APPROVED:

Aaron Roberts, Major ProfessorBarney Venables, Committee MemberThomas LaPoint, Committee MemberSam Atkinson, Chair of the Department

of BiologyMark Wardell, Dean of the Toulouse

Graduate School

PHOTOTOXIC EFFECTS OF TITANIUM DIOXIDE

NANOPARTICLES ON Daphnia magna

Charles M. Mansfield

Thesis Prepared for the Degree of

MASTER OF SCIENCE

UNIVERSITY OF NORTH TEXAS

December 2013

Mansfield, Charles M. Phototoxic effects of titanium dioxide nanoparticles on

Daphnia magna. Master of Science (Biology), December 2013, 48 pp., 3 tables, 8

figures, bibliography, 105 titles.

Titanium dioxide nanoparticles (TiO2-NP) are one of the most abundantly utilized

nanomaterials in the world. Studies have demonstrated the mechanism of acute toxicity

in TiO2-NP to be the production of reactive oxygen species (ROS) leading to oxidative

stress and mortality in exposed organisms. It has also been demonstrated that the

anatase crystalline conformation is capable of catalyzing the cleavage of water

molecules to further increase the concentration of ROS in the presence of ultraviolet

radiation. This photoenhanced toxicity significantly lowers the toxicity threshold of TiO2-

NP to environmentally relevant concentrations (ppb). The goal of this study was to

determine whether dietary uptake and accumulation of TiO2-NP in the aquatic filter

feeder Daphnia magna resulted in photoenhanced toxicity. D. magna and S.

caprincornatum were exposed to aqueous solutions of 20ppm and 200ppm TiO2-NP for

24hrs and then transferred to clean moderately hard water. Samples were taken at

various time points, dried, and TiO2 quantified using ICP-MS. Toxicity assays were run

on D. magna using three TiO2-NP (20ppm, 200ppm) exposure protocols and two

ultraviolet radiation treatments. The first exposure group was exposed to aqueous

solutions of TiO2-NP for the duration of the test. The second exposure group was

exposed to TiO2-NP for an hour and then transferred to clean water. The third exposure

group was fed S. capricornatum that had been allowed to adsorb TiO2-NP. All samples

were then placed in an outdoor UV exposure system and exposed to either full

spectrum sunlight (with UV) or filtered sunlight (no UV). Here we show that TiO2 uptake

peaked at one hour of exposure likely due to sedimentation of the particles out of

suspension, thus decreasing bioavailability for the duration of the test. Interetsingly,

when D. magna were moved to clean water, aqueous concentrations of TiO2 increase

as a result of depuration from the gut tract. Data also suggests these excreted particles

were bioavailable and re-consumed by D. magna. These data will contribute to the

understanding of TiO2-NP environmental fate and toxicity.

ii

Copyright 2013

by

Charles M. Mansfield

iii

ACKNOWLEDGEMENTS

I would like to thank several people. Dr. Aaron Roberts, thank you for taking me

on as a student and giving me the opportunity, direction and possible prodding through

my thesis. I would also like to thank the students of the Roberts’ lab for their help in

carrying out these experiments: Matt Alloy, Brianne Soulen, and Kristin Nielsen. I would

also like to thank my family, for their ongoing support and love throughout my research.

Last but not least, I would like to thank the members of my committee, Dr. Barney

Venables and Dr. Tom LaPoint, for their insight and assistance in method development

and data analysis.

iv

TABLE OF CONTENTS

Page ACKNOWLEDGEMENTS ............................................................................................... iii

LIST OF TABLES AND ILLUSTRATIONS ...................................................................... v

CHAPTER 1: INTRODUCTION ....................................................................................... 1

CHAPTER 2: THE PHOTOTOXIC EFFECTS OF TITANIUM DIOXIDE NANOPARTICLES ON Daphnia magna UNDER NATURAL SUNLIGHT ..................... 13

CHAPTER 3: METHODS AND MATERIALS ................................................................ 17

CHAPTER 4: RESULTS ................................................................................................ 23

CHAPTER 5: DISCUSSION .......................................................................................... 30

CHAPTER 6: CONCLUSIONS ...................................................................................... 36

BIBLIOGRAPHY ........................................................................................................... 39

v

LIST OF TABLES AND ILLUSTRATIONS

Page

Tables 1. Modeled Environmental Concentrations of TiO2-NP ............................................ 5

2. ICP-MS settings for TiO2 quantification protocol ................................................. 19

3. Particle Diameter and Zeta Potentials of test suspensions ................................. 24

Figures

1. Relationship of Particle Diameter to Surface Area................................................ 1

2. Crystalline structure of Rutile and Anatase TiO2 ................................................... 3

3. Water column concentrations of anatase TiO2 ................................................... 23

4. Survival comparison, Rutile v. Anatase phototoxicity………………………….24/25

5. Body burdens of anatase TiO2 during 48hr uptake/depuration ........................... 25

6. Waterborne v. body burden phototoxicity of anatase TiO2.................................. 27

7. Dose response of anatase TiO2 photoxicity ........................................................ 28

8. Phototoxic reciprocity model of anatase TiO2 ..................................................... 29

CHAPTER I

INTRODUCTION

Nanoparticles are defined as materials with at least one dimension between 1nm

and 100nm (Klaine et al., 2008; Moore et al., 2006). Nanoparticles demonstrate unique

physiochemical properties when compared to their corresponding bulk material

counterparts (EU-OSHA, 2005). As the particle size becomes smaller, the surface area

to mass ratio increases (Figure 1). The result is a large surface area of reactive particles

that are capable of increased rates of bio reactivity.

Nanotechnology is hailed as the next technical revolution. As of 2012, the world’s

governments invested $10 billion per year in nanotechnology development (Harper,

2011). Nanotech is already being used in industries such as; sporting goods, tires,

clothing, tooth paste, sunscreens (EPA, 2009), cosmetics (Muller et al.,2002),

electronics (Kachynski et al., 2008), solar energy production (Jiang et al., 2002),

0

10

20

30

40

50

60

70

1 10 100 1000 10000

Su

rface A

rea

Particle Diameter (nm)

Figure 1. Relationship of particle diameter and surface area. As particle diameter decreases, thisimplies an exponential increase in percent surface area. (Adapted from Oberdorster et al.,2005, Nelet al., 2006).

1

renewable energy (Pavasupree et al.,2006;Wei et al., 2008), waste disposal (Shan,

2009), environmental remediation (Zhang, 2003;Joo, 2006) and medicine (Sosonovik et

al., 2008, Corchero et al., 2009). Despite the global increase in the production and

utilization of nanoparticles little is known regarding their ultimate fate, accumulation or

transfer through food webs (Klaine et al., 2008; Handy et al., 2008). Development and

exploitation of these novel materials have vastly outpaced the understanding of the

toxicological mechanisms and the environmental fates thus raising concerns about the

safety of the nanotech industry (Whatmore, 2006). Titanium dioxide (TiO2), for example,

is one of the most utilized nanoparticles (NP) in the world, yet there is very little data

concerning the toxic effects of TiO2 nanoparticles (TiO2-NP) (Menard, 2011). It is more

than likely that despite current safeguards that the industrial wastes of waste

management, remediation, electronics, and cosmetics will inevitably be deposited in

aquatic systems (Moore, 2006).

