EPA/600/R-09/057F 

Nanomaterial Case Studies: 

Nanoscale Titanium Dioxide in Water 

Treatment and in Topical Sunscreen

November 2010 

National Center for Environmental Assessment–RTP Division 

Office of Research and Development 

U.S. Environmental Protection Agency 

Research Triangle Park, NC

 
Disclaimer 

This document has been reviewed in accordance with U.S. Environmental Protection Agency 

policy and approved for publication. Mention of trade names or commercial products does not 

constitute endorsement or recommendation for use. 

 

 ii
Table of Contents 

List of Figures _________________________________________________________ vi

List of Tables _________________________________________________________ vii

Authors, Contributors, and Reviewers ____________________________________ viii

Abbreviations ________________________________________________________ xiii

Foreword ____________________________________________________________ xix

Chapter 1. Introduction ________________________________________________ 1-1

1.1. Background _________________________________________________________________ 1-1

1.2. How to Read this Document _____________________________________________________ 1-3

1.3. Terminology _________________________________________________________________ 1-4

1.4. Conventional TiO2_____________________________________________________________ 1-7

1.5. Nano-TiO2 ___________________________________________________________________ 1-8

1.5.1. Drinking Water Treatment _______________________________________________ 1-12

1.5.2. Sunscreen ___________________________________________________________ 1-13

1.6. Analytical Methods ___________________________________________________________ 1-14

1.6.1. Methods for Laboratory Research _________________________________________ 1-15

1.6.2. Methods and Instrumentation to Assess Environmental Occurrence ______________ 1-16

1.6.3. Methods and Instrumentation to Assess Workplace Exposure ___________________ 1-18

1.6.4. Summary of Analytic Methods ____________________________________________ 1-19

Chapter 2. Life Cycle Stages ____________________________________________ 2-1

2.1. Feedstocks __________________________________________________________________ 2-1

2.2. Manufacturing ________________________________________________________________ 2-2

2.2.1. Drinking Water Treatment ________________________________________________ 2-3

2.2.2. Sunscreen ____________________________________________________________ 2-3

2.3. Distribution and Storage ________________________________________________________ 2-3

2.3.1. Drinking Water Treatment ________________________________________________ 2-4

2.3.2. Sunscreen ____________________________________________________________ 2-4

2.4. Use ________________________________________________________________________ 2-4

2.4.1. Drinking Water Treatment ________________________________________________ 2-4

2.4.2. Sunscreen ____________________________________________________________ 2-6

2.5. Disposal ____________________________________________________________________ 2-7

2.5.1. Drinking Water Treatment ________________________________________________ 2-7

2.5.2. Sunscreen ____________________________________________________________ 2-7

Chapter 3. Fate and Transport __________________________________________ 3-1

3.1. Water ______________________________________________________________________ 3-2

3.1.1. Drinking Water Treatment ________________________________________________ 3-4

3.1.2. Sunscreen ____________________________________________________________ 3-5

3.2. Soil ________________________________________________________________________ 3-5

3.2.1. Drinking Water Treatment ________________________________________________ 3-7

3.2.2. Sunscreen ____________________________________________________________ 3-7

3.3. Air _________________________________________________________________________ 3-7

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Chapter 4. Exposure–Dose Characterization ______________________________ 4-1

4.1. Biota _______________________________________________________________________ 4-2

4.1.1. Aquatic Species ________________________________________________________ 4-2

4.1.2. Terrestrial Species ______________________________________________________ 4-3

4.2. Humans ____________________________________________________________________ 4-3

4.2.1. General Population _____________________________________________________ 4-3

4.2.1.1. Drinking Water Treatment ________________________________________ 4-3

4.2.1.2. Sunscreen ____________________________________________________ 4-4

4.2.2. Occupational __________________________________________________________ 4-6

4.3. Aggregate Exposure to Nano-TiO2 from Multiple Sources and Pathways __________________ 4-8

4.4. Cumulative Exposure to Nano-TiO2 and Other Contaminants ___________________________ 4-9

4.5. Models to Estimate Exposure ____________________________________________________ 4-9

4.6. Dose ______________________________________________________________________ 4-10

4.6.1. Uptake in Aquatic Species _______________________________________________ 4-11

4.6.1.1. Bioaccumulation _______________________________________________ 4-11

4.6.1.2. Food Web ____________________________________________________ 4-12

4.6.1.3. Cumulative Dose of Nano-TiO2 and Other Pollutants __________________ 4-12

4.6.2. Respiratory (Inhalation and Instillation) _____________________________________ 4-14

4.6.3. Dermal ______________________________________________________________ 4-16

4.6.4. Ingestion ____________________________________________________________ 4-24

4.6.5. Blood Brain Barrier and Placental Transfer __________________________________ 4-24

4.6.6. Dose Metrics _________________________________________________________ 4-26

Chapter 5. Characterization of Effects ____________________________________ 5-1

5.1. Factors that Influence Ecological and Health Effects of Nano-TiO2 _______________________ 5-1

5.1.1. Nano-TiO2 Physicochemical Characteristics __________________________________ 5-2

5.1.1.1. Size __________________________________________________________ 5-2

5.1.1.2. Crystallinity ____________________________________________________ 5-3

5.1.1.3. Surface Chemistry ______________________________________________ 5-3

5.1.1.4. Recommended Characterization of Nanomaterial for Ecological and 

Toxicological Studies ____________________________________________ 5-4

5.1.2. Experimental Conditions _________________________________________________ 5-6

5.1.2.1. Medium/Vehicle ________________________________________________ 5-7

5.1.2.2. Dispersion Preparation ___________________________________________ 5-8

5.1.3. Environmental Conditions ________________________________________________ 5-9

5.1.4. Summary ____________________________________________________________ 5-10

5.2. Ecological Effects ____________________________________________________________ 5-10

5.2.1. Ecological Effects of Nano-TiO2 Exposure __________________________________ 5-10

5.2.1.1. Effects on Bacteria and Fungi (Terrestrial and Aquatic) ________________ 5-16

5.2.1.2. Effects on Aquatic Organisms ____________________________________ 5-17

5.2.1.3. Effects on Terrestrial Organisms __________________________________ 5-21

5.2.1.4. Indirect and Interactive Ecological Effects ___________________________ 5-23

5.2.1.5. Summary ____________________________________________________ 5-23

5.3. Health Effects _______________________________________________________________ 5-24

5.3.1. Noncarcinogenic Effects ________________________________________________ 5-24

5.3.1.1. Studies in Humans _____________________________________________ 5-24

5.3.1.2. Animal Studies ________________________________________________ 5-25

5.3.1.3. Summary of Noncarcinogenic Effects ______________________________ 5-51

5.3.2. Carcinogenic Effects ___________________________________________________ 5-51

5.3.2.1. Studies in Humans _____________________________________________ 5-52

5.3.2.2. Animal Studies ________________________________________________ 5-52

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5.3.2.3. Modes of Action for Carcinogenicity ________________________________ 5-56

5.3.2.4. Summary of Carcinogenic Effects _________________________________ 5-58

Chapter 6. Summary __________________________________________________ 6-1

6.1. Case Study Highlights _________________________________________________________ 6-1

6.1.1. Analytical Methods _____________________________________________________ 6-2

6.1.2. Life Cycle Characterization _______________________________________________ 6-3

6.1.3. Fate and Transport _____________________________________________________ 6-5

6.1.4. Exposure and Dose Characterization _______________________________________ 6-6

6.1.4.1. Exposure Characterization ________________________________________ 6-6

6.1.4.2. Dose Characterization ___________________________________________ 6-8

6.1.5. Ecological and Health Effects ____________________________________________ 6-10

6.1.5.1. Ecological Effects ______________________________________________ 6-10

6.1.5.2. Health Effects _________________________________________________ 6-11

6.2. Role of Case Studies in Research Planning and Assessment Efforts ____________________ 6-12

6.2.1. Workshop on Research Priorities for Nano-TiO2 ______________________________ 6-12

6.2.2. Implications for Research Planning ________________________________________ 6-13

6.2.3. Implications for Future Assessment Efforts __________________________________ 6-13

REFERENCES ______________________________________________________ 6-15

Annex A. Nano-TiO2 in Sunscreen: Background Information _________________ A-1

REFERENCES _______________________________________________________ A-8

Annex B. Nano-TiO2 in Sunscreen: Manufacturing Processes ________________ B-1

REFERENCES ______________________________________________________ B-10

Annex C. Nano-TiO2 Exposure Control in the Workplace and Laboratory ______ C-1

REFERENCES _______________________________________________________ C-4

 v
List of Figures 

Figure 1-1. Basic structure of CEA as a framework for identifying and prioritizing research efforts. _________________________1-2

Figure 4-1. Possible pathways of nano-TiO2 skin penetration. ____________________________________________________ 4-19

Figure 5-1. The pulmonary effects of TiO2 or nano-TiO2 exposure through inhalation or instillation. _______________________ 5-58

Figure B-1. Generic manufacturing process for nano-TiO2 for sunscreens. __________________________________________ B-1

Figure B-2. Sulfate and chloride processes for TiO2 manufacture. _________________________________________________ B-2

Figure B-3. Nano-TiO2 manufacturing process used by Altair Nanotechnologies, Inc. __________________________________ B-3

 vi
List of Tables 

Table 1-1. Examples of nano-TiO2 physicochemical properties ____________________________________________________ 1-9

Table 1-2. Characterization of three nano-TiO2 particle types _____________________________________________________ 1-11

Table 1-3. Analytical methods for characterizing nanomaterials in aerosol and in liquid_________________________________ 1-16

Table 1-4. Analytical methods for nanomaterials in soil, sediment, and ground water for size fraction and distribution, surface area, 

and phase and structure _________________________________________________________________________ 1-18

Table 4-1. Estimated dermal exposure to nano-TiO2 from sunscreen containing 5% nano-TiO2 for adults and 3-year-old children 4-5

Table 4-2. Tissue concentrations of various pollutants in fish after exposures to nano-TiO2 in water _______________________ 4-13

Table 4-3. Nano-TiO2 disposition in animals after inhalation or intratracheal instillation _________________________________ 4-16

Table 4-4. Overview of TiO2 skin absorption/penetration studies __________________________________________________ 4-20

Table 4-5. Animal studies that measured Ti concentrations in brain after nano-TiO2 exposures through injection or oral gavage_ 4-25

Table 5-1. Published recommendations for measuring nanomaterial parameters for exposure during characterization inhalation 

studies ______________________________________________________________________________________ 5-5

Table 5-2. Published recommendations for off-line nanomaterial characterization using noncontinuous techniques for toxicological 

studies ______________________________________________________________________________________ 5-6

Table 5-3. Summary of nano-TiO2 ecological effects____________________________________________________________ 5-11

Table 5-4. Summary of health effects of nano-TiO2 particles in mammalian animal models: dermal route __________________ 5-27

Table 5-5. Summary of health effects of nano-TiO2 particles in mammalian animal models: oral route _____________________ 5-30

Table 5-6. Summary of health effects of nano-TiO2 particles in mammalian animal models: respiratory route _______________ 5-35

Table 5-7. Summary of health effects of nano-TiO2 particles in mammalian animal models: other (injection, ocular) route ______ 5-50

Table 5-8. Treatments and pulmonary tumor incidences in rats exposed to fine and nano-TiO2 through intratracheal instillation in 

Pott and Roller (2005) study ______________________________________________________________________ 5-54

Table 5-9. Incidence of tumor in the abdominal cavity of rats intraperitoneally injected with photocatalytic nano-TiO2. _________ 5-56

Table 5-10. Results of nano-TiO2 carcinogenicity studies in animals ________________________________________________ 5-59

Table A-1. TiO2 content in various sunscreen products. _________________________________________________________ A-7

Table B-1. Selected list of nano-TiO2 particles used in sunscreen _________________________________________________ B-6

Table B-2. Formula SC-383-1 for “Weightless Morning Dew with Sun Protection” _____________________________________ B-8

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Authors, Contributors, and Reviewers 

Principal Authors 

J. Michael Davis (Project Leader)—National Center for Environmental Assessment, Office of 

Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Thomas C. Long—National Center for Environmental Assessment, Office of Research and 

Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Jo Anne Shatkin—The Cadmus Group (currently with CLF Ventures) 

Amy Wang—Oak Ridge Institute for Science and Education, Postdoctoral Fellow to National Center 

for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection 

Agency, Research Triangle Park, NC 

Co-Authors 

Judith A. Graham—Private Consultant 

Maureen Gwinn—National Center for Environmental Assessment, Office of Research and 

Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Brent Ranalli—The Cadmus Group 

Contributors 

Christian Andersen—National Health and Environmental Effects Research Laboratory, Office of 

Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Walter Cybulski—Office of Science Policy, Office of Research and Development, U.S. 

