining the 24-hour IC50 of benzethonium chloride

Benzethonium chloride (BTC) was purchased from Tokyo Chemical Industry

America Co. LTD (Portland, OR, USA). Stock suspensions of 1000 mg/L of BTC were prepared in 10 mL of Milli-Q water and <0.5% ethanol.

The benzethonium chloride concentration that inhibits the growth of the E. coli population tested to 50% of the control after 24 hours (24-hour IC50) was determined using a modified minimum inhibitory concentration (MIC) assay. BTC stock suspensions were diluted to 100 mg/L in 10 mL of LB media and vortexed. Using a micropipette, 200

µL of control LB media (no BTC added) was dispensed into each well in the first two columns (8 cells each) of a Falcon® clear, flat bottom, 96-well culture plate (Corning Inc.,

6 NY, USA) using a micropipette (Figure 1). Then 200 µL of 100 mg/L BTC in LB media was plated in each well in the third column of the plate for a starting concentration. Serial dilutions were performed, with every well in each subsequent row of the 96-well plate containing 200 µL of LB media with half the BTC concentration of the previous row. Final set-up of the plate is pictured in Figure 1.

Figure 1. Final Minimum Inhibitory Concentration assay 96-well plate set-up. Lane 1: negative control; Lane 2: positive control; Lane 3: 100 mg/L BTC in LB media; Lane 4: 50 mg/L BTC in LB media; Lane 5: 25 mg/L BTC in LB media; Lane 6: 12.5 mg/L BTC in LB media; Lane 7: 6.25 mg/L BTC in LB media; Lane 8: 3.125 mg/L BTC in LB media; Lane 9: 1.506 mg/L BTC in LB media; Lane 10: 0.75 mg/L BTC in LB media; Lane 11: 0.353 mg/L BTC in LB media; Lane 12: 0.153 mg/L BTC in LB media.

Plates were then scanned at l=600 nm on the UV-Vis Spectrophotometer (Molecular

Devices LLC, Silicon Valley, California) using SoftMax Pro software (Version 6.2). After this background reading, each well of the 96-well plate was then inoculated with 10 µL of E. coli culture, with the exception of the negative control (no bacteria) wells. Plates were then immediately placed in complete dark conditions, to avoid photocatalysis and isolate adsorption effects, and housed at 37 °C (± 0.5 °C – look up fluctuations in the machine) in MaxQ 4450 incubated shaker (Thermo Fisher Scientific, Waltham, MA,

7 USA). After 24 hours of incubation and growth, final readings were taken on the UV-Vis spectrophotometer.

Nanoparticle characterization

Titianium dioxide nanoparticle hydrodynamic diameter (HDD) and zeta potential for each material were measured using dynamic light scattering (DLS) in both Milli-Q water (18.2 W resistivity) and LB media on a Malvern Zetasizer Nano-ZS Dynamic Light

Scatterer (Malvern Instruments, Westborough, MA). The z- average was used as a measure of agglomerate size. Additional information was gathered from the specification sheets provided by the manufacturer for each material (Table 1).

Experimental Design for Combination Toxicity Study

Anatase and rutile titanium dioxide nanoparticles were purchased from Sigma

Aldrich (St Louis, MO, USA). Aeroxide® P25 titanium dioxide nanoparticles were purchased from Evonik Degussa Corporation (Parsippany, NJ, USA). Stock suspensions of 1000 mg/L were prepared by dispersing dry nanopowder in 10 mL of ultrapure water

(Milli-Q 18.2 W resistivity) and were ultra-sonicated for 10 min at 40% intensity using a

VCX 750 Vibra-Cell sonicator (Sonics & Materials Inc., Newtown, CT, USA).

Suspensions were then vortexed (VWR Scientific Vortex Genie 2) and diluted with LB media in autoclaved 1.5 mL centrifuge tubes to a final total volume of 1 mL.

Solutions of 1 mg/L, 2.5 mg/L, 5 mg/L, 10 mg/L, and 20 mg/L of titanium dioxide combined with 3.84 mg/L (the 24-hour IC50 of BTC) of BTC were prepared using this

8 method. Centrifuge tubes were then vortexed and stored in complete dark conditions for a 24-hour incubation period.