TiO2 is one of the most commonly used nanoparticles (Menard, 2010).TiO2 is

naturally occurring in three crystal formations: brookite, rutile and anatase. Rutile is the

most stable in bulk particles, while anatase is more stable at particle sizes between

2nm-14nm (Naicker et al., 2005; Chen 2009). At temperatures around 1,123K, brookite

and anatase conformations are capable of transforming into the rutile conformation.

Unlike the bulk form of TiO2, the physical and chemical properties of nano-TiO2 change

with the decrease in particle size (Donaldson and Tran, 2002; Oberdoster, 2001). For

example, the bond length of the Ti-O bond in bulk TiO2 is 0.196nm in a tetrahedral unit

cell. At particle diameters below 20nm the Ti-O bond length is 0.179nm (Sclafani, 1996)

and adopts a hexa-coordinated unit cell (Rajh, 2003), increasing both the affinity of

2

these uncoordinated defects to ortho-substituted enediol ligands and the overall surface

reactivity of the nanoparticle.

Rutile TiO2 (Figure 2a) is currently the most abundant form of TiO2 in nature

(EPA, 2009) and the most stable conformation. Rutile TiO2 is widely used as a white

pigment in paints, papers, inks, plastics, milk products, sunscreens, UV absorption,

(Mueller, 2008) and cosmetics (Gurr, et al., 2005). Anatase TiO2 (Figure 2b) is the

most reactive and demonstrates both catalytic and photocatalytic behavior. The

photocatalytic properties are unique to anatase and lead to the prolific use of anatase

TiO2 in self-cleaning surfaces, light-emitting diodes, solar cells, disinfectants, water

treatment, and sunscreens (EPA, 2009).

TiO2 is a naturally occurring ore that must be refined by either the sulfate process

or the chloride process to extract the TiO2 (Reck,1999). The pure forms of rutile and

anatase particles are then subject to numerous methods of processing, such as sol-gel,

sol, hydrothermal, solvo-thermal, physical vapor deposition, chemical vapor deposition,

and electro-deposition to produce purified nanoparticles (Chen, 2009). Precise and

careful manipulation of the temperatures and reagents of these processes can be

Figure 2. (A) Rutile crystal structure (B) Anatase crystal structure American Mineralogist Crystal Structure Database (2003)

A B

3

utilized to dictate the crystal formation produced through refinement as well as

producing TiO2 nanorods, nanowires, and ultrafine nanoparticles.

Titanium dioxide was one of the first nanoparticles to be utilized commercially

(Menard, 2011). Production of TiO2 is estimated at 5000 metric tons per year for the

years 2006-2010, and 10,000 metric tons for 2011 and 2014 (UNEP, 2007). TiO2

production is projected to increase to 2.5 million metric tons by 2025 (Robichaud et al.,

2009).

Environmental Concentrations of TiO2

Very little data exists concerning environmental concentrations of nano- TiO2

(Griffit et al., 2008). Kaegi et al. (2008) demonstrated surface paint runoff

concentrations of nano- TiO2 600ppb. Kiser et al. (2009) observed concentrations of

100-3000ppb in raw sewage and 5-15ppb in wastewater effluent. Gottschalk et al.

(2009) and Mueller and Nowack (2008) have developed models to predict the

distribution of TiO2-NP into environmental compartments (Table 1). Gottschalk et al.

(2009) used probabilistic material flow analysis based on inputs garnered from empirical

data and extrapolation to determine the flow of commonly used nanoparticles through

environmental compartments. These models showed that TiO2 concentrations would

reach such high concentrations in sewage effluent that it would merit further research to

evaluate the risk of TiO2-NP. The model also showed that when compared to nano-zinc,

nano-silver, carbon nanotubes and fullerenes, TiO2 showed the highest degree of

concentration in the sewage effluent and sewage treated soils (Gottschalk et al., 2009).

These results of sewage effluent concentration were echoed in a similar modeling

4

experiment performed by Mueller and Nowack (2008) using the same nanoparticles and

environmental compartments.

Compartment Predicted Environmental Concentration

United States Switzerland Europe

Water 0.002-0.01µg/L 0.7-0.16µg/L 0.012-0.057µg/L

Soil 0.42µg-2.3µg/kg 0.4µg/kg 1.01-4.45µg/kg

Sludge Treated Soil 34.5-170µg/kg ---------------------- 70.6-310µg/kg

Sediment 44-251µg/kg 426-2383µg/kg 273-1409µg/kg

Air 0.005µg/m3

0.002-0.042µg/m3

0.005µg/m3

Sewage Plant Sludge 1.37-6.7 mg/kg 3.5-16.3 mg/kg 2.5-10.8 mg/kg

Sewage Plant Effluent 107-523 µg/L 172-802 µg/L 100-433 µg/L

Due to the relative stability of both crystalline formations, it is likely that nano-

TiO2 will aggregate and settle out into sediments and adsorb to particulate matter in the

water column (Boxall et al., 2007). Factors such as the presence of humic acids

(Pettibone et al., 2008; Domingos et al., 2009), pH and ionic properties of the water

body (Navarro et al., 2008;Sharma et al., 2009) will affect the degree and rate of

aggregation of nano- TiO2. These sediment and particulate bound particles are then

capable of reacting with sediment dwelling organisms and filter feeders (Farre et al.,

2009). A study by Ferry et al. (2009) was able to demonstrate that NPs could pass from

the water into the aquatic food web. Nano TiO2 was found to not only pass into the food

web, but to accumulate in the primary consumer Daphnia.magna (Zhu et al., 2010).

Another metallic NP, quantum dots, was shown to transfer from Selenastrum

capricornutum to Ceriodaphnia dubia (Bouldin et al., 2008), indicating that trophic

transfer and bioaccumulation of nanoparticles was a possibility. Aggregation of

Table 1. Modeled Concentrations TiO2-NPreleased into environmental compartments in different countries.

(Mueller and Nowack, 2008; Gottschalk et al., 2009; Menard, 2010)

5

nanoparticles could cause suspended particles to settle out of solution (Fortner,

2005;Phenrat et al., 2007), thus reducing their bioavailability. The aggregation behavior

is based upon the physiochemical properties of the particle itself: geometry (Pal et al.,

2007), particle size (Morones et al., 2007) electrostatic repulsion (Phenrat et al., 2007),

crystal structure (Tiede et al., 2007), nature of surface coatings (Warheit et al., 2007)

Aggregation behavior is further impacted by the ionic strength, composition, availability

of natural organic matter (NOM) (Fabrega et al., 2011), pH (French et. al., 2009),

temperature and the concentration of the nanomaterial during exposure (Phenrat et al.,

2007;Dunphy et al., 2004).

Silver nanoparticles (Ag-NP) may shed some light as to the environmental fate

and behaviors of other metallic nanoparticles in aquatic systems. It has been

demonstrated that increased levels of NOM reduced bioavaibility and thus the toxicity of

Ag-NP (Gao et al., 2009). Liu and Hurt (2010) demonstrated that the humic and fulvic

acids present in NOM were able to reduce the release of Ag-NP through particle

aggregation. Increased aggregation would naturally lead to a reduced water column

bioavailability as the NP solubility decreased. However, the increase aggregation

behavior of the colloidal NP would increase the likelihood of the NP to sorb to organic

matter (Chen and Elimelch, 2007). As the particles aggregate and settle out in solution,

the nanoparticles were then show be taken up by mussels, oysters (Ward and Kach,

2009), and snails (Croteau et al., 2010) which bioaccumulated the Ag NP through

ingestion. It is presumed that these organisms feed primarily upon media in which the

Ag-NP may accumulate (Fabrega et al., 2011; Battin et al., 2009) such as surface

6

nanofilm produced by interactions between Ag-NP and NOM. Similar mechanisms of

humic and fulvic acid interaction has been shown with TiO2-NP (Lin, 2009).