Environmental Protection Agency, Washington, DC 

Genya Dana—Oak Ridge Institute for Science and Education, Postdoctoral Fellow to National 

Center for Environmental Assessment, Office of Research and Development, U.S. Environmental 

Protection Agency, Research Triangle Park, NC 

J. Allen Davis—National Center for Environmental Assessment, Office of Research and 

Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Steve Diamond—National Health and Environmental Effects Research Laboratory, Office of 

Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Kevin Dreher—National Health and Environmental Effects Research Laboratory, Office of Research 

and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Patricia Gillespie—Oak Ridge Institute for Science and Education, Postdoctoral Fellow to National 

Center for Environmental Assessment, Office of Research and Development, U.S. Environmental 

Protection Agency, Research Triangle Park, NC 

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Connie Meacham—National Center for Environmental Assessment, Office of Research and 

Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Emily Monosson—Private Consultant to the Cadmus Group 

Jeffery Morris—Immediate Office of the Assistant Administrator, Office of Research and 

Development, U.S. Environmental Protection Agency, Washington, DC 

Christine Ogilvie-Hendren—Oak Ridge Institute for Science and Education, Postdoctoral Fellow to 

National Center for Environmental Assessment, Office of Research and Development, U.S. 

Environmental Protection Agency, Research Triangle Park, NC 

Stephanie Rosch—The Cadmus Group 

Chon Shoaf—National Center for Environmental Assessment, Office of Research and Development, 

U.S. Environmental Protection Agency, Research Triangle Park, NC 

John Vandenberg—National Center for Environmental Assessment, Office of Research and 

Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Debra Walsh—National Center for Environmental Assessment, Office of Research and 

Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Sally White—Student Services Contractor to National Center for Environmental Assessment, Office 

of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

William Wilson—National Center for Environmental Assessment, Office of Research and 

Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

External Reviewers1

Pratim Biswas—Washington University 

Bernard Goldstein—University of Pittsburgh 

Fred Klaessig—Degussa 

Rebecca Klaper—University of Wisconsin 

Terry Medley—DuPont (with David Warheit, Gary Whiting, Scott Frerichs, and Brian Coleman) 

Srikanth Nadadur—National Institute of Environmental Health Sciences 

Gunter Oberdörster—University of Rochester 

John A. Small—National Institute of Standards and Technology (with Richard Holbrook) 

Jeffrey Steevens—U.S. Army Corps of Engineers 

Mark Wiesner—Duke University 

 

1

 Reviewers for the November 2007 draft of case study on nano-TiO2 in drinking water treatment. 

 ix
Peer Reviewers1

Steffen Foss Hansen—Technical University of Denmark 

Kiril D. Hristovski—Arizona State University 

Stephen J. Klaine—Clemson University 

Bernd Nowack—Swiss Federal Laboratories for Materials Testing and Research (EMPA) 

Annette B. Santamaria—ENVIRON International Corporation 

Kathleen E. Sellers—ARCADIS 

Workshop Participants2

David Andrews–Environmental Working Group 

Jeff Baker–TSI Incorporated 

Brenda Barry–American Chemistry Council 

Catherine Barton–DuPont 

Eula Bingham–University of Cincinnati 

Pratim Biswas–Washington University in St. Louis 

Jean-Claude Bonzongo–University of Florida 

Steven Brown–Intel Corporation 

Mark Bunger–Lux Research, Incorporated 

Carolyn Nunley Cairns–Consumers Union 

Richard Canady–McKenna, Long & Aldridge LLP 

Janet Carter–U.S. Occupational Safety and Health Administration 

Elizabeth Casman –Carnegie Mellon University 

Sylvia Chan Remillard–HydroQual 

Shaun Clancy–Evonik Industries AG 

Ramond David–BASF Corporation 

Joan Denton–California Environmental Protection Agency 

Gary Ginsberg–Connecticut Department of Public Health 

Pertti (Bert) Hakkinen–National Institutes of Health, National Library of Medicine 

Jaydee Hanson–International Center for Technology Assessment 

Patricia Holden–University of California, Santa Barbara, CA 

Paul Howard–U.S. Food and Drug Administration 

Sheila Kaplan–University of California, Graduate School of Journalism, Berkeley, CA 

 

1

 Reviewers of the April 2010 draft of Nanomaterial case Studies: Nanoscale Titanium Dioxide in Water Treatment and in Topical 

Sunscreen. 

2

 EPA’s “Nanomaterial Case Studies Workshop: Developing a Comprehensive Environmental Assessment Research Strategy for Nanoscale

Titanium Dioxide,” Research Triangle Park, NC, September 29-30, 2009 

 x
Fred Klaessig–Pennsylvania Bio Nano Systems, LLC 

Rebecca Klaper–University of Wisconsin, Great Lakes Water Institute 

Todd Kuiken–Woodrow Wilson International Center for Scholars, Project on Emerging 

Nanotechnologies 

John LaFemina–Battelle 

Thomas Lee–Minneapolis Star Tribune 

Shannon Lloyd–Concurrent Technologies Corporation 

Christopher Long–Gradient Corporation 

Margaret MacDonell–Argonne National Laboratory 

Fred J. Miller–Independent Consultant 

Nancy Monteiro-Riviere–North Carolina State University 

Paul Mushak–PB Associates 

Srikanth Nadadur–National Institutes of Health, National Institute of Environmental Health Sciences 

Michele Ostraat–Research Triangle Institute 

Anil Patri–Science Applications International Corporation, contractor with National Cancer Institute, 

Nanotechnology Characterization Laboratory 

Maria Victoria Peeler–Washington State Department of Ecology 

Richard Pleus–Intertox, Incorporated 

John Small–National Institute for Standards and Technology 

Jeff Steevens–U.S. Army Corps of Engineers Research and Development Center 

Geoffrey Sunahara–National Research Council, Canada, Biotechnology Research Institute 

Treye Thomas–U.S. Consumer Product Safety Commission 

John Veranth–University of Utah 

Donald Versteeg–The Procter & Gamble Company 

Nigel Walker–National Toxicology Program, National Institutes of Health, National Institute for 

Environmental Health 

William Warren-Hicks–EcoStat, Incorporated 

Paul Westerhoff–Arizona State University 

Mark Wiesner–Duke University 

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 xii

 

EPA Workgroup1

Christian Andersen# Kathryn Gallagher David Olszyk 

Rochelle Araujo Michael Gill Martha Otto 

Fred Arnold Michael Gonzalez Scott Prothero 

Ayaad Assaad Maureen Gwinn+ Kim Rogers 

Norman Birchfield Kathy Hart Nora Savage 

Deborah Burgin Tala Henry Phil Sayre 

Jim Caldwell Ross Highsmith Rita Schoeny 

David Cleverly Lee Hofmann Walter Schoepf 

Michele Conlon Marion Hoyer Najm Shamim 

Mary Ann Curran Joe Jarvis Deborah Smegal 

Walter Cybulski# Bernine Khan Jose Solar 

J. Michael Davis*+

 David Lai Neil Stiber 

Jane Denne Wen-Hsiung Lee Timothy Taylor 

Steve Diamond# Laurence Libelo Susan Thorneloe 

Jaimee Dong Diana Locke Dennis Utterback 

Kevin Dreher# Jacqueline McQueen* Amy Wang+

Jeremiah Duncan David Meyer Eric Weber 

Brian Englert Gregory Miller Randy Wentsel 

Patricia Erickson J. Vincent Nabholz Doug Wolf 

Cathy Fehrenbacher Nhan Nguyen 

Gina Ferreira Carlos Nunez 

U.S. EPA Editorial Support 

J. Sawyer Lucy—Student Services Contractor to National Center for Environmental Assessment, 

Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle 

Park, NC 

Deborah Wales—National Center for Environmental Assessment, Office of Research and 

Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 

Barbara Wright—Senior Environmental Employee to National Center for Environmental 

Assessment, Office of Research and Development, U.S. Environmental Protection Agency, Research 

Triangle Park, NC 

Contractor Editorial Support 

ICF International 

1

 *Co-Chair; +Co-author; #Contributor 
Abbreviations 

α-HBDH Alpha-hydroxybutyrate dehydrogenase 

ACGIH American Conference of Governmental Industrial Hygienists 

AFM Atomic force microscopy 

Al2(SO4)3 · 16H2O Alum 

Al2O3 Aluminum oxide, also known as alumina 

ALP Alkaline phosphatase 

ALT Alanine aminotransferase 

As(III) Arsenite 

As(V) Arsenate 

AST Aspartate aminotransferase 

BAL Bronchoalveolar lavage

BALF Bronchoalveolar lavage fluid 

BAuA German Occupational Safety and Health (Bundesanstalt für Arbeitsschutz und 

Arbeitsmedizin) 

BBB Blood brain barrier 

BET Brunauer, Emmett, Teller method of calculating surface area 

BrdU Bromo-deoxy-uridine 

BUN Blood urea nitrogen 

BW Body weight 

oC Degree(s) Celsius 

C60 Spherical fullerene composed of 60 carbon atoms; commonly “Bucky ball” 

Ca2+ Calcium cation 

CCOHS Canadian Centre for Occupational Health and Safety 

CE Capillary electrophoresis 

CEA Comprehensive environmental assessment 

CK Creatinine kinase 

cm2 Square centimeter(s), Centimeter(s) squared 

cm3 Cubic centimeter(s), Centimeter(s) cubed 

CMD Count median diameter 

CPC Condensation particle counter 

CREM Council for Regulatory Environmental Modeling 

CVD Chemical vapor deposition 

DIN Deutsches Institut für Normung (German Institute for Standardization) 

DLS Dynamic light scattering 

DMA(V) Dimethylarsinic acid 

DMEM Dulbecco’s Modified Eagle’s Medium 

DPPC Dipalmitoyl phosphatidylcholine 

EC3 Estimated concentration required to induce a threshold positive response, where stimulation 

index equals 3 

EC50 Half-maximal effective concentration, Effective concentration 50; the concentration at which 

50% of subjects show a response 

EDS Energy-dispersive X-ray analysis 

E-FAST V2.0 Exposure and Fate Assessment Screening Tool Version 2.0 

 xiii
EHS Environmental health and safety 

ELISA Enzyme-linked immunosorbent assay 

ELPI Electrical low pressure impactor 

EM Electron microscopy 

EN European Norm 

EPA U.S. Environmental Protection Agency 

EU European Union 

EWG Environmental Working Group 

oF Degree(s) Fahrenheit 

F344 Fischer 344 (Rat strain) 

FDA U.S. Food and Drug Administration 

FE-SEM Field emission-type scanning electron microscopy 

FeTiO3 Ilmenite 

FFF Field flow fractionation 

FHD Flame hydrolysis deposition 

γH2AX Phosphorylated form of histone H2AX (phosphorylation of H2AX at serine 139) 

g Gram(s) 

g/kg Gram(s) per kilogram 

GFAP Glial fibrillary acidic protein 

GGT γ–Glutamyltransferase 

GSD Geometric standard deviation 

GSH Reduced glutathione 

GSH-Px Glutathione peroxidase 

GST Glutathione-S-transferase 

H2O2 Hydrogen peroxide 

H2SO4 Sulfuric acid 

HBSS Hank’s Basic Salt Solution 

HCl Hydrochloric acid 

HEPA High efficiency particulate air 

HPLC High performance liquid chromatography 

hprt Hypoxanthine-guanine phosphoribosyltransferase (gene) 