Following incubation, titanium dioxide nanoparticles were removed from solution by centrifugation for 10 minutes at 14000 rpm. The supernatant from each sample was removed and placed in a fresh 1.5 mL centrifuge tube. Titanium dioxide nanoparticles were resuspended in 1 mL of LB media using a vortex. Samples were kept in complete darkness at all times between method steps. Supernatant solution and resuspended particle solutions were plated in Falcon® clear, flat bottom 96-well culture plates (Corning Inc., NY, USA). 200 µL of solution was pipetted into each well, with a total of 8 replicates of each sample per plate (Figure 2). Plating was done in mostly dark conditions, with low, indirect, artificial light used for visualization during plating.

Plates were then scanned at l=600 nm on the UV-Vis Spectrophotometer

(Molecular Devices LLC, Silicon Valley, CA, USA) using SoftMax Pro software (Version

6.2) to gain a preliminary reading of background absorbance from dispersed particles and the media. To eliminate confounding effects of particles or cells settling into only certain areas in each well, the well-scan setting was used, obtaining nine readings from nine different locations in each well that were then averaged by the SoftMax Pro software into one final absorbance reading. Following this “background” reading, each well was inoculated with 10 µL of E. coli culture, with the exception of the negative (no bacteria) control wells. After inoculation, UV-Vis Spectrophotometer “0-hour” readings were repeated with the same settings described previously. Plates were then immediately placed in complete dark conditions and housed at 37 °C in a MaxQ 4450

9 incubated shaker (Thermo Fisher Scientific, Waltham, MA, USA). After 24 hours of incubation and growth, final “24-hour” readings were taken on the UV-Vis spectrophotometer.

Figure 2. Final Combination Study 96-well plate set-up. Lane 1: negative control; Lane 2: positive control; Lanes 3-7: TiO2 NPs (1, 2.5, 5, 10, and 20 mg/L) and BTC Supernatant in

LB media; Lanes 8-12: TiO2 NPs (1, 2.5, 5, 10, and 20 mg/L) and BTC Resuspended Particles in LB media.

Statistics and Data Analysis

SigmaPlot version 13.0 (Systat Software, San Jose, CA, USA) was used to perform

IC50 statistical analyses. IC50s were determined using non-linear, 4-parameter sigmoidal regression. Background values were subtracted from the 0-hour and 24-hour readings to eliminate confounding effects of absorbance readings of LB media or suspended particles at l= 600 nm. Differences among bacterial growth for each titanium dioxide nanoparticle type and concentration were compared using one-way analysis of variance

(ANOVA) and Welch two-sided t-tests in R Version 3.4.1 (R Core Team 2017). Differences

10 were considered statistically significant when p ≤ 0.01. Error bars represent standard error of the mean. Box plots were visualized using ggplot2 (Wickham 2009).

RESULTS

Nanoparticle Characterization

Due to agglomerization in media, nanoparticles showed far larger diameters in water and in LB media than the primary particle size given by manufacturers (Table 1).

Characterization of Titanium Dioxide Nanoparticles

Hydrodynamic Hydrodynamic Zeta Zeta Primary Surface Material Diameter in Diameter in Potential Potential Particle Areab MQ Watera LB Mediaa in MQ in LB Sizeb (m2/g) (nm) (nm) Watera mediaa (nm)

P25 253.2 1152 19.3 -13.6 21 50 ± 15 (Degussa)

Anatase 491.4 629.3 18 -12.5 < 25 50 ± 5 (Sigma)

Rutile 626.7 2038 14.2 -11.2 < 100 50 (Sigma)

Table 1. Nanoparticle characterization showed increased diameter in water and LB media and increased agglomeration in LB media. Table displays (a) measurements collected on the Malvern Zetasizer DLS and (b) measurements reported by the manufacturer.

Due to agglomeration in LB media, rutile exhibited the largest decrease in surface area, followed by P25, then anatase. Although the zeta potential, the surface charge of all a

11 particle relative to the bulk of liquid it is immersed in, was positive in water, it was negative in LB media.

IC50 Analysis

A total of three benzethonium chloride minimum inhibitory concentration assays were run on E. coli to determine an overall IC50 value (Table 2). MIC data was plotted on a log concentration (mg/L) vs. OD 600 (optical density at l=600 nm) and analyzed using a non-linear regression (Figure 3) to determine the IC50 values for each assay. A combined analysis of these individual IC50s yielded an average 24-hour IC50 of 3.81 mg/L with a standard deviation of 0.0186. This concentration of benzethonium chloride was used in combination with titanium dioxide nanoparticles in the combination studies.