TiO2-NP Toxicity

TiO2 is one of the most tested nanoparticles (Cattaneo et al., 2009; Kahru and

Dubourguier, 2010) due to its prolific use in many industries. Recent research has

demonstrated that nanoparticles including TiO2 –NP, may accumulate within the

cytoplasm (Sakai et al., 1994; Wamer, 1997, Park et al., 2008; Singh et al., 2007). They

likely enter through some cellular transport mechanism, such as clathrin mediated

transport, phagocytosis, scavenger receptors, and caveolae (Geiser et al.,2005). As

with many other engineered nanoparticles, nano-TiO2 has demonstrated the ability to

generate reactive oxygen species (Reeves et al., 2008). Reactive oxygen species

(ROS) can be generated by the redox cycling and catalytic chemistry of the surface

defects of the particle itself. These ROS are capable of damaging proteins, lipids and

DNA leading to necrosis or apoptosis (Valko et al., 2004). Additionally, the crystal plane

of the nano crystal itself is able to interact with molecular oxygen to produce active

electronic configurations such as superoxide radicals, which then initiate a cascade of

free radical reactions. In addition to inducing a state of oxidative stress, TiO2 is also

capable of causing membrane destabilization through lipid peroxidation (Esterbauer et

al., 1992; Riley, 1994), signaling perturbation (Sies et al., 1986;Riley, 1994), DNA

damage (Riley, 1994; Sycheva et al., 2011, Trouiller et al., 2011), and the formation of

granulomas (Hamilton, 2009)

Significant research has implied that the surface defect present in the anatase

conformation results in increased toxicity when compared to the rutile structure.

7

Inhalation effects of rutile nanocrystals were observed to have an LC50 550ppm in lung

epidermal cells (Sayes et al., 2006). In a similar experiment, exposure via oral gavage

showed an increase in DNA strand breakage at concentrations of 40ppm -1000ppm of

160nm anatase TiO2 –NP in mice (Trouiller et al., 2012). Anatase TiO2 has also been

shown to be toxic to aquatic organisms. Federici et al. (2007) demonstrated oxidative

stress was present in rainbow trout gills, liver and intestinal epithelium at concentrations

ranging from 0.1-1ppm anatase TiO2. Additional studies by Patterson et al. (2011) on

developing japanese medaka (Oryzias latipes) showed that anatase TiO2-NP induced

early hatching with perturbed behavior and significantly reduced hatching rates in

exposure treatments at 10ppm. Palaniappan (2011) was able to demonstrate that

exposure to10ppm anatase TiO2-NP resulted in significant alterations in the biochemical

constituents of brain tissues in Danio rerio. Acute toxicity was demonstrated in

Caenorhabditis elegans significantly increased ROS generation, reduced growth and

behavioral perturbation by Roh et al. (2012) at 50ppm using 4nm-10nm anatase TiO2-

NP. In algal toxicological studies, Blaise et al. did not note any acute toxic effect of TiO2-

NP, while studies by Aruoja et al. (2009) show growth inhibition at an EC50 of 5.83ppm

in S. caprincornatum. This is supported by the work of Hartmann et al. (2010) on S.

caprincornatum where growth was inhibited EC50 levels of 241ppm for the formulation

mixture of 67.2% anatase, 32.8% unknown crystal, and 71ppm for the formulation

mixture 72.6% anatase, 18.4% rutile, 9% unknown.

The term phototoxicity is used to explain the phenomenon by which the toxicity of

a chemical is enhanced during the co-exposure to light energy, usually within the

ultraviolet wavelengths. (Landrum,1987). This chemical may be any chemical that

8

reacts or responds to high energy photons and is capable of transferring that energy

into chemical or biological systems (Diamond 2003). Plants have incorporated

phototoxic molecules into self defense mechanisms that prevent ingestion (Ivie, 1978)

or fungal colonization (Scheel, 1963). Anthropogenic chemicals such as polycyclic

aromatic hydrocarbons (PAHs), pharmaceuticals (Tetracycline) and pesticides

(erythrosine B) (Spikes, 1969) have all demonstrated phototoxic mechanisms.

The photocatalytic properties of the anatase nanocrystal is often utilized in

industries to photodegrade wastes and pollutants (Ma et al., 2012), as a disinfectant

(Kuhn et al., 2003), and shows promise as a tool to kill tumor cells (Fujishima et al.,

2000). When the anatase TiO2 absorbs a photon of light that possesses higher energy

than the band gap of TiO2 an electron is elevated from the valence band to the

conduction band, resulting in an electron hole. The interaction of anatase TiO2 with high

energy photons is capable of producing electron hole pairs that form free radicals from

the splitting of the covalent bonds in water molecules (Ma et al., 2012). This

photodecomposition process often produces ROS that are responsible for the

photocatalytic properties inherent in the anataste structure. This action paired with the

increase surface area to mass ratio of nanocrystals allows for an increased rate of

photocatalytic activity than would otherwise be found in bulk TiO2 (Donaldson and Tran,

2002; Oberdoster, 2001, Ma et al., 2012).

Early work exploring the phototoxic effects of anatase TiO2 and UV light showed

significant growth reduction at anEC50s of 44ppm in Desmodesmus subspicatus and no

toxic effects in UV negative controls at 50ppm (Hund-Rinke and Simon, 2006). LC50

values of 2.42ppm (with UV) and 155 ppm (without UV) were reported for medaka (Ma

9

et al., 2012; Brennan et al., 2012). Huang et al. (1999) examined the bactericidal effects

of anatase TiO2 and reported mortality at concentrations of 100ppm in the presence of

UV. Further work by Maness determined the mechanism of toxicity to be lipid

peroxidation and a decrease in cellular function of Escherichia coli. This work was

supported in the experiments of Matsunuga et al. (1994), which examined E. coli

mortality and the cellular leakage of β-D galactosidase to extracellular fluids, an

indicator of cellular membrane breakdown. A phototoxic effect was demonstrated in

Artemia salina and Chattonella Antigua at 1ppt anatase TiO2 under UV exposure in

work performed by Matsuo (2001). Bar-Ilan (et al. 2013) showed an LC50 of ~8ppb of

anatase TiO2 in developing Danio rerio co-exposed to UV. In addition to mortality, Bal-

Ilan also noted survivors of the UV/Tio2 exposure showed significant growth

malformations when compared to UV-negative controls. . Ma et al. (2012) reported an

LC50 of 29.8ppb under simulated solar radiation (SSR) and a calculated LC50 of 500ppm

in UV negative controls in D. magna. The SSR for this experiment was measured at

1700µW/cm2, which is approximately 25% of the total solar radiation on a sunny day. To

date, no study examining TiO2 phototoxicity under natural sunlight with full spectrum

conditions has been conducted.

Goals and Hypothesis

The phototoxic effects of anatase TiO2 and natural sunlight has not been

adequately studied. Previous experiments have examined a relatively narrow set of UV

conditions and have not represented natural sunlight. Such information must be

reported in order to provide more accurate data for TiO2 phototoxicity in ecological risk

assessments. This study seeks to examine the phototoxic effects of TiO2 upon a

10

grazing primary consumer using aqueous and uptake exposure methodologies. In this

study, I (1) assess the uptake of anatase TiO2-NP via two exposure methods (dietary v.

waterborne) in D. magna, (2) determine whether toxicity is affected by exposure

method, and (3) compare the photoenhanced toxicity of rutile and anatase crystal

structure under varying natural sunlight conditions.