HRTEM High resolution transmission electron microscopy 

Hz Hertz 

i.p. Intraperitoneal 

i.v. Intravenous 

IAEA International Atomic Energy Agency 

IARC International Agency for Research on Cancer 

IC20, IC25 Concentration that results in a 20% (or 25%) change (inhibition) from the control response, 

Inhibitory concentration at which organisms show 20%, 25% inhibition in measured 

endpoints 

ICP Inductively coupled plasma 

ICP-AES Inductively coupled plasma atomic emission spectrometry 

ICP-MS Inductively coupled plasma-mass spectrometry 

IEP Isoelectric point 

IFN-γ Interferon-gamma 

IL-10 Interleukin-10 

 xiv
IL-1β Interleukin-1β

IL-4 Interleukin-4 

IL-6 Interleukin-6 

IL-8 (KC) IL-8 = interleukin-8, KC = chemokine (CXC motif) ligand 1 (CXCL1) 

ILSI International Life Sciences Institute 

IOAA (U.S. EPA) Immediate Office of the Assistant Administrator 

ISO International Organization for Standardization 

ITT Isopropyl titanium triisostearate 

K+ Potassium cation 

kg Kilogram(s) 

L Liter(s) 

LC50 Concentration of a chemical that kills 50% of a sample population, Lethal concentration 50; 

the concentration at which 50% of subjects died 

LDH Lactate dehydrogenase 

LIBD Laser-induced breakdown detection 

LOEC Lowest observed effect concentration 

LOEL Lowest observed effect level 

LPS Lipopolysaccharide 

4-MBC 4-methylbenzylidene camphor 

μg Microgram(s) 

μg/g Microgram(s) per gram 

μg/kg Microgram(s) per kilogram 

μg/L Microgram(s) per liter 

μL Microliter(s) 

μm Micron(s), Micrometer(s) 

μm2

/cm3 Square microns per cubic centimeter, Micrometer(s) squared per centimeter cubed 

m2 Square meter(s), Meter(s) squared 

m2

/g Square meter(s) per gram, Meter(s) squared per gram 

m3 Cubic meter(s), Meter(s) cubed 

MARA Microbial array for risk assessment (assay) 

MCL Maximum contaminant level 

mg Milligram(s) 

mg/cm2 Milligram(s) per square centimeter, Milligram(s) per centimeter squared 

mg/kg Milligram(s) per kilogram 

mg/L Milligram(s) per liter 

mg/m3 Milligrams per cubic meter, Milligram(s) per meter cubed 

mg/mL Milligram(s) per milliliter 

Mg2+ Magnesium cation 

MgCl2 Magnesium chloride 

micro-TiO2 Microscale titanium dioxide 

mL/kg/day Milliliter(s) per kilogram per day 

mm Millimeter(s) 

mM Millimolar 

MMA(V) Monomethylarsonic acid 

MMAD Mass median aerodynamic diameter 

MMPD Multiple Path Particle Dosimetry (name of a computer model) 

 xv
MPPS Maximum penetrating particle size 

mSv Milliseviert 

MT Metric ton(s) 

MTC Microbial Toxic Concentration, in microbial array for risk assessment (MARA) assay 

MTP Microsomal triglyceride 

Na+ Sodium cation 

NaCl Sodium chloride 

NAG Nacetyl-β-glucosaminidase 

Nano-TiO2 Nanoscale titanium dioxide 

Nano-TiO2 F-1R Nanoscale titanium dioxide a formula containing nano-TiO2 that is 3% anatase and 97% rutile 

NCEA (U.S. EPA) National Center for Environmental Assessment 

Nano-TiO2 Nanoscale titanium dioxide 

ng/mL Nanogram(s) per milliliter 

NHEERL (U.S. EPA) National Health and Environmental Effects Research Laboratory 

NIOSH National Institute for Occupational Safety and Health 

nm Nanometer(s) 

NMR Nuclear magnetic resonance 

NMRI Naval Medical Research Institute 

NOEC No observed effect concentration 

NOM Natural organic matter 

NOSH Nanoparticle Occupational Safety and Health (Consortium) 

O2

- Superoxide radical anion 

OC Octocrylene 

OECD Organization for Economic Co-operation and Development 

OH Hydroxyl 

· OH Hydroxyl radical(s) 

OM Octyl methoxycinnamate 

· OOH Hydroperoxyl radical(s) 

OPC Optical particle counter 

OPPT (U.S. EPA) Office of Pollution Prevention and Toxics 

ORD (U.S. EPA) Office of Research and Development 

ORISE Oak Ridge Institute for Science and Education 

OSHA PEL Occupational Safety and Health Administration permissible exposure limit 

OSP (U.S. EPA) Office of Science Policy 

π Pi, approximately equal to 3.14159 

p Pink-eyed dilution (gene) 

P25 Degussa Aeroxide® P25 (uncoated nano-TiO2) 

P805 Degussa Aeroxide® P805 (hydrophobic nano-TiO2) 

PAM Pulse amplitude modulation 

PBS Phosphate buffered saline 

PEC Predicted environmental concentration 

pH Measure of acidity or alkalinity of a solution 

pHpzc pH at the point of zero charge 

PGF Placenta growth factor 

PMN Polymorphonuclear neutrophil 

PNEC Predicted no-effect concentration 

 xvi
ppb Part(s) per billion 

PPE Personal protective equipment 

ppm Part(s) per million 

PTFE Polytetrafluoroethylene 

Pt Platinum 

PTM Particle tracking model 

pun Pink-eyed unstable (locus) 

RLE-TN Rat alveolar type II epithelial cell line 

ROS Reactive oxygen species 

rPTM Radius particle tracking model 

RT-PCR Reverse transcription polymerase chain reaction 

σg Geometric standard deviation 

s.c. Subcutaneous 

SAXS/WAXS Small- and wide- angle X-ray scattering 

SCCNFP (European Commission) Scientific Committee on Cosmetic Products and Non-Food Products 

Intended for Consumers 

SCCP (European Commission) Scientific Committee on Consumer Products 

SCID Severe combined immunodeficiency 

SEC Size exclusion chromatography 

SEM Scanning electron microscopy 

SiO2 Silicon dioxide 

SMPS Scanning mobility particle sizer 

SOD Superoxide dismutase 

SPF Sunburn protection factor 

SPM Scanning probe microscopy 

St-C n Sunscreen standard C from the Japan Cosmetic Industry 

SWCNT Single-walled carbon nanotube(s) 

T805 Degussa Aeroxide® T805 (hydrophobic nano-TiO2) 

TEC Threshold effect concentration 

TEM Transmission electron microscopy 

TEOM® Tampered element oscillating microbalance 

TFF Tangential-flow ultrafiltration 

TGA (Australian) Therapeutic Goods Administration 

TGF-β Transforming growth factor-beta 

THF Tetrahydrofuran 

Ti Titanium 

TiCl4 Titanium tetrachloride 

TiO2 Titanium dioxide 

TiOSO4 Titanyl sulfate 

TLV Threshold limit value 

TNF-α Tumor necrosis factor-alpha 

TRAIL Tumor necrosis factor-related apoptosis-inducing ligand 

TS Technical Specification 

TUNEL Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling 

USP U.S. Pharmacopeia 

UV Ultraviolet (light/radiation), wavelengths in the range of 10 to 400 nm 

 xvii
UV-A Ultraviolet A, wavelengths in the range of 320 to 400 nm 

UV-B Ultraviolet B, wavelengths in the range of 290 to 320 nm 

VEDIC Video-enhanced differential interference contrast 

WHMIS (Canadian) Workplace Hazardous Materials Information System 

Wt% Weight percent 

XAS X-ray absorption spectroscopy 

XPS X-ray photon spectroscopy 

XRD X-ray diffraction 

ZnO Zinc oxide 

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Foreword 

Nanoscale materials (nanomaterials) have been described as having at least one dimension on 

the order of approximately 1 to 100 nanometers (nm) (National Nanotechnology Initiative, 2006, 

091186). Such materials often have unique or novel properties that arise from their small size. This 

document is a starting point to determine what is known and what needs to be known about selected 

nanomaterials as part of a process to identify and prioritize research to inform future assessments of 

the potential ecological and health implications of these materials. Two specific applications of 

nanoscale titanium dioxide (nano-TiO2) are considered: (1) as an agent for removing arsenic from 

drinking water; and (2) as an active ingredient in topical sunscreen. These “case studies” do not 

represent completed or even preliminary assessments, nor are they intended to serve as a basis for 

risk management decisions in the near term on these specific uses of nano-TiO2. Rather, the intent is 

to use this document in developing the scientific and technical information needed for future 

assessment efforts. 

The case studies are organized around the comprehensive environmental assessment (CEA) 

approach, which combines a product life-cycle framework with the risk assessment paradigm. Risk 

assessment relates exposure and effects information for a substance or stressor; CEA expands on this 

paradigm by including life-cycle stages and considering both indirect and direct ramifications of the 

substance or stressor. The organization of the document reflects the CEA approach: after Chapter 1 

(Introduction), Chapter 2 highlights stages of the product life cycle (feedstocks, manufacturing, 

distribution, storage, use, disposal), followed by Chapter 3 on fate and transport processes, Chapter 4 

on exposure-dose characterization, and Chapter 5 on ecological and health effects. Chapter 6 

highlights the information that is currently available in each of these areas, and it describes 

information gaps and research questions identified in the case studies. It also discusses the role of the 

case studies in informing research planning and future assessment efforts. Appendices A through C 

provide supplementary information on the use of nano-TiO2 in topical sunscreens, manufacturing 

processes for nano-TiO2 and sunscreen formulations, and examples of laboratory and workplace 

exposure control practices, respectively. 

The intent of these case studies is to characterize the current state of knowledge on the 

environmental impacts of nano-TiO2 as used in these two specific applications, as well as areas 

where information is missing. Note that some information gaps are specific to nano-TiO2, either as a 

drinking water treatment agent or as an ingredient in topical sunscreen. Other gaps may pertain more 

broadly to nano-TiO2 irrespective of its application, and still other gaps may pertain even more 

widely to nanomaterials in general. In this way, the case studies may be used in developing research 

strategies that will support comprehensive environmental assessments of nanomaterials. 

The case studies document has undergone a formal external peer review performed by 

scientists in accordance with Environmental Protection Agency (EPA) guidance on peer review 

(U.S. EPA, 2006, 194566). Six external peer reviewers reviewed the April 2010 draft of this 

document and provided responses to charge questions on the extent to which the case studies 

accurately and sufficiently characterize the state of understanding regarding the use of nano-TiO2 in 

drinking water treatment and sunscreens. This final document incorporates revisions in response to 

the peer review comments. 
Chapter 1. Introduction 

1.1. Background 

Nanoscale materials (nanomaterials) have been described as having at least one dimension on 

the order of approximately 1-100 nm (National Nanotechnology Initiative, 2006, 091186). 

Engineered nanomaterials are intentionally made, as opposed to being an incidental by-product of 

combustion or a natural process such as erosion, and they often have unique or novel properties that 

arise from their small size. These materials are being used in an expanding array of consumer 

products (The Project on Emerging Nanotechnologies, 2009, 196052), and, like all technological 

developments, nanomaterials offer the potential for both benefits and risks. The assessment of such 

risks and benefits relies on information, and given the nascent state of nanotechnology, much 

remains to be learned about the characteristics and impacts of nanomaterials before such assessments 

can be completed. This document is a starting point to identify what is known and, more importantly, 

what needs to be known about selected nanomaterial applications – in this case, for nanoscale 

titanium dioxide (nano-TiO2) – to assess their potential ecological and health implications.