Determination of Benzethonium Chloride IC50

2 Trial IC50 value R of Non-Linear Standard Error (mg/L) Regression Line

1 3.78 0.9951 0.0336

2 3.84 0.9916 0.0514

3 3.83 0.9921 0.0495

Mean 3.81 ----- 0.0186

Table 2. Summary table of all benzethonium chloride IC50 assays.

12 Figure 3. Example 24-hour IC50 curve for one E. coli benzethonium chloride minimum inhibitory concentration assay (Trial 1). Error bars represent standard error from n=8.

Combination Study

The toxicity to E.coli of benzethonium chloride in combination with titanium dioxide nanoparticles was evaluated in the second half of the study. When combination study data was plotted on a box plot, two extreme outlying points (values more than 3 times the upper quartile) were identified in the P25 and rutile supernatant treatments

(Figure 4). These points were eliminated from all subsequent statistical analyses.

Toxicity was evaluated by exposing cells to both the BTC-titanium dioxide supernatant

(Figure 5) and resuspended titanium dioxide nanoparticles (Figure 6) separately and measuring cell growth.

13

Figure 4. E. coli cell growth when treated with BTC and P25, anatase, and rutile titanium dioxide nanoparticle supernatant and resuspended particles. Horizontal black bars represent median values for each treatment and the top and bottom of each vertical bar signifies the maximum and minimum value, excluding outliers (black dots). “Extreme outliers” were identified at 20 mg/L in both P25 and rutile supernatant treatments. Note that the range of the y-axis differs between the resuspended particles and supernatant treatments. Larger version of Figure 4 in Appendix 1.

In Welch’s Two-Sided t-tests, for all crystalline structures and all concentrations in the supernatant treatment, there was a significant difference between cell growth in the treatment media and cell growth in BTC alone at the IC50 (p<0.01, Table 3). Cell growth at 5 mg/L of resuspended anatase titanium dioxide nanoparticles was significantly higher than BTC alone at the IC50 (p<0.01, Table 3). For the same treatment at 20 mg/L, cell growth was significantly lower than BTC alone (p<0.01, Table 3). For all other titanium dioxide nanoparticle crystalline structures and all concentrations of resuspended nanoparticles, there was no significant difference between cell growth

14 with the treatment (titanium dioxide) and cell growth in BTC alone at the IC50 (p>0.01,

Table 3).

Figure 5. Cell density of E. coli after 24-hour exposure to BTC-TiO2 nanoparticle supernatant. 0 mg/L incubated titanium dioxide concentration values are BTC alone, at the IC50. Red circles are samples incubated with P25 titanium dioxide nanoparticles. Orange circles are samples incubated with anatase titanium dioxide nanoparticles. Yellow triangles are samples incubated with rutile titanium dioxide nanoparticles. Error bars represent the standard error from n=16 samples.

Some titanium dioxide nanoparticle structures showed significant differences in cell counts among different concentrations. In the P25 and rutile supernatant treatment groups, cell count differed significantly (p<0.01, Table 4) among concentrations, with a roughly decreasing trend from 1 mg/L titanium dioxide nanoparticles to 20 mg/L

15 titanium dioxide nanoparticles. In the anatase resuspended treatment, cell counts among different concentration were significantly different (p<0.01, Table 4); however, results did not follow a clear dose response relationship. Instead, cells appeared to grow most at 2.5 mg/L anatase titanium dioxide nanoparticles added to BTC.

Figure 6. Cell density of E. coli after 24-hour exposure to resuspended TiO2 nanoparticles. 0 mg/L incubated titanium dioxide concentration values are BTC alone, at the IC50. Red circles are samples incubated with P25 titanium dioxide nanoparticles. Orange circles are samples incubated with anatase titanium dioxide nanoparticles. Yellow triangles are samples incubated with rutile titanium dioxide nanoparticles. Error bars represent the standard error from n=16 samples.