Hypothesis 1:

The uptake of TiO2 NP by D. magna will be significant via both dietary and aqueous exposure.

Rationale:

The interactions of water chemistry and the TiO2-NP will favor particle

aggregation that will increase the likelihood of uptake. The pH ranges of the anterior

and posterior gut tracts of D. magna are 6.0-6.8 and 6.6-7.2, respectively. The zero

point charge of TiO2-NP is between 6 > pH > 7. In these conditions, the TiO2-NP are at

zero charge and non-polar. With their small size and non-polar charge, the particles

theoretically are able to cross the gut membrane into the tissues of the organism.

Hypothesis 2:

The aqueous exposures of TiO2 NP will demonstrate a higher degree of mortality under phototoxic conditions than the dietary uptake of similar suspensions of TiO2 NP.

Rationale: Previous experiments by Diamond et al. (2012) have suggested that aqueous

suspensions of TiO2 NP will demonstrate a greater toxic effect than the body burden of

TiO2 NP within the organism due to the increase in reactive surface area on the outside

of the organism as opposed to the gut-tract accumulation within the organism.

Hypothesis 3:

11

Pure mixtures of the anatase crystalline structure will demonstrate a higher degree of photo-enhanced toxicity than mixed preparations containing higher percentages of rutile.

Rationale:

The surface defect responsible for the surface reactivity of the anatase crystal

structure is absent in the rutile crystal form, rendering the rutile form relatively inert in

the presence of UV.

12

CHAPTER 2

PHOTOTOXIC EFFECTS OF TITANIUM DIOXIDE NANOPARTICLES ON DAPHNIA

MAGNA UNDER NATURAL SUNLIGHT

Manufactured nano-scale particles are a global market garnering in excess of

$10 billion worth of investments for research and development (Harper, 2011). Of this,

titanium dioxide nanoparticles (TiO2 NP) are one of the most utilized nanoparticles in the

global industry, being utilized in renewable energy (Pavasupree et al., 2006) , cosmetics

and paints (Mueller and Nowack, 2008), waste disposal (Weisner, 2007), environmental

remediation (Zhang, 2003) and self-sterilizing surface coatings (Lopez, 2002). The

current usage of TiO2 NP is 10,000 metric tons (UNEP, 2013) and is projected to

increase to 2.5 million metric tons by the year 2025 (Robichaud et. al, 2009). The

industrial use of is dependent upon the crystalline conformation of the TiO2-NP. Rutile is

utilized primarily as a pigment in paints, sunscreens, and cosmetics due to its

absorbance of ultraviolet (UV) radiation (Mueller and Nowack, 2008). Anatase, the more

reactive form of TiO2, is used in many industrial and consumer processes that capitalize

on its unique form of photo-reactivity. This photo-reactivity allows anatase to cleave

water molecules and produce reactive oxygen species (ROS) that allow it to be used in

environmental remediation (Zhang, 2003), solar energy production (Jiang et al., 2002),

waste disposal (Weisner, 2007), and self-sterilizing surfaces (Lopez, 2002). However,

the rapid development and deployment of these technologies has vastly outpaced our

understanding of the environmental fate and toxicity of these particles, which leads to

concern about the safety of the nanotech industry as a whole (Whatmore, 2006). With

its global use and high degree of industrial application, it is inevitable that all forms of

TiO2 NP will end up within aquatic ecosystems (Moore, 2004).

13

TiO2 is a naturally occurring and can be refined from ore or synthetically

produced. By controlling the chemical substrates, reaction temperatures and cooling

temperatures, industries are capable of determining the dominant crystal confirmation of

the end product (Chen, 2009). The most common forms of TiO2 are rutile, brookite and

anatase (Reyes-Coronado et al., 2008). Of the three, anatase is the most reactive,

owing its reactivity to a unique surface defect in the crystalline structure. It is this

surface defect that is believed to be to be responsible for the understood mechanism of

toxicity, reactive oxygen species (ROS) generation. The mechanism for this generation

of ROS is well understood, with the defect allowing for the generation of an electron-

hole pair, allowing anatase TiO2 to interact with oxygen and water to produce hydroxyl,

hydrogen and superoxide radicals (Reeves et al., 2008; Ma et al., 2012). This ROS

production is increased when anatase is exposed to high energy radiation (UV, X-ray)

as it increases the generation of electron-hole pairs which paired with the surface area

of the NPs, exponentially increasing the production of ROS. These ROS can then react

with organic substrates and initiate damaging chain reactions such as lipid peroxidation

that lead to the destabilization of cellular membranes (Esterbauer et al., 1988; Riley,

1994). Elevated ROS are capable of damaging cellular proteins (Wolffe et al., 1986),

forming DNA adducts and strand breakage (Riley, 1994, Trouiller et al., 2011), and

slowly exhausting cellular resources leading to cell death (Valko et al., 2004).

Despite being one of the most studied NP on the market today, anatase TiO2 NP

has been observed to exhibit a wide range of LC50 ranging from 5.83ppm in P.

subcapita (Aruoja et al., 2009) to 241ppm in P. subcapita (Hartmann et al., 2010) and

10ppm in D. magna (Kim et al., 2010) to 20,000ppm in D. magna (Heinlaan et al.,

14

2008). This is due in part to the large variability in the characterization of the TiO2 NPs

themselves, dispersion methods, and exposure protocols. Many toxicology studies have

focused on the toxic effects associated with particle size of the nanocrystal. More

recently, toxicity has been linked to the surface reactivity of the nanocrytsal itself

(Warheit et al., 2007). Surface reactivity is of particular importance in regard to the

phenomenon of photo induced toxicity.

Photo induced toxicity is a phenomenon in which the toxicity of a given

compound is amplified in the presence of light, most often from the UV spectrum

(Diamond, 2013; Santamaria, 1964). Early studies exploring the phototoxic effects of

co-exposure to anatase TiO2 and UV light reported growth inhibition at 44ppm using

Desmodesmus subspicatus while no toxic effects were reported at similar

concentrations without UV light (Hund-Rinke and Simon, 2006). Diamond et al. (2012)

reported a 48hr LC50 of 29.8ppb in Daphnia magna when organisms were co-exposed

to TiO2 NP and UV, while UV negative controls (TiO2 only) had calculated LC50 of

500ppm. Diamond et al. (2012) reported a 96hr LC50 of 2.19ppm in O. latipes co-

exposed to UV and anatase TiO2 NP with a UV negative control LC50 of 155ppm. The

studies by Diamond showed significantly lower LC50 of anatase TiO2 NP at simulated

solar radiation of output 1700µW/cm2, which the authors estimated to be 25% of natural

solar radiation on a sunny day. To date, there has been little to no research looking at

the phototoxic effects of anatase TiO2 NP under full spectrum solar radiation (natural

sunlight).

This study was designed to examine the effects of natural solar radiation on the

photoenhanced toxicity of anatase TiO2 NP. Body burdens of anatase TiO2 NP were

15

determined in 48hr uptake/depuration studies and quantified using inductively coupled

mass spectrometry (ICP-MS) in order to more accurately compare concentrations of

waterborne TiO2 and body burdens present in test organisms. Phototoxicity in

organisms with body burden TiO2 vs. waterborne TiO2 was compared to examine

different exposure pathways. Phototoxic effects of anatase TiO2 NP were compared to

that of a mixture of anatase and rutile TiO2 under natural solar radiation to compare

effects of crystal structure.