This document focuses on two specific uses of nano-TiO2: as a drinking water treatment agent, 

and as an active ingredient in topical sunscreen. These “case studies” do not represent completed or 

even preliminary assessments; rather, they present the structure for identifying and prioritizing 

research needed to support future assessments of nano-TiO2 and an approach to study other 

nanomaterials. 

Part of the rationale for focusing on specific applications of selected nanomaterials is that such 

materials and applications can have highly varied and complex properties that make considering 

them in the abstract or in generalities quite difficult. Different materials and different applications of 

a given material could raise unique questions or issues, as well as some issues that are common to 

various applications of a given nanomaterial or even to different nanomaterials. After several 

individual case studies have been examined, refining a strategy for nanomaterials research to support 

long-term assessment efforts should be possible. 

The process for selecting case studies of nano-TiO2 in drinking water treatment and in topical 

sunscreen involved a workgroup representing several EPA program offices, regional offices, and 

Office of Research and Development (ORD) laboratories and centers. The EPA workgroup 

considered several candidate nanomaterials and identified their preferences based on, among other 

things, apparent relevance of the nanomaterial to EPA programmatic interests. The choice of specific 

applications was determined by a smaller team directly involved in the production of the case studies 

document. Among the factors guiding the selection process at each stage was the potential for 

exposure of ecological receptors and human populations to the nanomaterial as a function of a 

particular application. This is not to say, however, that the selection of these case studies signifies a 

determination that they present the greatest potential for exposure of all possible applications, or, for 

that matter, that any exposure actually occurs. Rather, the case studies simply provide a means to 

focus thinking about the types of information that would be instructive in assessing the potential 

ecological and health implications of selected nanomaterials. 

The case studies follow the CEA approach, which combines a product life-cycle framework 

with the risk assessment paradigm (Davis, 2007, 089803; Davis and Thomas, 2006, 089638). In 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments. 

 1-1
essence, risk assessment relates exposure and effects information for a given substance or stressor, 

and CEA expands on this paradigm by including life-cycle stages and considering both indirect and 

direct ramifications of the substance or stressor. Figure 1-1 illustrates the principal elements in the 

CEA approach. The first column of Figure 1-1 lists typical stages of a product life cycle: feedstocks, 

manufacturing, distribution, storage, use, and disposal (including reuse or recycling, if applicable). 

The second column lists environmental pathways or media (e.g., air, water, sediment, soil) to which 

nanomaterials or associated materials (e.g., manufacturing by-products) might be released at various 

stages of the life cycle. Within these media, nanomaterials or associated materials can be transported 

and transformed, as well as interact with other substances in the environment, both natural and 

anthropogenic. Thus, a combination of primary (e.g., manufacturing by-products) and secondary 

(e.g., environmental transformation products) contaminants can be spatially distributed in the 

environment (Column 3, Figure 1-1). 

Source: Adapted from Davis and Thomas (2006, 089638) and Davis (2007, 089803). 

Figure 1-1. Basic structure of CEA as a framework for identifying and prioritizing 

research efforts. 

The fourth column of Figure 1-1, exposure-dose, goes beyond characterizing the occurrence of 

contaminants in the environment, as exposure refers to actual contact between a contaminant and 

organisms (i.e., biota1

 as well as human populations). Under the CEA approach, exposure 

characterization can involve aggregate exposure across routes (e.g., inhalation, ingestion, dermal); 

 

1

 The term biota is used here to refer to all organisms other than humans. 

 1-2
cumulative exposure to multiple contaminants (both primary and secondary); and various 

spatiotemporal dimensions (e.g., activity patterns, diurnal and seasonal changes). Dose is the amount 

of a substance that actually enters an organism by crossing a biological barrier. Conceptually, dose 

links exposure with the last column of Figure 1-1, which refers to ecological and human health 

effects that can result when an effective dose reaches a target cell or organ in a receptor organism or, 

in an ecological context, when a stressor is at a sufficient level to cause an adverse response in a 

receptor. “Effects” encompass both qualitative hazards and quantitative exposure-response 

relationships. 

The CEA framework is highly simplified in Figure 1-1. Among the many direct and indirect 

impacts that could conceivably be included in a CEA are effects on other materials (e.g., damage to 

surfaces of structures, statuary, vehicles), hedonic or aesthetic qualities (e.g., alterations in visibility, 

taste, odor), and other possible large scale impacts such as energy consumption, resource depletion, 

and global climate change. Although none of these effects are being excluded a priori from 

consideration here, their inclusion would depend on having a plausible premise for expecting a 

discernible impact. If such a premise can be articulated for additional types of effects, the case study 

can be expanded to encompass their consideration within the CEA framework. 

CEA involves the elaboration and synthesis of information from the elements in all five 

columns depicted in Figure 1-1 to systematically evaluate the direct and indirect ramifications of a 

nanomaterial and its by-products. Underlying the CEA elements are analytical methods that make 

detection, measurement, and characterization of nanomaterials in the environment and in organisms 

possible. Not reflected in Figure 1-1 is an essential ingredient in making CEA effective – the 

inclusion of diverse technical and stakeholder perspectives to ensure that a holistic view is 

maintained. As either an assessment tool or as a framework for developing a research strategy, CEA 

is also a process that draws upon formal, structured methods to reach collective judgments by a 

diverse group of participants and contributors. 

Other efforts have been made to assess the potential risks of nanomaterials by incorporating a 

life-cycle perspective (e.g., Environmental Defense-DuPont Nano Partnership, 2007, 090565; 

Shatkin, 2008, 180065; Thomas and Sayre, 2005, 088085) or by using collective expert judgment 

methods (e.g., Kandlikar et al., 2007, 091626; Morgan, 2005, 088831), primarily in a risk 

management context. Although the present document differs somewhat from these other efforts in its 

purpose, namely to aid in developing a research strategy for the CEA of nanomaterial risks, all of 

these endeavors complement and reinforce one another. 

1.2. How to Read this Document 

The intent of this document is to identify systematically what is known and what needs to be 

known about nano-TiO2 to conduct an adequate assessment of nano-TiO2 in the future. The goal is 

not to provide an actual CEA or to state conclusions regarding possible ecological or health risks 

related to nano-TiO2, but to enable decisions on prioritizing research that would support future 

efforts to provide the input to policy and regulatory decision-making 

Although the differences between the applications of nano-TiO2 as a drinking water treatment 

agent versus a topical sunscreen are important, the information currently available does not allow 

complete differentiation between the two. For example, the ecological and health effects of 

nano-TiO2 are described in a single chapter without regard to whether the source of nano-TiO2 is 

drinking water treatment or sunscreen. However, where distinctions are possible or seem likely (e.g., 

in life-cycle stages such as manufacturing and use), the discussion of drinking water treatment is 

presented first, followed by discussion of sunscreen. In some sections, the discussions are not strictly 

parallel, reflecting differences in the availability of data. 

 1-3
Also important to note is that these case studies have been developed without a specific 

regulatory objective in mind. Although the topics selected for consideration, drinking water 

treatment and sunscreen, might be of interest in various policy and regulatory contexts, this 

document is not intended to serve as a basis for risk management decisions in the near term on these 

specific uses of nano-TiO2. Rather, the intent is to use this document in developing the scientific and 

technical information needed for future assessment efforts as input to policy and regulatory decisionmaking. 

Focusing on only two examples of nano-TiO2 applications obviously does not represent all the 

possible ways in which this nanomaterial could be used or all the issues that different applications 

could raise. Rather, by considering the commonalities and differences between two applications of 

nano-TiO2, research needs can be identified that apply not only to these specific applications but 

generally to nano-TiO2 and perhaps even more broadly to other nanomaterials. Also, additional case 

studies will be developed for other applications and nanomaterials so that this process can continue 

and research strategies to support assessment efforts can be further refined. 

When implemented, a CEA is intended to be comparative, examining the relative risks and 

benefits of different technological options, for example. The focus of a comparative CEA would be 

guided by risk management objectives. For example, the use of nano-TiO2 for arsenic removal in 

drinking water might be compared to one or more current methods for arsenic removal; use of 

nano-TiO2 for topical sunscreen might be compared to sunscreen containing conventional TiO2 or to 

sunscreen with organic ultraviolet (UV) radiation blocking agents. Given that a number of different 

options could be of interest to risk managers, considering every potential option in the present case 

study is not feasible. Therefore, this document focuses solely on nano-TiO2 in drinking water 

treatment and topical sunscreen, which is also consistent with the fact that the case studies are not 

intended to be assessments at this time, but rather are meant to assist in identifying and prioritizing 

research needs related to nano-TiO2. Readers seeking comparative assessments of topical sunscreen 

products, with or without nano-TiO2, may wish to consult evaluations by the Scientific Committee 

on Consumer Products (SCCP) (2007, 196826) and the Environmental Working Group (EWG) 

(2009, 196367). The EWG analysis in particular takes a broad view that is consistent with the CEA 

approach in referring to the product life cycle and noting potential ecological as well as human 

health considerations. 

This document is not intended to provide an exhaustive review of the literature, and focuses 

instead on findings most clearly relevant to assessment objectives. The information presented in this 

document was obtained from a variety of published and unpublished sources, including corporate 

Web sites and personal communications, as well as inferences based on information about other 

materials or applications. Such information sources are used because of the limited amount of 

published materials on nano-TiO2 and its applications in the peer-reviewed literature, coupled with 

the limited mechanisms for making manufacturer-specific data publicly available. This document is 

not an assessment but, rather, a means to identify information gaps and research questions, thus a 

range of information sources was used. 

1.3. Terminology 

A number of terms used in the field of nanotechnology have specialized meanings, and 

definitions of certain terms could have important legal, regulatory, and policy implications. Not 

surprisingly, perhaps, defining such words, including the term nanomaterial itself, has often been a 

matter of considerable interest and debate. For the purposes of this document, however, it is not 

deemed necessary to have a connotative definition that states the necessary and sufficient conditions 

that define a nanomaterial. Instead, a denotative approach is used; that is, the term “nanomaterial” in 

the case study denotes something that most persons would agree is (or at least appears to be) an 

 1-4
example of a nanomaterial or a product that incorporates a nanomaterial, regardless of whether a 

consensus exists regarding what properties or characteristics qualify it as such. 

Although this case study focuses on “nano-TiO2,” readers should note that this term 

encompasses a variety of materials that might possess a range of physicochemical properties. As a 

result, not all materials referred to as nano-TiO2 will necessarily behave in the same manner and 

exert the same biological effects. Thus, caution in extrapolating from one nano-TiO2 formulation to 

another when assessing hazards is appropriate. Conversely, until more information is available to 

discern more precisely how various formulations differ in behavior and effects, pooling information 

from multiple sources can be useful for the purposes of this document, namely to identify potential 

research directions to pursue.

Some other terms used throughout this document are discussed below, primarily to explain 

how the terms are used here rather than to attempt to provide a formal definition of them. 

Nano-TiO2 

This document focuses primarily on engineered nano-TiO2, which usually is in the form of 

particles in the 1 to 100 nm size range. The term “nano-TiO2,” as it is used in this document, refers to 

a variety of formulations containing titanium dioxide particles that meet this size-based definition. 

When reading this document, it is important to understand that the general use of this term 

encompasses specific formulations that can display a range of characteristics and behaviors 

depending on the properties of the particle, the experimental or environmental conditions, and other 

factors.1

 Where information is not specific to nano-TiO2, the term “titanium dioxide” (TiO2) is used 

without the “nano-” prefix. 

Conventional TiO2 

To make an explicit distinction between the nanoscale material and other forms of TiO2 not 

having the special characteristics of nano-TiO2, the term “conventional” is used in this document. 

Even so, materials described as conventional often contain a range of particle sizes, including some 

with nanoscale dimensions. In the scientific and technical literature, the terms “bulk” and 

“pigmentary” also are often used to distinguish conventional from nano-TiO2. Additionally, terms 

such as “ultrafine,” “PM-0.1” (which means particulate matter up to 0.1 micrometer [μm] diameter), 

and “micronized grade” have been used to denote nanoscale particles, but typically in a particular 

context or field of specialization such as aerosols and air pollution. 