In ANOVA tests for all titanium dioxide nanoparticle crystalline structures at all concentrations, cell growth in BTC-titanium dioxide supernatant was significantly higher

16 than cell growth in corresponding concentrations of resuspended nanoparticles (p<0.01,

Table 5). There was no significant difference among cell growth when treated with different crystalline structures of nanoparticles (p>0.01, Table 6) at any concentration in the supernatant treatment group except 2.5 mg/L. In the resuspended nanoparticle treatment group, cell growth showed significant difference among the three different crystalline structures tested, but only at 2.5 mg/L and 20 mg/L (Table 6).

Differences Between Combinations and BTC Alone at the IC50

Titanium Dioxide Treatment P Value for Given Concentration Structure

1 mg/L 2.5 mg/L 5 mg/L 10 mg/L 20 mg/L

P25 Supernatant 2.60 x 10-3 4.77 x 10-3 3.29 x 10-3 2.22 x 10-3 3.39 x 10-8

Anatase Supernatant 3.04 x 10-13 1.58 x 10-7 4.93 x 10-11 1.65 x 10-14 7.48 x 10-16

Rutile Supernatant 1.14 x 10-13 3.50 x 10-13 1.88 x 10-11 6.73 x 10-23 3.95 x 10-10

P25 Resuspended 2.21 x 10-1 3.60 x 10-1 2.35 x 10-1 2.69 x 10-1 9.86 x 10-1

Anatase Resuspended 4.10 x 10-1 6.15 x 10-6 1.38 x 10-1 3.29 x 10-1 1.22 x 10-7

Rutile Resuspended 2.71 x 10-2 7.83 x 10-1 7.93 x 10-1 7.44 x 10-1 2.10 x 10-2

Table 3. Statistical analysis of variance between all treatments and BTC alone. Bolded values are statistically significant.

17 Differences in Concentration Across Structure

Titanium Titanium Treatment Dioxide P Value Treatment Dioxide P Value Structure Structure

Supernatant P25 5.44 x 10-4 Resuspended P25 8.14 x 10-2

Supernatant Anatase 7.37 x 10-1 Resuspended Anatase 9.79 x 10-5

Supernatant Rutile 8.08 x 10-3 Resuspended Rutile 2.00 x 10-1

Table 4. Statistical analysis of variance among all concentrations tested within the same titanium dioxide nanoparticle treatment. Bolded values are statistically significant.

Differences Between Supernatant and Resuspended Particle Treatments

Titanium Dioxide P Value for Given Concentration Structure

1 mg/L 2.5 mg/L 5 mg/L 10 mg/L 20 mg/L

P25 1.77 x 10-13 2.31 x 10-16 1.27 x 10-13 2.50 x 10-13 5.26 x 10-11

Anatase 5.36 x 10-15 1.80 x 10-4 2.80 x 10-13 8.04 x 10-15 2.49 x 10-16

Rutile 4.08 x 10-15 8.43 x 10-14 1.03 x 10-12 2.59 x 10-16 5.54 x 10-13

Table 5. Statistical analysis of variance between supernatant and resuspended particle treatments across concentrations. Bolded values are statistically significant.

18 Differences in Crystalline Structure Across Concentrations

Treatment Concentration P Value Treatment Concentration P Value (mg/L) (mg/L)

Supernatant 1 7.06 x 10-1 Resuspended 1 1.47 x 10-2

Supernatant 2.5 1.34 x 10-6 Resuspended 2.5 7.95 x 10-11

Supernatant 5 3.63 x 10-2 Resuspended 5 6.11 x 10-1

Supernatant 10 3.32 x 10-2 Resuspended 10 6.96 x 10-1

Supernatant 20 9.34 x 10-1 Resuspended 20 8.75 x 10-5

Table 6. Statistical analysis of variance among titanium dioxide crystalline structures within nanoparticle concentration. Bolded values are statistically significant.

DISCUSSION

Due to the prevalence of antibacterials and titanium dioxide nanoparticles in consumer products, these two materials have a high potential for interaction in wastewater and aquatic ecosystems. Currently, toxicity data and models exist for each material individually, but combination studies are uncommon, and the way crystalline structure affects the behavior of titanium dioxide in adsorption of antimicrobials remains unclear. Characterization and analysis of environmentally relevant combinations of materials can provide a greater understanding of the effective toxicity of each chemical in the environment and facilitate the development of new methods to remove materials from wastewater and drinking water.