16

CHAPTER 3

METHODS AND MATERIALS.

Organism Culture

Daphnia magna were obtained from obtained from existing cultures at the

University of North Texas. Organisms were cultured according to standard US EPA

methods (600/4-90). Organisms were cultured in the reconstituted hard water (RHW)

(4800mg NaHCO3, 3000mg MgSO4, 3,000mg CaSO4∙ 2H2O, 200mg KCl dissolved in

50L Milli-q H20 stock) and at 26°C. Daphnia were fed a mixture of S. capricornutum and

yeast:cerophyl:tetramin (YTC) that was purchased through Aquatic Ecosystems

(Apopka, Fl) at a 3:1 ratio of YTC to algae and kept at a light:dark cycle of 16:8 hours.

TiO2-NP Preparation

Anatase TiO2 was purchased through Fisher Scientific (Acros Organics

Titanium(IV) dioxide, 98%+ anatase powder, CAS:12463-67-7). Titanium dioxide was

mixed with RHW and sonicated using a Fisher Scientific Sonic Dismembrator (model

500) at 60% intensity for fifteen minutes with thirty second pulses on/off cycles.

Suspensions were allowed to cool to room temperature before dilution for organism

exposures. Mixed ratio 70:30 (anatase:rutile) TiO2 was purchased through Sigma-

Aldrich(CAS: 13463-67-7).

Characterization of TiO2-NP

Suspensions of TiO2 were prepared from stocks of 98% anatase and 70:30

anatase:rutile suspension were prepared at concentrations of 2ppb, 20ppb, 200ppb,

2ppm, 20ppm, and 200ppm. These suspensions were placed in 250mL amber vials and

shipped to Clemson University for analysis of zeta potentials and mean particle

17

diameter and particle size/distribution. TiO2-NP suspensions were brought to a pH of

3.6 by adding three drops of .1M HCl and sonicated to ensure that particles were

separated prior to analysis. Particle size and distribution was analyzed on a Wyatt

QELS and a Brookhaven 90 plus particle size analyzer. Two mixes of TiO2-NP were

analyzed: a 90:10 (anatase:rutile) and 70:30 (anatase:rutile).

Uptake of TiO2-NP by D. magna

Adult D. magna were placed in 250mL glass crystallization dishes (15 adults per

dish) containing one of three test suspensions (0, 20ppm, 200ppm) with five replicates

per concentration per time point, total 35 replicates per concentration. Throughout the

experiment, five dishes of each concentration were collected at time points of 0, 1, 8, 24

hours to determine the rate of uptake. At 24 hours, D. magna were removed from the

test suspension and placed in control RHW, without TiO2, and sampled at 25, 32 and 48

hours for depuration. At each sampling point, D. magna were collected and frozen (-

80°C). The D. magna were then lyophilized (Labconco 7753027) and dry weights were

measured on a microbalance (Mettler H51AR). Samples were then digested using

143µL 70% metals free nitric acid (HNO3) and 60µL 30% hydrogen peroxide and

microwaved at 30% power on a Magic-chef microwave. After samples were then diluted

back up to 5mL yielding a suspension with an acid content of <2% so that it could be

directly injected onto a Varian 820 ICP-MS set to detect the titanium ion (47.99amu) .

Values were compared to a nine point standard curve to determine concentration.

Uptake data was analyzed by two-way analysis of variance(ANOVA) in Microsoft

excel followed by a Tukey’s post hoc test. TiO2-NP uptake was the dependent variable

and time and TiO2-NP concentrations were the independent variable.

18

ICP-MS settings:

Flow Parameters

(L/min) Third Extraction Lens -211

Plasma Flow 16.5 Corner Lens -336

Auxiliary Flow 1.65 Mirror Lens Left 31

Sheath Gas 0.16 Mirror Lens Right 25

Nebulizer Flow 0.92 Mirror Lens Bottom 26

Torch Alignment (mm) Entrance Lens 1

Sampling Depth 5 Fringe Bias -2.3

Other Entrance Plate -30

RF Power (kW) 1.5 Pole bias 0

Pump Rate (rpm) 17 CRI (mL/min)

Stabilization delay (s) 5 Skimmer Gas Source OFF

Ion Optics (volts) Sampler Gas Source OFF

First Extraction Lens -1 Skimmer Flow 0

Second Extraction Lens -174 Sampler Flow 0

Stability Testing of TiO2-NP

Anatase TiO2-NP were suspended in RHW at experimental concentrations

(Control, 20ppm and 200ppm) and analyzed for stability in aqueous solution at a pH of

7.8, 356µs. 250mL of test suspension were placed in 300mL beakers and sampled

every twenty minutes for three hours. Samples were drawn using a 5mL pipette,

drawing off 5mL of the suspension at each time point. The samples were taken at the

same depth at every sampling, using the 1.5mL mark as a guideline for four

centimeters. Samples were then diluted into 50mL samples so that they could be

directly injected onto the ICP-MS in order to determine the concentration of Ti (47.99)

ions in the water column. A nine point standard curve was created to determined

concentration/counts using TiO2-NP suspended in RHW. Counts were then used to

Table 2. ICP-MS protocol used for the detection of Ti (47) under high attenuation.

19

back calculate the concentration suspended in order to demonstrate a representation of

concentration over time to determine the rate of sedimentation of anatase TiO2-NP.

Stability data was analyzed by a two-way Analysis of variance(ANOVA) in

Microsoft excel followed by a Tukey’s post hoc test. Mean water column concentration

was the dependent variable and the anatase TiO2-NP initial exposure concentrations

and time were the independent variables.

Phototoxic Mortality

All photo-enhanced toxicity bioassays were run in the following manner with

variation in exposure pathway/concentration. D. magna neonates (<48hr) were

harvested from brood boards and placed into 250mL glass crystallizing dishes

containing test suspensions which were then split among three experimental groups; a

UV-10% intensity, UV-50% intensity and UV-100% intensity in a full factorial design.

Natural sunlight was used as the source of UV radiation for outdoor exposures in

Denton, Texas (33.2147° N, 97.1328° W, 642 ft elevation) during the daytime hours. UV

opaque (SACROP3. 125x48000X96.000CEP) and transparent (SACRUVT.

187x63.000X88.000CP) film was obtained from Professional Plastics (Fullerton, CA)

and used to control the UV exposure in temperature controlled water baths. UV

exposure measurements were evaluated using the A PUV 2500 Biospherical radiometer

(Biospherical Instruments, San Diego, CA). Crystallization dishes were suspended in a

100 gallon water bath on a recirculation system in order to maintain an average

temperature of ~25°C +/- 2°C throughout the UV exposure.

Phototoxic mortality was analyzed by two-way analysis of variance (ANOVA) in

Microsoft excel followed by a Tukey’s post hoc test. Mean survival was the dependent

20

variable and anatase TiO2-NP concentration and UV concentration were the

independent variables. LC50s were calculated using TRAP-TOX statistical program

provided by the Environmental Protection Agency (EPA).