Aggregate and Agglomerate 

As discussed in Chapter 3, in many circumstances primary nanoscale particles can aggregate

or agglomerate into secondary particles with dimensions greater than 100 nm (a cluster that is 

sometimes referred to as a colloid, as described below). Specifically, the terms “aggregate” and 

“agglomerate” are used in the literature on nanomaterials and other fields to indicate the clustering of 

particles into a single entity of such particles. These two terms can have specific meanings. For 

example, the British Standards Institution (BSI, 2007, 202162) defines aggregate as a “particle 

comprising strongly bonded or fused particles where the resulting external surface area may be 

significantly smaller than the sum of calculated surface areas of the individual components” and 

notes that “the forces holding an aggregate together are strong forces, for example, covalent bonds, 

or those resulting from sintering or complex physical entanglement.” The BSI defines agglomerate 

as a “collection of loosely bound particles or aggregates or mixtures of the two where the resulting 

external surface area is similar to the sum of the surface areas of the individual components” and 

 

1

 Where sources have provided documentation on size, surface coating, extent of aggregation or agglomeration, and other salient properties 

or characteristics, this information is included in the case study with sources referenced appropriately. 

 1-5
notes that “the forces holding an agglomerate together are weak forces, for example van der Waals 

forces, as well as simple physical entanglement.” However, the meanings of aggregate and 

agglomerate have sometimes been interchanged, as noted by Nichols et al. (2002, 202114). This lack 

of consistency in terminology usage across, and sometimes within, the various fields that contribute 

to nanomaterials research exemplifies the challenges posed by the multidisciplinary nature of the 

nanotechnology field. The nanotechnology community is an amalgam of investigators who all study 

nanoscale materials but whose scientific roots are in various other mature fields spanning toxicology, 

ecology, colloid science, materials science, and many other disciplines. The customary terminology 

for aggregates and agglomerates may be well established within one field, but use of these terms 

may elicit different interpretations within another; as a result, the definitions for these terms are not 

specific, nor are they consistent. Given this inconsistency in usage and, more importantly, the 

frequent lack of adequate information to determine which term might be more appropriately applied 

in a particular study or report, the term “cluster” is used in this document to subsume both 

aggregates and agglomerations of nanoparticles. This term has precedent within multiple disciplines 

and avoids confusion between potentially inconsistent connotations of the other terms. Note that, in 

addition to being used as a noun (as explained above), the word “aggregate” is used as an adjective 

(primarily in Chapter 5) to refer to exposure to a given material from multiple sources, pathways, 

and routes. 

Colloid 

The term “colloid” is used in the literature to refer to a particle or cluster of particles 

suspended within a given medium and that are smaller than microscale (i.e., <10-6 m). Luoma (2008, 

157525) describes a colloidal particle as containing multiple atoms of a substance measuring 

between 1 nm and 1,000 nm, and thus a colloid might or might not be a nanoparticle in that context. 

In this case study, although the term “colloid” is used at times to refer to a sub-microscale particle 

(especially if a cited publication uses this terminology), either the more specific term “nanoscale” or 

a specific size range is used when the particle size is salient to the discussion. 

The extent to which the properties of a cluster of primary nano-TiO2 particles that exceeds 

100 nm are similar to the properties of conventional TiO2 is unclear. For example, inhalation of 

nano-TiO2 (20-nm diameter) induced more pulmonary inflammation in the rat than inhalation of fine 

TiO2 (approximately 250-nm diameter) at a similar mass concentration, even though particles in both 

groups had similarly sized agglomerates (0.71 μm mass median aerodynamic diameter [MMAD] 

nano; 0.78 μm MMAD fine) (Oberdörster, 2000, 036303; Oberdörster et al., 1994, 046203). 

Additional analysis revealed that effects were similar when expressed on the basis of surface area. 

Also unclear is the extent to which changes in conditions might initiate the formation, 

decomposition, or dissolution of a cluster; and there is uncertainty as well regarding what specific 

factors drive important changes in conditions. As discussed in Chapter 3 (Fate and Transport), 

disaggregation can occur under some conditions. Given these considerations, this document does not 

use 100 nm as the essential and exclusive criterion for considering what might be relevant to an 

evaluation of nano-TiO2. This view is consistent with a statement by the European Commission 

(2008, 196378) that extends the term “nanomaterial” to encompass “nanostructured materials,” 

defined by the International Organization for Standardization (ISO) (2004, 190006) as “[a]ggregates 

and agglomerates, often existing at a micro size, [that] may have some of the behaviour and effects 

of their smaller sub units, e.g., due to an increased surface area.” 

Naturally Occurring, Incidental, and Engineered Nanoparticles 

In addition to distinctions based on size of particles, The Project on Emerging 

Nanotechnologies (2009, 196052) divides nanoscale materials into three classes based on the origin 

of the particles. The first class, naturally occurring nanosized particles, includes, for example, 

particles that originate from volcanic explosions, ocean spray, and soil and sediment weathering and 

 1-6
biomineralization processes (which can result in crystals of aluminum and iron oxides with 

nanometer-scale dimensions). The second class is incidental nanosized particles, which are generated 

as by-products of processes such as combustion, cooking, or welding. The focus of this report is on 

the third class of nanoscale materials, engineered nanomaterials. This class comprises materials 

purposely generated for a specific function, such as the carbon nanotubes used in tennis rackets to 

make them lighter and stronger. In this case study, unless otherwise specified, references to nanoTiO2 indicate engineered nanoscale materials. Nonengineered types of nanosized TiO2 (from the first 

or second class) are referred to as “nanoscale TiO2.” 

Degussa Aeroxide® P25 (hereafter referred to as P25) is a commercial-grade, uncoated 

nano-TiO2 product (Evonik, 2008, 157578) that has been studied extensively and referenced in the 

literature and is therefore often mentioned in later sections of this document. As discussed below, 

however, P25 does not represent all nano-TiO2 preparations and should not be equated with the 

generic term nano-TiO2. 

1.4. Conventional TiO2

Although this document focuses on nano-TiO2, highlighting some facts about conventional 

TiO2 first is instructional. Also known as “titania,” TiO2 has been used commercially since the early 

1900s in numerous consumer and industrial applications, particularly coatings and pigments. TiO2 is 

a naturally occurring mineral that can exist in three crystalline forms, known as rutile, anatase, and 

brookite, and in amorphous form. Rutile is the most common form of TiO2 found in nature. 

Elemental Ti is also found in ilmenite (FeTiO3) and other minerals and ores, and TiO2 can be 

produced by processing of these minerals and ores. TiO2 is insoluble in water, hydrochloric acid, 

nitric acid, and ethanol, but soluble in hot concentrated sulfuric acid, hydrogen fluoride, and alkali 

(NRC, 1999, 091188). TiO2 is used to increase the whiteness or opacity of many consumer products, 

such as paints, coatings, plastics, paper, printing inks, roofing granules, food, medicine, toothpaste, 

cosmetics, and skin care products, including topical sunscreens. In the U.S., surface-mining 

operations in Virginia and Florida produce concentrated Ti-containing minerals (ilmenite and rutile) 

suitable as feedstock for TiO2 production (USGS, 2009, 157454). Other countries that produce 

significant amounts of Ti ores include Australia, Canada, China, India, Norway, and South Africa 

(USGS, 2009, 157454). 

With exposure to ultraviolet (UV) radiation (wavelengths less than ~400 nm), pure TiO2 is 

photocatalytic.

1

 Studies suggest anatase and rutile have different photocatalytic properties, with 

anatase being the more reactive (Sayes et al., 2006, 090569; Uchino et al., 2002, 090568). In 

applications such as paints, coatings, and cosmetics, where chemical stability is required, the 

photocatalytic properties of TiO2 are often suppressed by coating the particles with silica and 

alumina layers. On the other hand, the photocatalytic properties of TiO2 are increasingly exploited in 

a number of other experimental and commercial applications, including degradation of organic 

compounds, microbiological organism destruction, and conversion of metals to less soluble forms in 

wastewater, drinking water, and indoor air. For more information on conventional TiO2, see the 

article by Diebold (2003, 193342) and the Current Intelligence Bulletin published by the National 

Institute for Occupational Safety and Health (NIOSH) (2005, 196072). 

 

1

 Photocatalysis is the phenomenon by which a relatively small amount of material, called a photocatalyst, increases the rate of chemical reaction 

without itself being consumed. (adj. photocatalytic). 

 1-7
1.5. Nano-TiO2

One of the main differences between nano-TiO2 and conventional TiO2 is the much greater 

surface area of a given mass or volume of nanoparticles compared to an equivalent mass or volume 

of conventional TiO2 particles. To illustrate, a 5-nm particle would have a volume of 65 nm3

(4/3 π r3

) whereas a 500-nm particle would have a volume of 65,000,000 nm3. Therefore, one million 

5-nm particles would be required to equal the volume of a 500-nm particle. The surface area of a 

5-nm particle equals approximately 80 nm2

 (4 π r2), whereas the surface area of a 500-nm particle 

equals approximately 800,000 nm2. Multiplying the surface area of the 5-nm particle by one million 

(the number of 5-nm particles needed to equal the volume of a 500-nm particle) yields a total surface 

area of approximately 80,000,000 nm2

, which is 100-fold greater than the surface area of the 500-nm 

particle. This greater relative surface area of the nano-TiO2 particles affords a greater potential for 

properties such as catalytic activity and UV absorption at certain wavelengths (Shao and 

Schlossman, 1999, 093301). 

Such properties have led to the development or use of nano-TiO2 for a wide variety of 

applications, including self-cleaning surface coatings, light-emitting diodes, solar cells, disinfectant 

sprays, sporting goods, and the subjects of this document, drinking water treatment agents and 

topical sunscreens. Before considering specific applications of nano-TiO2, some fundamental issues 

related to characterization of this material should be noted. 

Surface areas of nano-TiO2 primary particles, aggregates, and agglomerates can be expressed 

as total (inner and outer) surface area and external surface area. The total surface area includes the 

inner surface area of porous or aggregated or agglomerated nanoparticles (Scientific Committee, 

2007, 157639), and can be measured by the Brunauer, Emmett, Teller method (BET) and other 

methods. The external surface area, which is insensitive to particle porosity, can be measured 

indirectly by microscopy, diffusion chargers, scanning mobility particle sizers, and other methods 

(LeBlanc, 2009, 625209). Whether the total surface area or external surface area is more relevant for 

nanoparticle effects has not been determined. In one instance, humic acid caused nano-TiO2

micropore blockage and consequently decreased the total surface area, but not the external surface 

area (Yang et al., 2009, 190513). Humic acid-coated nano-TiO2 had lower zeta potential (i.e., 

increased electrostatic repulsion), which leads it to be more easily dispersed and suspended than 

uncoated nano-TiO2 (Yang et al., 2009, 190513). External surface area alone, however, does not 

always predict the nature and magnitude of effects. When possible, the method of measuring surface 

area is provided when discussing studies in this document. 

Several types of nano-TiO2 are available with differing physicochemical properties. 

Commercially available brands of nano-TiO2 can vary in particle size1

, surface area, purity (e.g., due 

to doping, coating, or quality control), surface characteristics, crystalline form, chemical reactivity, 

and other properties (Table 1-1). Nano-TiO2 is available in pure anatase, pure rutile, and mixtures of 

anatase and rutile. In general, anatase nano-TiO2 is more photocatalytic than the rutile form, and 

nanoscale rutile is less photoreactive than either anatase and rutile mixtures or anatase alone. 

However, a mixture of 79% anatase and 21% rutile nano-TiO2 (P25) was found to be more 

photocatalytic than 100% anatase nano-TiO2 in some instances (Coleman et al., 2005, 089849; 

Uchino et al., 2002, 090568), but less effective in others (Nagaveni et al., 2004, 090578). Such 

contrasts point to the role of other factors in accounting for the behavior and effects of nano-TiO2. 