19 Co-Contaminant Effects

In this study, E. coli cell growth was measured in solutions containing antimicrobial BTC and combinations of P25, anatase, or rutile titanium dioxide nanoparticles to identify whether they could be used to mitigate the toxicity of BTC through physical adsorption. Analysis of the hydrodynamic diameter and zeta potential of the materials examined in this study provided the basis for predicting the outcome of the combination study. Based on the agglomeration of the particles, and therefore corresponding surface area for adsorption of BTC onto the particles, it was hypothesized that anatase titanium dioxide nanoparticles would most effectively remove the antibacterial from solution. Through MIC assays and UV-Vis spectrophotometry, we found that all crystalline structures of titanium dioxide nanoparticles effectively neutralized the toxicity of BTC. In previous studies, it was demonstrated that BTC adsorbed onto the surface of bulk titanium dioxide (Yaremko & Petryshyn, 2013) and the present study confirmed that this behavior holds true at the nanoscale as well. In general, resuspended nanoparticles with BTC adsorbed to their surface did not display growth significantly different than BTC alone, indicating that all BTC in solution was removed from the supernatant and instead present in the resuspended particle media.

Crystalline Structure Effect

No clear trend or significant difference in adsorption emerged when examining the differences in cell growth among different crystalline structures. Differences between P25 and anatase at 2.5 mg/L and 20 mg/L and between rutile and anatase at

20 2.5 mg/L were statistically significant. However, without a higher sample size, no definitive conclusion can be made as to why. It is possible that human error contributed to the unexpected values. These results that follow no clear trend warrant more investigation in a larger study.

Concentration Effect

We hypothesized several explanations for the lack of a dose-response relationship between toxicity and the amount of titanium dioxide nanoparticles added to the solution. First, it is possible that the materials do not follow the traditional, sigmoidal dose-response relationship at all. Second, and far more likely, only a very small amount of titanium dioxide is required to adsorb the low concentration of BTC used in this study, and the dose response curve occurs at lower concentrations than were used in this study. This would have profound impacts on the potential of titanium dioxide nanoparticles for remediation of BTC, as at lower concentrations, several of the reported toxic impacts of titanium dioxide nanoparticles are not observed (Sharma,

2009). Further study at very low concentrations of titanium dioxide nanoparticles could reveal more about this potential.

Implications for Environmental Fate and Transport

Though the data show little difference in toxicity between media containing only

BTC and media containing resuspended particles and adsorbed BTC, it is possible that the particles themselves had a toxic effect on the E. coli, contributing to the decreased

21 growth observed. Previous studies have demonstrated the toxicity of titanium dioxide nanoparticles in aquatic ecosystems (Adams et al., 2016; Sharma, 2009), though the effects on E. coli seem to be negligible at the concentrations and dark conditions (no photocatalysis) used in this particular study, with the possible exception of anatase at 20 mg/L. Despite these potential effects, the supernatant treatment data clearly showed that the presence of titanium dioxide nanoparticles with BTC in incubation led to significant increase in cell growth compared to BTC alone, indicating that adsorption of

BTC to the particle surface and its subsequent removal from solution did occur.

This result has several implications for how combinations of these materials in aquatic ecosystems must be considered in regulation and further study. First, titanium dioxide still has the potential to cause toxic effects to some organisms in the environment, and any use of it for remediation should be carefully considered and controlled. It is worth noting that even at very low concentrations, titanium dioxide nanoparticles were capable of adsorbing BTC, making it likely that high concentrations of particles would not be necessary to see remediatory effects for this specific case.

Second, for combinations of titanium dioxide nanoparticles and BTC found in aquatic ecosystems, special attention must be paid to organisms that spend the most time in sediment or near the bottom of bodies of water. While titanium dioxide nanoparticles present in these waters are likely adsorbing and removing BTC from the overlying surface waters, they have the potential to settle out in still waters and cause the same growth inhibition effects seen in the resuspended particle treatment in this study.

Furthermore, in fast-moving waters where these sediments are frequently stirred up,

22 the remediatory effects of co-contamination with titanium dioxide nanoparticles could be negated. Finally, these co-contaminants clearly interact in a way that alters the toxicity of BTC and therefore must be considered in the creation of future regulations according to its properties in a mixture, rather than an antimicrobial agent alone.