Body Burden versus Waterborne Exposure Pathways

Ten D. magna neonates (<48hr) were placed in 250mL glass crystallization with

five replicates per exposure pathway/concentration per UV exposure. One set of dishes

contained waterborne TiO2-NP (control, 20ppm, and 200ppm). Organisms in these

dishes were placed in the UV exposure system with waterborne TiO2-NP. A second set

of dishes contained waterborne TiO2-NP (control, 20ppm, 200ppm) were also prepared

in which organisms were exposed to waterborne TiO2-NP for one hour and then

transferred to control water before being placed in the UV exposure system. Organisms

were counted every two hours for mortality, which was determined by immobility of the

organism.

Phototoxic mortality was analyzed by two-way analysis of variance (ANOVA) in

Microsoft excel followed by a Tukey’s post hoc test. Mean survival was the dependent

variable and anatase TiO2-NP concentration and UV concentrations were the

independent variables.

Comparison of Anatase Phototoxicity and Rutile Photo-enhanced Toxicity

Using the same outdoor exposure system as the phototoxic mortality experiment,

test organisms were suspended in exposure concentrations (control, 2ppb, 20ppb,

200ppb, 2ppm, of three TiO2-NP suspensions: 99:1 (anatase:rutile) TiO2-NP, 85:15

(anatase:rutile) TiO2-NP, 70:30 (anatase:rutile) TiO2-NP. As with the phototoxic

mortality experiment, samples were split among three experimental groups; a UV-10%

21

intensity, UV-50% intensity and UV-100% intensity in a full factorial design. All

organisms were then exposed to eight hours of natural sunlight in Denton, Texas from

10am-6pm. Organisms were counted every two hours for mortality, being determined by

the immobility of the organism.

22

CHAPTER 4

RESULTS

Stability Testing of TiO2-NP

It was shown that TiO2-NP concentration in the water column reduced by 50%

after 1.5 hours and by another 50% after two and a half hours. Over the period of three

hours, anatase TiO2-NP could visibly be seen settling out of solution and accumulating

at the bottom of the 300mL beaker in a thin layer of white powder. In the 20ppm dishes,

approximately ~2% of the TiO2-NP settled out of solution by the end of the two and a

half hour period (p = 0.03) when compared to time zero. In the 200ppm dishes, a much

higher rate of loss was observed with ~75% settling out of solution over two and a half

hours when compared to time zero (p < 0.01).

0

50

100

150

200

250

0 0.5 1 1.5 2 2.5

Wat

er

Co

lum

n {

} in

pp

m

Time (hours)

200PPM

20ppm

Figure 3. Mean water column concentration of anatase TiO2-NP suspended in moderately hard water (pH 7.8) over a 2.5 hour time period.

23

Analysis of the zeta potentials of the experimental suspensions determined that

the higher concentration resulted in a lower zeta potential, which increased the

likelihood of agglomeration of the NP, thus increasing the diameter of the NP. In

samples where the anatase concentration was higher, there was an increase in zeta

potential and a reduction in particle size.

Comparison of Anatase Phototoxicity and Rutile Phototoxicity

0

0.2

0.4

0.6

0.8

1

1.2

0 0.5 1 1.5 2 2.5

% s

urv

ival

Log TiO2 [ ]

UV-10

UV-50

UV-100

Concentration of Suspension 0.20ppm 2.00ppm 20.00ppm 200.00ppm

Particle Diameter (nm)

99 anatase: 1 rutile 47.75 46.95 24.40 112.71

70 anatase: 30 rutile 53.95 10.46 36.45 222.14

Zeta Potential (mV)

99 anatase: 1 rutile -16.77 -23.51 -19.79 -20.78

70 anatase: 30 rutile -13.33 -25.74 -24.63 -28.50

Table 3. Mean particle diameter and mean zeta potential of TiO2-NPsuspensions at experimental suspension concentrations.

A

24

Anatase TiO2-NP demonstrated a significantly greater degree of phototoxicity

than rutile TiO2-NP (p < 0.01). The highest degree of mortality (25%) was noted in the

rutile concentrations occurred at 200ppm. LC50’s for both formations were calculated

using EPA TRAP tox software. The rutile LC50 after eight hours of 100% UVR exposure

was 282ppm (177-296ppm), which was higher than the calculated LC50 of the anatase

structure under the same exposure calculated at 180ppb (43-467ppb).

Daphnia Anatase TiO2-NP Uptake

0

0.2

0.4

0.6

0.8

1

1.2

0 0.5 1 1.5 2 2.5

% S

urv

ival

Log TiO2 [ ]

UV-10

UV-50

UV-100

0

1

2

3

4

5

6

7

0 1 2 16 24 25 48

ug

TiO

2-N

P/m

g D

ap

hn

ia (

DW

)

Time (Hours)

200ppm

20 ppm

CON

Figure 5. Mean concentration of anatase TiO2-NP (±SD) in Daphnia magna exposed to TiO2-NP for 24 hours followed by a 24 hour depuration period.

B Figure 4. Comparison of %survival ofD. Magna exposed to 70:30 (A) (anatase:rutile) to 99:1 (B) (anatase:rutile) over 8 hours of UVR exposure.

25

Body burdens of TiO2-NP in D. magna increased significantly during the first hour

of exposure compared to controls (p < 0.01). It was noted that the TiO2-NP

agglomerated and settled out of solution after two hours and was observed to coat the

bottom of the crystallization dishes. Body burdens in test organisms were at their peak

just prior to this this period (1-2 hour), when the highest concentration of TiO2-NP was

suspended in the water column. The degree of adsorption was such that it was visible

with the naked eye as test organisms became coated with TiO2-NP. Through the uptake

portion of the exposure, Daphnia exposed to TiO2-NP were noted to have distinctly

outlined gut tracts laced with TiO2-NP when compared to controls.

The TiO2-NP suspensions (20ppm and 200ppm) and their measured body

burdens did not significantly vary from each other (p = 0.38). When the depuration

began at the 24 hour mark, there was a noticed increase in body burden of TiO2-NP

measured at 150ppb. This trend was not seen in the 200ppm exposure, which declined

steadily over both the uptake exposure and the depuration period. There was no

mortality noted in any of the exposures during this experiment.

26

Phototoxic Effects of Waterborne versus Body Burden TiO2

Mortality was both UV and TiO2 concentration dependent following exposure to

waterborne TiO2-NP (p = 0.01; two way ANOVA). Toxicity was highest in the

waterborne 200ppm concentration, with 100% mortality in the 50 and 100% UV

treatments and 50% mortality even in the 10% UV treatment. Waterborne exposures

demonstrated significantly greater toxicity (p < 0.01) at a given TiO2 concentration/UV

intensity compared to their dietary exposure counterparts. Survival for both 20ppm and

200ppm body burden organisms was ≥80% in all UV exposures.

*

0

0.2

0.4

0.6

0.8

1

1.2

UV- UV-50 UV-100

% S

urv

ival

UV Treatment

Control

Uptake-20

Uptake-200

Aqueous-20

Aqueous-200

Figure 6. Comparison of the phototoxic effect of waterborne and body burden anatase TiO2 after UV exposure (8hrs). Figure reports mean survival (+/- 1 SD/SE). * = significantly different from controls. (α = 0.05)

*

27

Photo-enhanced Toxic Effect of Waterborne TiO2

Because nearly all toxicity was observed in the waterborne TiO2-NP exposures,

a second dose-response experiment was conducted using a wider range of waterborne

TiO2-NP concentrations. Mortality was both UV and TiO2 concentration dependent

following exposure to waterborne TiO2-NP (p < 0.01). Toxicity was greatest in the

200ppm concentration, with 100% mortality in the UV-50 and UV-100 treatments and

10% in the UV-10 treatments. Duration of UV exposure significantly impacted mortality

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0 0.5 1 1.5 2 2.5

% S

urv

ival

Log [TiO2-NP]

UV-10

UV-50

UV-100

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0 0.5 1 1.5 2 2.5

% S

urv

ival

Log [TiO2-NP

UV-10

UV-50

UV-100

Figure 7. Dose response of D. magna co-exposed (4hrs) to anatase TiO2-NP and UVR for a period of eight hours. Results are shown for 4hrs (A) and 8hrs (B) with ±SD in ppm.