For example, surface treatment of nano-TiO2 can change nano-TiO2 activity, including 

photoreactivity. Aeroxide® T805, which is P25 nano-TiO2 that has been treated with trialkoxyoctyl 

silane on the surface to increase hydrophobicity, has lower surface reactivity than P25 as indicated 

 

1

 The sizes of nanoparticles may be reported either as crystallite size (sometimes called primary crystallite size, such as size determined by 

X-ray Diffraction analysis) or as primary particle size, which is typically larger than crystallite size. (Barton, personal communication, 

2009, 625563). 

 1-8
by a vitamin C oxidation assay (Degussa, 2003, 193916). Similarly, surface coatings of silicone and 

other compounds are used to decrease nano-TiO2 photoreactivity so that nano-TiO2 can be used to 

protect human skin, plastic, and other objects from UV radiation. This document presents 

information on both coated and uncoated nano-TiO2, recognizing that differences in properties and 

environmental behavior may limit the applicability of information from one particle type to another. 

Table 1-1. Examples of nano-TiO2 physicochemical properties 

Agglomeration/aggregation status in 

the relevant media 

Bulk density/particle density 

Composition/surface coatings 

Crystal structure/crystallinity

(crystalline phase, crystallite size) 

Dustiness 

Octanol-water partition coefficient 

Particle size and size distribution 

Photocatalytic activity 

Pore density 

Porosity 

Purity of sample 

Radical formation potential 

Redox potential 

Shape/aspect ratio (e.g., width and 

length) 

Surface area/specific surface area 

Surface charge/zeta potential 

Surface chemistry 

Surface contamination 

Surface reactivity 

Water solubility 

Source: Data from U.K. Department for Environment, Food, and Rural Affairs (2007, 195461); Used with permission from Oxford University 

Press, Powers et al. (2006, 088783); Used with permission from Informa Healthcare, Powers et al. (2007, 090679); Used with permission from 

Informa Healthcare, Warheit et al. (2007, 091305); and data from Organisation for Economic Co-operation and Development (OECD) (2008, 

157512). 

External factors can also influence photoreactivity. Krishna and co-authors (2006, 193482), for 

example, found that the presence of fullerenes, which scavenge photogenerated electrons, enhances 

the photocatalytic efficacy of nano-TiO2. Likewise, Komaguchi and colleagues (Komaguchi et al., 

2006, 193479) saw significant increases in photocatalytic efficiency of P25 after exposure to an 

oxidizing environment. 

Photocatalytic nano-TiO2 is preferred for drinking water treatment, and photostable nano-TiO2

is preferred for sunscreen use. Some sunscreens, however, contain photoreactive nano-TiO2. 

Although pure uncoated and undoped anatase TiO2 is photocatalytic, and uncoated and undoped 

rutile TiO2 is generally photostable, there is no quick way to identify the photoreactivity of 

nano-TiO2. For example, although doped rutile nano-TiO2 can be extremely photostable, rutile 

nano-TiO2 produced by a certain specific powder-preparation method can be highly photocatalytic 

(Kim et al., 2003, 157861). Similarly, not all coatings decrease nano-TiO2 photoreactivity. 

A report by Barker and Branch (2008, 180141) has noted that nano-TiO2 in some sunscreens 

might not be photostable. The investigators studied the weathering of paint in contact with 

sunscreen. Of five nano-TiO2 sunscreens tested, four released photocatalytically generated hydroxyl 

radicals that accelerated the weathering of the paint. All four of those sunscreens used an 

anatase/rutile mix. The one nano-TiO2 sunscreen formulation that showed no appreciable effect on 

paint weathering used 100% rutile doped with manganese rather than surface coating with 

manganese (Barker and Branch, 2008, 180141). 

Due to various degrees of porosity, nano-TiO2 particles with the same diameter can differ in 

surface area. Because nano-TiO2 reactivity and consequently behavior and effects are influenced by 

many nano-TiO2 physicochemical properties, two nano-TiO2 products with the same values for a 

limited set of parameters should not be assumed in fact to be equivalent. For instance, a 

manufacturer might use the same core nano-TiO2 for surface-treated and untreated nano-TiO2, and 

both might have the same particle size and surface area, but differ in reactivity, as in the case of P25 

and Aeroxide® T805 (T805). 

Another characteristic of significance is the aggregation or agglomeration of nano-TiO2

particles. According to one industrial manufacturer of nanoscale titania produced through flame 

 1-9
hydrolysis (see Section 2.2 for a description of this manufacturing technique and others), “tests and 

calculations have shown that free primary particles with dimensions of less than 100 nm only exist in 

[flame] reactors for a few milliseconds” (Degussa, 2009, 193919). Aggregates of nano-TiO2, 

sometimes referred to as “colloidal,” are often roughly an order of magnitude greater in size than 

primary particles (Dunphy Guzman et al., 2006, 090584; Kormann et al., 1988, 090582; Lecoanet et 

al., 2004, 089258). The mean aggregated particle diameter of the product P25 is claimed to be 

approximately 3.6 μm, with the smallest 4% of aggregated particles reported to have an average 

diameter of 160 nm (Klaessig, personal communication, 2006, 196041). After being subjected to 

bath sonication for 10 minutes, the smallest 15% of P25 particles averaged an agglomerate diameter 

of 160 nm, while the 50th percentile diameter was 1.6 μm, roughly two orders of magnitude larger 

than the reported primary particle size of P25, which is 21 nm (Degussa, 2007, 193917; Wahi et al., 

2006, 090580). Ridley et al. (2006, 090599) observed that a suspension of uncoated nano-TiO2

anatase from Ishihara Techno Corporation (Osaka, Japan) with primary particles of 4-nm diameter 

consisted mainly of aggregates in the 1- to 30-μm diameter range, and that these size ranges 

persisted even under sonication and other conditions that would favor disaggregation. Reported 

particle size values for aggregates and agglomerates are influenced by factors such as initial 

concentration, sonication power input, pH, and measurement technique. Such variables are germane 

to interpreting the results of reported size distributions. 

Despite the presence, and sometimes the predominance, of larger aggregates and 

agglomerates, several researchers investigating laboratory-synthesized anatase and commercial 

nano-TiO2 products such as P25 have also found free particles or aggregates with diameters <100 nm 

in varying amounts. The quantity of such particles has been found to depend on synthesis method, 

temperature, solution pH, and the presence of buffers (Jiang et al., 2009, 193450). Moreover, some 

preparations are specifically designed to generate dispersed particles (e.g., Seok et al., 2006, 091198) 

which would be important in using nano-TiO2 as a catalyst. 

The pHpzc of a nanoparticle (the pH at the “point of zero charge,” where the net electric charge 

at the particle surface is zero) has important ramifications for aggregation, because at that pH 

particles will fail to electrostatically repel each other. In laboratory studies, the size range of 

aggregates and the presence of free nano-TiO2 particles (synthesized on-site, ranging from 5 to 

50 nm) were found to be pH-dependent; when the solution pH differed from the pHpzc of the 

particles, the aggregates tended to be smaller (Dunphy Guzman et al., 2006, 090584; 

Dunphy Guzman, personal communication, 2007, 091184). Sampled aggregates ranged up to 

150 nm in size, and contained an estimated 8-4,000 nanoparticles (Dunphy Guzman et al., 2006, 

090584). The pHpzc also depends at least in part on the crystallinity of the nano-TiO2 particles; 

Finnegan et al. (2007, 193379) reported pHpzc values of approximately 5.9 for rutile and 

approximately 6.3 for anatase. 

Coatings and surface treatments also affect particle aggregation/agglomeration behavior. A 

preliminary report by Wiench and colleagues (2007, 090635) indicated that coated nano-TiO2

particles (rutile, size 50 × 10 nm, surface area of 100 square meters per gram [m2

/g]; coatings 

included combinations of aluminum hydroxide, hydrated silica, and various polymers) had slower 

agglomeration and sedimentation rates and a larger fraction of primary nanoparticles remaining in 

the sample compared with uncoated particles (20-30 nm, anatase/rutile 80/20, surface area 

48.6 m2

/g). 

A recent study showed that a type of coated nano-TiO2 used in sunscreens quickly lost its 

outermost coating and became easily dispersed soon after contact with water (Auffan et al., 2010, 

625063). The tested nano-TiO2, T-LiteTM SF (manufactured by BASF company, Germany) had a 

mainly rutile TiO2 core, coated with Al(OH)3 and an outermost hydrophobic coating with 

polydimethylsiloxane (PDMS). The structural changes were seen within 30 minutes of aging 

(stirring) when the initially hydrophobic nano-TiO2 started to disperse into the aqueous phase of the 

suspension. The PDMS coating was dissolved, and the dissolution rate was accelerated by UV 

exposure. Aluminum was not detected, suggesting that the Al(OH)3 outer layer was more stable. 

 1-10
The complexity of nano-TiO2 characterization is illustrated in Table 1-2, from Warheit et al. 

(2007, 091075). The chemical composition of three different types of ultrafine TiO2 manufactured by 

DuPont was determined by X-ray fluorescence. The cores of all three types of nano-TiO2 were TiO2, 

but the crystalline form and the surface coating of alumina or silica differed. Each type of particle 

was said to exhibit a mean diameter of approximately 140 nm but with (unspecified) fractions of the 

size distributions below 100 nm as measured by dynamic light scattering. The chloride ions on the 

surface of the particles were neutralized during production (Other effects these materials cause are 

described in Chapter 5). As shown in Table 1-2, the surface area, crystallinity, chemical reactivity, 

surface coating, particle size distribution, and pH varied for the materials, all three of which were 

nominally nano-TiO2. Even with detailed information such as provided here, additional details may 

be necessary to fully characterize the complex nature of nano-TiO2 nanomaterials. In particular, the 

presence of a surface coating changes the nature of the interface between the nano-TiO2 particle and 

the environment or an organism, and it is not clear whether the surface coating or the core material 

dominates particle-environment and particle-organism interactions. 

Table 1-2. Characterization of three nano-TiO2 particle types 

Median Particle Size and 

Size Rangeb pH in 

Deionized 

Water 

Particle 

Type 

BET Surface 

Area (m2/g) 

Chemical 

Reactivity Chemical Composition a

in Waterc in PBS 

Uf-A 18.2 98% TiO2 (100% rutile), 2% alumina 10.1 136 nm ± 35% 1,990 nm ± 25% 5.64 

Uf-B 35.7 88% TiO2 (100% rutile), 5% alumina, 

7% silica 

1.2 149.4 nm ± 50% 2,669 nm ± 25% 7.14 

Uf-C 38.5 92% TiO2 (79% rutile; 21% anatase), 

7% alumina, 1% silica 

0.9 140 nm ± 44% — 4.80 

BET – Brunauer, Emmett, Teller method of calculating surface area 

PBS – Phosphate buffered saline 

a

Chemical reactivity was tested using a Vitamin C (antioxidant) yellowing assay. 

b

After sonication for 15 min at 60 Hertz (Hz). 

c

0.1% tetrasodium pyrophosphate solution. 

Source: Modified with permission from Elsevier, Warheit et al. (2007, 091075). 

The characteristics of a nano-TiO2 product might change over time. Using a custom-made 

anatase nano-TiO2 formulation (uncoated) with a range of particle sizes, Kolář et al. (2006, 193478) 

found that average particle sizes increased over time, due to both agglomeration and 

re-crystallization (smaller particles dissolving in the aqueous medium and their constituent 

molecules then adding to the mass of the larger particles). Over the course of 8 years, average 

(mode) particle size increased from approximately 10 nm to approximately 14 nm. The investigators 

also observed that over time relative surface area decreased, light energy absorbance characteristics 

changed, and perhaps most surprisingly, photocatalytic performance improved, even as relative 

surface area decreased. 