Limitations and Future Research

One limitation of this study is that it does not account for the variation in relevant parameters of natural waters. For example, the influence of organic matter present in natural waters could alter the behavior of the nanoparticles. In the environment, nanoparticles can adsorb this natural organic matter, which could decrease surface area for BTC adsorption and change the surface charge of the particles.

This has been shown to alter the fate and transport of nanoparticles in the environment

(Hyung et al., 2007; Ghosh et al., 2008). Additionally, the physical characteristics of titanium dioxide nanoparticle such as primary particle size, agglomeration, and zeta potential have been shown to change in solutions of varying pH (Suttiponpamit et al.,

2011). Overly acidic or basic waters, or those containing high amounts of other metals and ions, could cause titanium dioxide nanoparticles to behave differently in relation to

BTC.

This study produced some unclear and unexpected results in that there was no observable dose-response relationship. Though this could be indicative of the way the materials behave together, it could also be the result of the limited size of the study.

Future studies could include more samples to make any trends clearer. Additionally, in

23 utilizing UV-Vis spectrophotometry as the primary analytical method, it was impossible to distinguish whether the cells measured were alive or dead. Future study could introduce flow cytometry and live/dead stains to give a more complete picture of the ways these co-contaminants affect not only bacterial population growth as a whole, but also whether the mechanism is inhibition of mitosis, excessive death of live cells, or a combination of multiple effects.

Finally, this study examined only one effect of BTC against only one species of its intended target, E.coli. Bacteria are incredibly diverse, and different species could interact with BTC in unique ways. Bacterial communities play an important role in nutrient cycling and trophic webs of ecosystems and can, in some instances, be used as an indicator of overall ecosystem health (Lau et al., 2015). However, in order to gain an understanding of how antibacterials affect ecosystems as a whole, it is important to examine their off-target effects as well. Future studies must be done in to analyze the overall ecosystem effects of the relationship between titanium dioxide nanoparticles and BTC. Analysis through tools such as the Harper nanocosm (Wu, Harper, & Harper,

2017) could be beneficial in modeling the ways these co-contaminants behave in environmentally relevant media and multiple communities of organisms. Furthermore, adsorption is just one way that titanium dioxide nanoparticles can reduce the toxicity of

BTC. The photocatalytic effects of titanium dioxide nanoparticles on various environmental contaminants are explored in other studies (Yu et al., 2006; Diebold,

2002). Future research could examine the behavior of these two materials in

24 combination when allowed to be under UV light, rather than only in the dark conditions used to isolate adsorption effects in this study.

Without understanding the ways co-contaminants interact within a system, scientific understanding of the toxicity of chemicals in the environment will never be complete. With so many materials constantly entering the world’s sources of drinking water, effluent streams, and natural aquatic ecosystems, characterizing the interactions between multiple materials has never been more important to the study of toxicology.

P25, anatase, and rutile titanium dioxide nanoparticles are equally capable of mitigating the toxicity of benzethonium chloride, and this finding could lead to novel methods for removing these potentially harmful chemicals from different water sources.

ACKNOWLEDGEMENTS

I thank Dr. Stacey Harper, professor at Oregon State University and Primary Investigator of my lab for her mentorship during the writing of my thesis, and for serving as a strong role model of all that a woman in science can achieve. During my many years in the lab, she helped me design experiments, interpret my results, and seek opportunities for funding for my research. She encouraged me to grow in not only my research, but all my extracurricular activities and studies during my time at Oregon State, constantly pushing me to pursue more opportunities and deepen my learning. Dr. Davida Brown, researcher and professor at George Fox Univeristy, closely advised my thesis project, especially during the data collection phase. She aided in experimental design and facilitated my learning and deeper thinking about my project throughout the thesis

25 process. I also thank her for her constant support and for letting me talk her ear off about Hamilton. I thank Dr. Kate Field, the director of BioResource Research, for serving on my committee and editing my manuscript. I thank her for broadening my learning in

BRR over the past 5 years, and for her quick wit and brilliant sense of humor. Wanda

Crannell, M.S., supervisor and academic advisor of the BioResource Research program, served as a constant inspiration and source of strength and direction throughout my research experience. I cannot thank her enough. I thank Fan Wu, Lauren Crandon, and

Lindsay Denluck, Ph.D. candidates in the Harper lab at the time of this project for their constant support in bench work and interpreting my results, and for always answering my questions patiently. Their mentorship was invaluable to my understanding of the scientific process, backwards and forwards, during the course of this project. Finally, I would like to thank my friends and family who supported me emotionally, physically, and spiritually throughout the research and writing process. Without my support system, I would not be where I am today.