A

B

28

with a four hour LC50 of 1.2ppm (95% CL .210-247ppm) and an eight hour LC50 of

180ppb (95% CL 43-467ppb) TiO2-NP (p < 0.01).

Dose response data demonstrated a reciprocal relationship between UVR

exposure and the waterborne concentration of anatase TiO2-NP. In the 100% UVR

treatment at 200ppm anatase TiO2-NP there was 100% mortality, whereas in the 10%

UVR treatment at 200ppm there was 20% mortality.

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5

% S

urv

ival

Log (UV * TiO2 [ ]

UV-10

UV-50

UV-100

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5

% S

urv

ival

Log (UV * TiO2 [ ] )

UV-10

UV-50

UV-100

Figure 8. Reciprocity model for UVR/anatase TiO2-NP over four hours (A) and eight hours (B) for D. magna.

B

A

29

CHAPTER 5

DISCUSSION

Results of these studies demonstrates that anatase TiO2-NP exerted an acute

toxic effect (mortality) after four hours of natural UVR exposure, which is a significantly

reduced time of exposure when compared to non-UV exposure work with D. magna.

Work done by Wiench et al. (2009) showed an EC50 of 100ppm, Heinlann et al. (2008)

report an LC50 of ~20,000ppm, and Kim et al. (2010) with an LC50 of >10ppm. This

study supported findings by Diamond et al. (2012) which showed the photo enhanced

toxic effect of anatase TiO2-NP reporting a 48 hour LC50 of 29.8ppb after 8 hours of

UVR. Work done in this study dealt with higher concentrations of anatase TiO2-NP and

increased concentrations of UVR, which are responsible for the reduced time of toxicity

seen in our results.

Stability Testing of TiO2-NP

Peak body burden concentrations were measured at time points at which the

waterborne TiO2-NP were also at their highest concentration. As the waterborne

concentration decreased, the body burden of TiO2-NP did as well. Suspensions of

20ppm and 200ppm fall within the sewage plant effluent concentrations (107-802ppm)

modeled by Gottschalk et al. (2009) and Mueller and Nowack (2008), implying that it

may be likely that aquatic organisms will be exposed to similar concentrations in aquatic

ecosystems. Toxicity studies by Lover and Klaper (2006) and Hund-Rinke and Simon

(2006) are lacking in stability information of their suspended TiO2-NP thus making it

difficult to compare results directly to this study. Stability and sedimentation rates are

30

crucial in determining the period as to which the organisms are exposed to waterborne

TiO2-NP.

Characterization of the particles supports the stability data as it shows that the

zeta potential decreases as the concentration of 99:1 (anatase:rutile). The increased the

hydrophobicity favors agglomeration behavior and increases the likelihood of the TiO2-

NP settling out of solution. Characterization data demonstrates that as the TiO2-NP

concentration in the water column decreases there is an increase in zeta potential,

reducing the hydrophobicity, which would lead to a greater stability of suspensions and

prolonged concentrations of TiO2-NP within the water column itself.

Comparison of Anatase Phototoxicity and Rutile Phototoxicity

Due to the surface defect found in its crystal structure, anatase TiO2-NP

demonstrated higher degrees of phototoxicity when compared to the 30% rutile mix.

The photocatalytic reaction between anatase TiO2-NP was capable of increasing ROS

generation to concentrations high enough to induce acute toxic effects in the D. magna.

This ROS generation was likely absent in the rutile mix due to the absence of the

surface defect. Additionally, the rutile formulation is often exploit for its UV absorbent

properties, which may have been capable of attenuating the UVR within the samples,

further reducing the ROS generation and thus the toxic effect.

Daphnia Anatase TiO2-NP Uptake

We observed a rapid increase in daphnid TiO2 body burden followed by a decline

after two hours. This is likely the result of increased particle aggregation of the TiO2-NP

themselves that led to increase sedimentation rates, and reduced the bioavailability of

TiO2-NP to the organisms. This is supported by the results of the stability experiment,

31

which also demonstrated a rapid loss of TiO2-NP out of the water column. At the

beginning of the depuration period there is a noticeable increase in concentration of

TiO2-NP in the body burdens of the test organisms. During the depuration phase, the

test organisms were fed fresh cultures of algae, which resulted in the excretion of TiO2-

NP present in the gut tract. The depuration of TiO2-NP from D. magna into the

surrounding environment occurred to such a degree that waterborne concentration of

TiO2-NP in these depuration chambers was measured between 200-900ppb and

allowed the organism to re-take up the suspended TiO2-NP.

Experiments performed here were single exposures in still water, allowing

organisms a brief time frame to be exposed to suspended TiO2-NP in the water column

before it settled out of solution. In real world exposures, the exposure is likely to be

periodic or constant. Additionally, many aquatic environments are turbulent, which will

increase the time TiO2-NP will be suspended in the water column. These conditions

would allow organisms, such as D. magna, to be continually exposed to TiO2-NP.

Previous work by Zhu et al. (2010) has demonstrated the ability for TiO2-NP to be

tropically transferred from D. magna to Danio. rerio. Further research is needed to

examine the waterborne uptake of TiO2-NP in D. rerio to determine the ability of TiO2-

NP to bioaccumulate through the food web. However, the results of this study

demonstrate that body burdens of anatase TiO2-NP demonstrate little to no phototoxic

effect within organisms.

Phototoxic Effects of Waterborne TiO2 and TiO2 Body Burdens

This experiment demonstrated significantly higher phototoxicity of waterborne

TiO2-NP suspensions to D. magna compared to body burden TiO2. Any TiO2-NP bound

32

within the tissues of the daphnid are encapsulated by the carapace of the organism

containing the pigment melanin, which is reported to protect organisms from damage

done by UV exposure (Hessen, et al., 1999). It is possible that the melanin present in

the carapace of the organism was capable of blocking enough UVR that it reduced the

production of ROS within the tissues of the organism to a sub-lethal level. Furthermore,

body burden TiO2 would have less opportunity with which to react with water, reducing

the photocatalytic production of the highly reactive hydroxyl radical.

After determining that aqueous TiO2-NP exerted a greater toxic effect than TiO2-

NP body burdens, dose response curves were made using a variety of concentrations

of waterborne TiO2-NP. LC50 were calculated to be 180ppb at eight hours of 100% UVR

and 1.2ppm at four hours 100% UVR. These values are orders of magnitude lower than

toxicity values reported by Wiench et al. (2009), Heinlann et al. (2008) and Kim et al.

(2010). All of these studies were done in the absence of UV irradiation, thus

demonstrating the photoenhanced toxicity mechanism. Studies by Diamond et al.