As discussed in greater detail in Chapter 5 (Section 5.1.1), these and other issues have been 

noted in various recommendations for improving the physicochemical characterization of 

nanomaterials in exposure and ecological as well as health effects studies. In general, however, 

reports of toxicity and exposure studies of nano-TiO2, especially those conducted prior to the year 

2000, have not been sufficiently attentive to the issues described above. As discussed in 

Section 1.6.1, ideal characterization to support toxicological testing would include analysis of the 

raw material (as received from the manufacturer or supplier), analysis of nanomaterials in the testing 

 1-11
media for the duration of the experiment; and analysis of nanomaterials (and possibly degraded 

products or biotransformed products) in biological samples. Manufacturers’ literature often has been 

accepted as having described their products under all conditions – an oversimplification at best. 

Additionally, attempts to characterize nanoscale particle sizes and size distributions in relation to 

toxicity and exposure evaluations have been prone to errors involving nonrepresentative sampling, 

agglomeration during sample preparation, contamination and degradation during product storage, 

measurement methods, and conditions under which the study was conducted (Powers et al., 2007, 

090679). Further, different particle characterization techniques can produce different estimates of 

particle size, suggesting that more than one technique might be necessary to describe particle sizes 

accurately. Accurate characterization is clearly important if the behavior and effects of nano-TiO2 are 

to be understood, predicted, and related to other materials (both nanoscale and conventional), and the 

type and extent of characterization is an important consideration in interpreting the results of nanoTiO2 research. 

1.5.1. Drinking Water Treatment 

This document assumes that one use of nano-TiO2 would be specifically for arsenic removal in 

a drinking water treatment facility. In addition to arsenic removal (Li et al., 2009, 193506), however, 

nano-TiO2 could be used for disinfection of pathogens (Alrousan et al., 2009, 157461; Coleman et 

al., 2005, 089849; Li et al., 2008, 157538; Rincon and Pulgarin, 2003, 157856) or for remediation of 

ground water or wastewater contaminated with various organic and inorganic pollutants (Adams et 

al., 2004, 193250; Chen and Ray, 2001, 193310; Han et al., 2009, 193407; Kim et al., 2003, 193472; 

Lee et al., 2008, 098739; Lin and Valsaraj, 2003, 193511; Ryu and Choi, 2008, 157501). The latter 

use would pose rather different scenarios of environmental releases and fate and transport, and 

would add considerably to the complexity of this document. Therefore, the case study of nano-TiO2

for water treatment has been limited to the consideration to arsenic removal in drinking water 

treatment facilities. 

Most of the relevant literature has reported laboratory tests of nano-TiO2 as a photocatalytic 

treatment for conversion of arsenite [As(III)] to arsenate [As(V)], a species that is more easily 

removed in water treatment because of its lower solubility in typical drinking water treatment 

conditions (e.g., Dutta et al., 2004, 157845; Ferguson et al., 2005, 090572; Pena et al., 2006, 

090573). Although neither conventional TiO2 nor nano-TiO2 is known to have been used in a fullscale drinking water treatment plant, both conventional TiO2 and nano-TiO2 as photocatalytic agents 

have been pilot-tested in drinking water treatment plants (Dionysiou, personal communication, 2009, 

193921; Pichat, 2003, 196037; Purifics, 2008, 196040; Richardson et al., 1996, 193612). 

For arsenic removal from water, both conventional and nanoscale TiO2 have been developed to 

photocatalytically oxidize arsenic and adsorb arsenic. Studies have shown that TiO2 can oxidize 

As(III) to As(V) and adsorb inorganic arsenic (Dutta et al., 2004, 157845; Fostier et al., 2008, 

193381; Hristovski et al., 2007, 193436). The mechanism for TiO2 photocatalytic oxidation of 

As(III) has been suggested to be through the generation of superoxide ions, and the major oxidant 

species might be hydroxyl radicals (·OH) (Sharma and Sohn, 2009, 193641). Recently, nano-TiO2

was shown to mineralize methylated arsenic and to adsorb methylated arsenic (Xu et al., 2007, 

193725; Xu et al., 2008, 193727). Both dimethylarsinic acid [DMA(V)] and monomethylarsonic 

acid [MMA(V)] were readily mineralized to As(V) by transforming the methyl group into organic 

compounds such as methanol, formaldehyde, and formic acid. Dimethylarsinic acid was 

photocatalytically oxidized into MMA(V), which was subsequently oxidized into As(V). Hydroxyl 

radicals could be the primary oxidant (Xu et al., 2007, 193725; Xu et al., 2008, 193727). 

The mechanism of arsenic adsorption onto TiO2 surfaces has been demonstrated to be through 

the formation of bidentate inner sphere complexes for As(V), As(III), and MMA(V), and forming 

monodentate inner sphere complexes for DMA(V) (Jing et al., 2005, 193452; Jing et al., 2005, 

193454; Pena et al., 2006, 090573). In ground water containing As(III), As(V), MMA(V), and 

 1-12
DMA(V), nano-TiO2 adsorbed As(III) and As(V) most, followed by MMA(V), but almost no 

DMA(V) (Jing et al., 2009, 193453). The difference in competitive adsorption could be due to the 

increased thermodynamic stability of the bidentate ligands formed by other arsenic species with TiO2

compared with the monodentate surface structure formed between TiO2 and DMA(V). However, in 

the presence of high concentrations of competing ions, the other arsenic species may be forced to 

form monodentate rather than bidentate ligands. 

Photocatalytic oxidation is also the mechanism for TiO2 degradation of organic pollutants in 

wastewater. Photocatalytic degradation is based on the formation of radicals (hydroxyl radicals 

[·OH], superoxide radical anions [·O2

− ], and hydroperoxyl radicals [·OOH]), which serve as 

oxidizing species in the photocatalytic oxidation process (Lu et al., 2009, 193528). Hydroxyl 

radicals, the most powerful oxidants TiO2 produces in the photocatalysis, can act on organic 

contaminants present at or near the surface of TiO2 (Bianco Prevot et al., 1999, 193278). 

One generally accepted mechanism of nano-TiO2 antimicrobial action is the generation of 

reactive oxygen species (ROS), which can cause cell wall or cell membrane damage (Kuhn et al., 

2003, 090597; Neal, 2008, 196069), such as lipid peroxidation (Maness et al., 1999, 193538). 

Although UV illumination increases photocatalytic nano-TiO2 toxicity to bacteria and fungi, 

photocatalytic nano-TiO2 is also toxic in the dark (Adams et al., 2006, 157782; Coleman et al., 2005, 

089849). Because TiO2 generates ROS (mainly highly reactive hydroxyl radicals) in the presence of 

UV light and oxygen (Reeves et al., 2008, 157506), mechanisms other than oxidative stress might 

also contribute to nano-TiO2 toxicity in the dark (and possibly also under UV), as suggested by a 

recent study indicating that anatase nano-TiO2 can generate carbon-centered free radicals in the dark 

in the presence of dissolved oxygen (Fenoglio et al., 2009, 180383). 

1.5.2. Sunscreen 

Nano-TiO2 formulations of sunscreen have proven popular because they appear transparent on 

the skin; formulations using conventional TiO2 or other inorganics such as zinc oxide (ZnO) 

(Schlossman et al., 2006, 093309) create a milky white appearance. Nano-TiO2 serves as a sunscreen 

in two ways, by absorption and scattering, depending on the wavelength of UV light. UV-B 

wavelengths are in the range of 290 to 320 nm, and are primarily absorbed by nano-TiO2; UV-A 

wavelengths are in the range of 320 to 400 nm, and are primarily scattered by nano-TiO2 (Shao and 

Schlossman, 1999, 093301). Optimal scattering is thought to occur when the diameter of the 

particles is approximately half the wavelength of the light to be scattered (Fairhurst and Mitchnick, 

1997, 196248) also see Appendix A for more information on how nano-TiO2 particle size relates to 

UV-A and UV-B protection). Information on chemical and other properties of topical sunscreens 

containing nano-TiO2 can be found in Appendix A. 

Conventional TiO2 absorbs and scatters UV radiation, making it an effective active ingredient 

in sunscreens. Like ZnO, TiO2 is a “physical blocker” of UV radiation, as opposed to many 

chemically active ingredients that serve as “chemical filters,” such as avobenzone and 

benzophenone, which in some individuals can cause adverse skin reactions, including blisters, 

itching, and rash (U.S. FDA, 2006, 157728). Thus, sunscreens containing physical blockers have 

long been an attractive option to those with sensitive skin. Apart from this niche market, the use of 

TiO2 in sunscreen was historically limited because of aesthetic considerations. Because conventional 

TiO2 scatters visible light, it remains visible as a white film when applied on skin. With the advance 

of technology to produce transparent nanoscale TiO2 particles, which scatter very little visible light 

and therefore appear transparent when applied on skin, nano-TiO2 has entered the mainstream as an 

active ingredient in sunscreens and has also been added to numerous other cosmetic products to 

provide UV protection. With exposure to UV radiation (wavelengths less than ~400 nm), pure 

anatase nano-TiO2 is photocatalytic. In sunscreen, however, photocatalysis is an undesirable property 

that can be addressed by applying surface treatments to the crystals, selecting a less photoreactive 

form (rutile), or adding antioxidant ingredients to the formula. 

 1-13
The maximum concentration (by weight) of TiO2 in sunscreen that the U.S. Food and Drug 

Administration (FDA) allows is 25% (U.S. FDA, 1999, 196374), but this limit does not distinguish 

between conventional and nano-scale TiO2, between anatase and rutile, or between coated and 

uncoated particles. The concentrations actually used, according to product labels, typically range 

from 2% to 15% (Table A-1, Appendix A). Europe, Australia, Canada, and South Korea also have 

approved the use of TiO2 as a UV filter in sunscreen with a maximum concentration of 25%. Japan 

does not regulate TiO2 as a UV filter in sunscreen (Comparative study on cosmetics legislation in the 

EU and other principal markets with special attention to so-called borderline products, 2004, 

157826; Oxonica, 2005, 157793; Steinberg, 2007, 193656). 

Some TiO2-bearing sunscreens are explicitly labeled as containing nanoparticles. Others are 

labeled as containing “micronized” TiO2, a grade commonly used in cosmetics. “Micronized” 

implies a particle size of approximately 1 micron (or μm, which is one order of magnitude larger 

than 100 nm), but how precisely manufacturers use the term is unclear. Sometimes “micronized” is 

taken to imply a nano-size range (e.g., Shao and Schlossman, 1999, 093301), as it was formerly 

considered distinct from nano (e.g., EWG, 2008, 196343) but such a distinction is no longer made by 

the European Working Group (EWG, 2009, 196367). In the latter case, TiO2 with a mean particle 

size of several micrometers is still very likely to include a significant fraction of particles in the 

nano-size range. Even sunscreens using pigment-grade TiO2 likely contain a proportion of 

nano-sized particles. When Consumer Reports tested seven leading national sunscreens labeled as 

containing ZnO or TiO2 or both, but with no indication on the container regarding the presence of 

nanoparticles (with at least one dimension less than 100 nm (La Farge, personal communication, 

2007, 196047), they found nanoparticles in all seven products (La Farge, personal communication, 

2007, 196047; Sunscreens: Some are short on protection, 2007, 155618). They also confirmed the 

presence of nanoparticles in an eighth brand labeled as containing nanoparticles. No information was 

available, however, on the quantities or sizes of the nanoparticles detected in any of these sunscreens 

(La Farge, personal communication, 2007, 196047). FDA does not specify the use of nano or other 

terms to describe TiO2 (64 FR 27671), and some nano-TiO2 sunscreens are therefore simply labeled 

as containing “titanium dioxide.” 

1.6. Analytical Methods 

Sensitive and accurate analytical methods for nanomaterials are critical tools for nanomaterial 

risk assessment, because measurement and characterization of nanomaterials, alone and in various 

media, are required for properly assessing exposure, conducting toxicological studies, estimating 

dose-response relationships, and understanding the behavior and effects of nanomaterials. The 

standardization of characterization method and sample preparation protocols will also greatly 

facilitate the physicochemical characterization of the nanomaterials. 