REFERENCES

Adams, L.K., Lyon, D.Y., Alvarez, P.J.J., 2006. Comparative eco-toxicity of nanoscale TiO2,

SiO2, and ZnO water suspensions. Water Research 40, 3527–3532.

https://doi.org/10.1016/j.watres.2006.08.004

Adesina, A.A., 2004. Industrial exploitation of photocatalysis: progress, perspectives and

prospects. Catalysis Survey Asia 8, 265–273. https://doi.org/10.1007/s10563-

004-9117-0

26 Bertani, G., 1951. Studies on lysogenesis I. J Bacteriol 62, 293–300.

Chen, X., Mao, Y., 2007. Titanium dioxide nanomaterials: synthesis, properties,

modifications, and applications. Chemical Reviews 107, 2891–959.

https://doi.org/10.1021/cr0500535

Chitose, N., Ueta, S., Seino, S., Yamamoto, T.A., 2003. Radiolysis of aqueous phenol

solutions with nanoparticles. 1. Phenol degradation and TOC removal in

solutions containing TiO2 induced by UV, gamma-ray and electron beams.

Chemosphere 50, 1007–1013.

Commissioner, Office of the. Press Announcements - FDA issues final rule on safety and

effectiveness of antibacterial soaps [WWW Document]. URL

https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm51747

8.htm (accessed 1.21.18).

Diebold, U., 2003. The surface science of titanium dioxide. Surface Science Reports 48,

53–229. https://doi.org/10.1016/S0167-5729(02)00100-0

Ghosh, S., Mashayekhi, H., Pan, B., Bhowmik, P., Xing, B., 2008. Colloidal behavior of

aluminum oxide nanoparticles as affected by pH and natural organic matter.

Langmuir 24, 12385–12391. https://doi.org/10.1021/la802015f

Giammar, D., Maus, C., Xie, L., 2006. Effects of particle size and crystalline phase on lead

adsorption to titanium dioxide nanoparticles | Environmental Engineering

Science. Environmental Engineering Science. 24:1, 1506-1518.

27 Guzmán, K.A.D., Taylor, M.R., Banfield, J.F., 2006. Environmental risks of

nanotechnology: National Nanotechnology Initiative funding, 2000-2004.

Environmental Science and Technology 40, 1401–1407.

Hyung, H., Fortner, J.D., Hughes, J.B., Kim, J.-H., 2007. Natural organic matter stabilizes

carbon nanotubes in the aqueous phase. Environmental Science and Technology

41, 179–184.

Jiang, J., Oberdörster, G., Biswas, P., 2009. Characterization of size, surface charge, and

agglomeration state of nanoparticle dispersions for toxicological studies. Journal

of Nanoparticle Research 11, 77–89. https://doi.org/10.1007/s11051-008-9446-4

Jiang, X., Manawan, M., Feng, T., Qian, R., Zhao, T., Zhou, G., Kong, F., Wang, Q., Dai, S.,

Pan, J.H., 2018. Anatase and rutile in evonik aeroxide P25: Heterojunctioned or

individual nanoparticles? Catalysis Today 300, 12-17.

https://doi.org/10.1016/j.cattod.2017.06.010

Jomini, S., Clivot, H., Bauda, P., Pagnout, C., 2015. Impact of manufactured TiO2

nanoparticles on planktonic and sessile bacterial communities. Environmental

Pollution 202, 196–204. https://doi.org/10.1016/j.envpol.2015.03.022

Jones, R.D., 1999. Bacterial resistance and topical antimicrobial wash products.

American Journal of Infectious Disease Control 27, 351–363.