(2012) demonstrate a lower 48 hour LC50 (29.8ppb) after eight hours of UVR

demonstrating the photoenhanced toxicity of anatase TiO2-NP under simulated solar

radiation (SSR). The Diamond study exposed organisms to seven TiO2 (86:14,

antase:rutile, zp=16.5) suspensions (5, 15, 25, 50, 75, 100, 250, 500 ppb) and exposed

to four hours of UVR (1700µW/cm2/s) every 24 hours for 48 hours. Experimental

solutions we changed out after the first 24 hour period. This study exposed organisms

to higher concentrations of suspended (99:1, anatase:rutile) TiO2-NP (200ppb, 2ppm,

20ppm, 200ppm) and higher concentrations of UVR (2700µW/cm2/s) allowing for a four

hour LC50 (1.2ppm) and an eight hour LC50 (128ppb) to be calculated. Our exposure

33

protocol differed from Diamond et al. due to the fact that we had one suspension of

TiO2-NP exposed without interruption over an 8 hour period to higher intensity UVR. As

organisms were subject to higher concentrations of TiO2-NP and UVR for a longer

period of time, we saw LC50s earlier than in the Diamond study.

The work done in this experiment also demonstrates a consistent, reciprocal

relationship between the intensity of UV exposure, concentration of TiO2-NP and time of

UV exposure. This demonstration of reciprocity in the dose response phototoxicity is

missing in the work of Hunde-Rinke and Simon (2006) and Diamond et al. (2012). The

data gathered in this experiment was able to show significant differences (p<0.01)

between 100%, 50% and control samples in the same concentrations of TiO2 NP at the

same time, demonstrating that the variation in UV intensity has a direct impact on the

phototoxic effect seen in the organism. When the UV intensity remains constant, we see

significantly different degrees of toxicity across doses varying by orders of magnitude.

Lastly, as you increase both the TiO2 NP concentration and the UV exposure, there is

also a significant increase in the phototoxic effect with rates of 80-100% mortality in

highest exposure concentrations.

The polycyclic hydrocarbon, anthracene, has been extensively studied for its

photoenhanced toxicity. Without UVR, anthracene had been show to exhibit no acutely

toxic effect (immobilization) in daphnia (Herbes et al., 1976). When Herbes at al. co-

exposed with natural UVR, anthracene showed acute toxic effects (immobilization) at

18.9ppb after thirty minutes. Additional research demonstrated that PAHs exert acute

photoxicity upon algae (Cody et al., 1984, Gala and Giesy, 1994), amphipods (Ankley et

al. 1994), mosquito larvae (Kagan and Kagan, 1986), and bluegill sunfish (Oris and

34

Giesy, 1985). Oris and Geisy went on to also demonstrate that not only was the

concentration of PAHs/UVR important, but the time of UV exposure was also a

determining factor in PAH toxicity (1986). Both PAHs and TiO2 present themselves as

wide spread environmental contaminants that are both capable of exhibiting acute

toxicity that is not seen in the absence of UV exposure.

35

CHAPTER 6

CONCLUSIONS

In these experiments, it was shown that aqueous suspensions of TiO2 NP were

more phototoxic than TiO2 NP found in body burdens in D. magna. Furthermore, these

experiments have demonstrated effect levels at or below concentrations presented in

the current TiO2-NP environmental fate models. Depending on the physiochemical

properties of the water and the effects upon UV attenuation, it is likely that aquatic

organisms will be co-exposed to anatase TiO2-NP and natural UVR. This mechanism is

likely most relevant to alpine aquatic, estuarine, and shallow fresh water ecosystems

where UV penetration is likely highest.

TiO2 NP has demonstrated acute mechanisms of toxicity due to the ROS

generation when co-exposed with UVR. As nanoparticles, they also possess low

particle diameters and molecular weights. Taken together, these properties of TiO2 NP

bear many similarities to another class environmental contaminant, PAHs. Both

compounds possess the capability for large scale environmental exposure, low

molecular weights and a mechanism of phototoxicity that greatly increases the acute

toxic effect of the compound in aquatic environments exposed to UVR. Further research

would be necessary to compare the phototoxicity of both compounds to determine if

PAHs could be used as a template with which how to address TiO2 NP contamination.

Due to the lipophilic nature and lipid peroxidation during ROS generation, body burdens

are an accurate predictor of PAH toxicity. TiO2-NP however, waterborne concentration

is a better predictor of toxicity when compared to body burdens.

36

Uptake data has demonstrated that D. magna are not only are capable of

building up body burdens of TiO2-NP, they demonstrate the potential to re-suspend

TiO2-NP in solution at ppb concentrations. Stability data showed that water column

concentrations of TiO2-NP dropped drastically after two hours of exposure, which is

when the peak body burden of TiO2-NP was measured. In a more natural setting, such

as a flowing river, the TiO2-NP would be less likely to settle out of solution and remain

suspended for longer periods of time. While the NPs themselves are non-polar in

nature, but research has demonstrated the ability for NP to be transported into the

cytoplasm, which could lead to an accumulation in tissue under constant exposure.

Further researcher is needed to determine a body burden model for a constant flow

through exposure and how this would relate to work by Zhu et al. and how this body

burden would be transferred through the aquatic food web.

TiO2 NP uptake is directly proportional to the amount of NP suspended in the

water column at the time of contamination. Thus far, models have accounted for

industrial runoff, accidental exposure and sewage plant operation as mechanisms of

exposure. One likely mechanism of constant exposure and rexposure might be the

drying and sale of solid sewage sludge as organic fertilizer. These fertilizers are applied

in commercial and personal irrigation settings. In this small environment, terrestrial

plants and organisms may become exposed to TiO2 NP in a phototoxic manner. If not,

the possibility exists by which agricultural runoff may function as a constant source of

TiO2 NP in aquatic ecosystems that could possibly increase concentrations of TiO2 NP

in the water column. Previous research with PAHs has shown that the phototoxic effect

during co-exposures to different compounds of PAH were additive in nature and not

37

synergistic. When examining TiO2 NP concentrations in large bodies of water such as

rivers, lakes, estuaries, and Shallow Ocean environments it would be pertinent to

determine the nature of phototoxicity when organisms were exposed to TiO2 NP, PAH,

and UVR as it is likely that this will occur due to the widespread nature of environmental

contaminants.

It was observed during the exposure that the organisms demonstrated photo-

avoiding behavior. Organisms in control dishes would seek the ‘shadiest’ part of the

exposure dish and descent to the lowest part of the dish itself. TiO2-NP exposed

organisms also relocated to the bottom of the dish, but they did not attempt to seek out

the shaded areas of the crystallization dish. Many of them organisms remained resting,

covered in the TiO2-NP. Due to the surface area of the NP themselves, the waterborne

exposures were exposed to a greater bio-reactive surface area (Nel et al., 2006) than

their uptake and control counterparts. This increased surface area would increase the

interface between the oxygen rich water and the TiO2-NP exponentially increasing the

organism’s exposure to ROS, which may be responsible for the increased toxicity seen

in waterborne concentrations. One explanation for the reduced photo-avoidance is the

fact that the light sensing abilities of the daphnid’s eye were reduced due to ROS

damage from exposure to TiO2-NP. Further research could be done examining if TiO2-

NP organisms demonstrate a reduced ability to daphnid to sense incoming light in order

to avoid it.

38

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Bar-Ilan, O., Louis, K. M., Yang, S. P., Pedersen, J. A., Hamers, R. J., Peterson, R. E.,& Heideman, W. (2012). Titanium dioxide nanoparticles produce phototoxicity in thedeveloping zebrafish. Nanotoxicology, 6(6), 670-679.

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