Kung, H.H., Kung, M.C., 2004. Nanotechnology: applications and potentials for

heterogeneous catalysis. Catalysis Today, Heterogenous and Bio Catalysis in

Commodity and Fine Chemical Synthesis. 97, 219–224.

https://doi.org/10.1016/j.cattod.2004.07.055

28 Lan, Y., Lu, Y., Ren, Z., 2013. Mini review on photocatalysis of titanium dioxide

nanoparticles and their solar applications. Nano Energy 2, 1031–1045.

https://doi.org/10.1016/j.nanoen.2013.04.002

Lau Kelvin E. M., Washington Vidya J., Fan Vicky, Neale Martin W., Lear Gavin, Curran

James, Lewis Gillian D., 2015. A novel bacterial community index to assess

stream ecological health. Freshwater Biology 60, 1988–2002.

https://doi.org/10.1111/fwb.12625

Liu, L., Liu, H., Zhao, Y.-P., Wang, Y., Duan, Y., Gao, G., Ge, M., Chen, W., 2008. Directed

synthesis of hierarchical nanostructured TiO2 catalysts and their morphology-

dependent photocatalysis for phenol degradation. Environmental Science and

Technology 42, 2342–2348. https://doi.org/10.1021/es070980o

Liu, X., Chen, G., Su, C., 2011. Effects of material properties on sedimentation and

aggregation of titanium dioxide nanoparticles of anatase and rutile in the

aqueous phase. Journal of Colloid and Interface Science 363, 84–91.

https://doi.org/10.1016/j.jcis.2011.06.085

Mueller, N.C., Nowack, B., 2008. Exposure modeling of engineered nanoparticles in the

environment. Environmental Science and Technology 42, 4447–4453.

https://doi.org/10.1021/es7029637

Reyes-Coronado, D., Rodríguez-Gattorno, G., Espinosa-Pesqueira, M.E., Cab, C., Coss, R.

de, Oskam, G., 2008. Phase-pure TiO 2 nanoparticles: anatase, brookite and

rutile. Nanotechnology 19, 145605. https://doi.org/10.1088/0957-

4484/19/14/145605

29 Savage, N., Diallo, M.S., 2005. Nanomaterials and water purification: opportunities and

challenges. Journal of Nanoparticle Research 7, 331–342.

https://doi.org/10.1007/s11051-005-7523-5

Sharma, V.K., 2009. Aggregation and toxicity of titanium dioxide nanoparticles in aquatic

environment--a review. Journal of Environmental Science and Health 44, 1485–

1495. https://doi.org/10.1080/10934520903263231

Su, R., Bechstein, R., Sø, L., Vang, R.T., Sillassen, M., Esbjörnsson, B., Palmqvist, A.,

Besenbacher, F., 2011. How the anatase-to-rutile ratio influences the

photoreactivity of TiO2. Journal of Physical Chemistry Part C 115, 24287–24292.

https://doi.org/10.1021/jp2086768

Suttiponparnit, K., Jiang, J., Sahu, M., Suvachittanont, S., Charinpanitkul, T., Biswas, P.,

2011. Role of surface area, primary particle size, and crystal phase on titanium

dioxide nanoparticle dispersion properties. Nanoscale Research Letters 6, 27.

https://doi.org/10.1007/s11671-010-9772-1

Tan, L., Nielsen, N.H., Young, D.C., Trizna, Z., 2002. Use of antimicrobial agents in

consumer products. Archives of Dermatoligy 138, 1082–1086.

https://doi.org/10.1001/archderm.138.8.1082

US EPA, 2018. TSCA Chemical Substance Inventory [WWW Document]. US EPA. URL

https://www.epa.gov/tsca-inventory (accessed 5.4.18).

Wu, F., 2017. Novel approaches to evaluate the environmental impacts of

nanomaterials (PhD Dissertation). Oregon State University.

30 Yaremko, Z.M., Petryshyn, R.S., 2013. Adsorption of benzethonium chloride from

aqueous solutions on dispersed adsorbents. Colloid Journal 75, 741–746.

https://doi.org/10.1134/S1061933X13050177

Yu, J.C., Kwong, T.Y., Luo, Q., Cai, Z., 2006. Photocatalytic oxidation of triclosan.

Chemosphere 65, 390–399. https://doi.org/10.1016/j.chemosphere.2006.02.011

31 Appendix 1. Enlarged version of Figure 4 from Results section of text, rotated. th P25

treatments.

axis differs between - he y . Note that the range of t cell growth when treated with BTC and P25, anatase, and rutile titanium dioxide nanoparticle supernatant and particles. Horizontal black bars represent median values for each treatment and the top and bottom of each vertical bar

E. coli

rutile supernatant treatments

Figure 4. resuspended signifies the maximum and minimum value, excluding outliers (black dots). “Extreme outliers” were identified at 20 mg/L in bo and

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