Section 1.5 addressed some general aspects of characterization of nanomaterials, particularly 

nano-TiO2. This section provides a brief overview of analytical methods that could be suitable for 

nano-TiO2, with a focus on currently available methods. Different methods for measuring the same 

parameter may yield different results for the same material (Hewitt, 2009, 625212), and therefore 

stating the testing method is important. Because nano-TiO2 is not radio-labeled and does not 

fluoresce, analytical methods based on these two attributes are not relevant. Additionally, the 

importance of chemical analysis of nanomaterials is acknowledged (such as methods used for 

identifying their molecular components and characterizing certain surface properties), but these 

methods also are not discussed in Section 1.6. Some of the chemical analysis methods suitable for 

nanomaterials are discussed in Powers et al. (2006, 088783) and U.S. EPA (2008, 157480). Methods 

for analyzing nanoparticles in the environment are summarized by Hoyt (2009, 633900). For detailed 

 1-14
comparison of various methods, see review articles by Maynard and Aitken (2007, 090674), Powers 

et al. (2006, 088783; 2007, 090679), and Domingos et al. (2009, 193346). 

1.6.1. Methods for Laboratory Research 

The physicochemical properties of nano-TiO2 can change over time (Kolár et al., 2006, 

193478) and in various milieux; therefore, the characteristics of engineered nanomaterials at the 

point of production could be vastly different after transport, storage, and preparation for testing. 

Nanomaterials used in toxicological testing ideally would be characterized by analyzing the raw 

material (as received from the manufacturer or supplier); nanomaterials in the testing media for the 

duration of the experiment; and nanomaterials (and possibly degraded products or biotransformed 

products) in the biological samples being collected and tested, such as in urine, organs, and cells. 

The equipment and methods for measuring nanomaterials in the laboratory are numerous and 

are evolving. Table 1-3 summarizes methods that can be used for characterizing nanomaterials in 

aerosols and liquids (including biological fluids) (Maynard and Aitken, 2007, 090674; Nanosafe, 

2008, 594868; Powers et al., 2007, 090679). Methods for properties relating to chemical reactivity or 

charge, such as surface charge and pHpzc, are not included in the basic characterization methods 

summarized in Table 1-3. Specialized methods are also available that are specific for radio-labeled or 

fluorescent nanomaterials. The following methods have been used to measure various characteristics 

of nanomaterials in biological samples: dark field microscopy, transmission electron microscopy 

(TEM), energy-dispersive X-ray analysis (EDS), and inductively coupled plasma mass spectroscopy 

(ICP-MS) for presence and location, with additional information on size, shape, and 

aggregation/agglomeration state available from analysis of TEM images; dynamic light scattering 

(DLS) in conjunction with TEM for size (both core and shell); high resolution transmission electron 

microscopy (HRTEM) for crystalline structure; inductively coupled plasma atomic emission 

spectroscopy (ICP-AES) for elemental composition and quantitative nanomaterial uptake; videoenhanced differential interference contrast (VEDIC) microscopy for uptake and localization 

(Marquis et al., 2009, 193539); and scanning probe microscopy (SPM) for size and threedimensional images. ICP, X-ray diffraction (XRD), and nuclear magnetic resonance (NMR) can be 

used to determine chemical composition. The combination of flow FFF and ICP-AES has been used 

to detect nano-TiO2 in tested commercial sunscreen, with information on mass-size distribution and 

Ti content of extracted nano-TiO2 from sunscreen (Contado and Pagnoni, 2008, 157585). Although 

combinations of these methods can be used to infer the presence of nanomaterials in tissue (e.g., by 

metal content), no broad-spectrum techniques are currently available to measure the total amount of 

nanomaterials in tissues. 

 1-15
Table 1-3. Analytical methods for characterizing nanomaterials in aerosol and in liquid 

Metric Method Aerosol Liquid 

Number Condensation particle counter (CPC) Yes No 

Scanning mobility particle sizer (SMPS) Yes No 

Electrical low pressure impactor (ELPI) Yes No 

Optical particle counter (OPC) Yes Maybe 

Electron microscopy (EM) Yes Yes 

Surface areaa Scanning mobility particle sizer (SMPS) Yes No 

Electrical low pressure impactor (ELPI) Yes No 

SMPS and ELPI used in parallel Yes No 

Diffusion charger Yes No 

Specific surface area (BET, titration, diffusion charging) Yes Titration techniques only 

Mass Size selective personal sampler Yes No 

Size selective static sampler Yes No 

Tapered element oscillating microbalance (TEOM®) Yes No 

Scanning mobility particle sizer (SMPS) Yes No 

Electrical low pressure impactor (ELPI) Yes No 

Size Dynamic light scattering (DLS) Maybe Yes 

Centrifugal sedimentation No Yes 

Laser diffraction/static light scattering Yes Yes 

Low pressure impactor and electrical low pressure impactor (ELPI) Yes No 

Scanning/differential mobility analysis Yes No 

Field flow fractionation (FFF) No Yes 

Size exclusion chromatography (SEC) No Yes 

Acoustic techniques No Yes 

Electron microscopy (EM) No Possible with 

cryotechniques 

Scanning probe microscopy (SPM) Maybe Yes 

Time of flight mass spectroscopy Yes No 

Atomic force microscopy (AFM) No Maybe 

a

For some particle shapes, electron microscopy can be used to estimate surface area (LeBlanc, 2009, 625209). SMPS, ELPI, diffusion charger, and electron microscopy measure 

external surface area and may underestimate total surface area for porous particles. BET, on the other hand, will measure total surface area, which includes the inner surface 

area of porous or aggregated or agglomerated particles (LeBlanc, 2009, 625209). 

Source: Modified with permission from Informa Healthcare, Maynard and Aitken (2007, 090674); Used with permission from Oxford University Press, Powers et al. (2006, 

088783); Used with permission from Informa Healthcare, Powers et al. (2007, 090679); and data from Nanosafe (2008, 594868) 

1.6.2. Methods and Instrumentation to Assess Environmental 

Occurrence 

Detecting nanoparticles in the environment can be difficult because available analytical 

methods often are not sensitive enough for current environmentally relevant concentrations and 

cannot distinguish natural materials in the nanoscale size range from manufactured nanomaterials 

(Domingos et al., 2009, 193346; Englert, 2007, 193367; Simonet and Valcárcel, 2009, 193648). 

Also, many analytical methods require sample treatment and extraction (Englert, 2007, 193367), 

 1-16
which may include solvent evaporation, and consequently could cause nanoparticle aggregation and 

salt precipitation (Simonet and Valcárcel, 2009, 193648). Detecting nanoparticles in water or soil is 

further complicated by the heterogeneous nature of the samples. Ideally such measurements would 

be done in situ to avoid changes in nanoparticles (such as agglomeration) due to differing conditions 

in the immediate milieu, but portable equipment sufficiently sensitive to detect nanoparticles at 

environmentally relevant concentrations has not yet been developed (Simonet and Valcárcel, 2009, 

193648). 

Analytical methods that are currently available for nanomaterials in soil, sediment and ground 

water were summarized in a recent EPA State of Science Review (U.S. EPA, 2008, 157480) 

(Table 1-4). Methods can be coupled to enable detection of more than one parameter at a time. For 

example, flow FFF can be coupled with ICP-MS for both size and chemical analysis. Methods for 

properties relating to chemical reactivity or charge, such as surface charge and pHpzc, are not 

included in the basic characterization methods summarized in Table 1-4. 

In a study comparing six analytical methods for determining nanomaterial sizes (TEM, atomic 

force microscopy [AFM], DLS, fluorescence correlation spectroscopy, nanoparticle tracking 

analysis, and flow FFF), Domingos et al. (2009, 193346) concluded that the two most commonly 

used techniques reported in the literature (electron microscopy [EM] on air-dried samples and DLS) 

were also the two techniques that appear to be most prone to artifacts that can interfere with 

interpretation of results. Using multiple analytical techniques or multiple preparation techniques, or 

both, has been recommended (Domingos et al., 2009, 193346; Englert, 2007, 193367). 

The measurement and detection of engineered nanoparticles across a variety of environmental 

media is an active and growing area of research, though an extensive review of analytical methods 

falls outside the scope of this case study. A growing body of research focuses on developing methods 

to detect and characterize nanomaterials and their behavior within environmentally relevant matrices 

(Boxall et al., 2007, 157712; Hassellöv et al., 2008, 157559; Stone et al., 2010, 633898; Tiede et al., 

2008, 196278; Tiede et al., 2009, 633895; Tiede et al., 2009, 193680). 

 1-17
Table 1-4. Analytical methods for nanomaterials in soil, sediment, and ground water for size 

fraction and distribution, surface area, and phase and structure 

Metric Analytical method Sample type 

Size fractionation Centrifugation Aquatic colloids and particles extracted from 

soil and sediment samples. Nanoparticles Ultrafiltration – direct-flow ultrafiltration or tangential-flow ultrafiltration (TFF) must be in solution. 

Field flow fractionation (FFF) 

Capillary electrophoresis (CE) 

Size exclusion chromatography (SEC) 

Size distribution Transmission electron microscopy (TEM) 

Scanning electron microscopy (SEM) 

Scanning probe microscopy (SPM) 

Dynamic light scattering (DLS) 

Laser-induced breakdown detection (LIBD) 

Small- and wide-angle X-ray scattering (SAXS/WAXS) 

Surface area Brunauer, Emmett, Teller method (BET) 

Calculation from TEM (length and width) and atomic force microscopy 

(AFM) (height) measurements, and particle nanocrystalline geometrics 

Only nanomaterials with a regular or pseudoregular geometry and without significant 

porosity 

Phase and crystalline 

structure 

Electron diffraction 

X-ray diffraction (XRD) 

X-ray absorption spectroscopy (XAS) 

Raman spectroscopy 

Source: Data from U.S. EPA (2008, 157480). 

1.6.3. Methods and Instrumentation to Assess Workplace Exposure 

Workplace exposure monitoring thus far has focused on measuring nanoparticles in the air. 

Instruments that can be used for aerosol sampling are available, but most are designed for laboratory 

use (Nanosafe, 2008, 594868) and lack one or more of the following desired attributes: portability, 

ease of use, capacity to distinguish nanoparticles from non-nanoparticles, different size bins in the 

1-100 nm range, or ability to sample personal breathing zones (Ostraat, 2009, 196077). 

Several governmental and nongovernmental organizations have begun addressing the need for 

instrumentation and methods for monitoring nanomaterials, particularly nanoaerosols, in the 

workplace. For example, NIOSH recently published a document titled Approach to Safe 

Nanotechnology–Managing the Health and Safety Concerns Associated with Engineered 

Nanomaterials (NIOSH, 2009, 196073), in which sampling and monitoring methods and equipment 

are discussed. The Nanoparticle Occupational Safety and Health (NOSH) Consortium, an industry 

led consortium of participants from academia, governmental, and nongovernmental organizations, is 

helping to define best practices for working safely with engineered nanoparticles (Ostraat et al., 

2006, 667690; Ostraat et al., 2008, 193591). The NOSH Consortium has developed portable air 

monitoring methods intended for daily monitoring in nanoparticle research and development or in 

manufacturing settings. 

Maynard and Aitken (2007, 090674) summarized available devices and approaches for 

evaluating particle number, surface area, and mass concentration of nanoparticles for use in 

monitoring aerosol exposure. In 2008, the NanoSafe2 project, a European Community-sponsored 

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 1-19

project for safe production and use of nanomaterials, released a report that highlighted findings in 

measurement methodologies for nanoparticle detection and measurement that use various types of 

on-line and off-line monitoring instruments (Nanosafe, 2008, 594868). The report provided 

examples of new nano-aerosol measurement equipment that is easy to transport and use. No 

commercially available equipment, however, is currently available for long term monitoring. The 

report also recommended that monitoring at workplaces include not only personal sampling and 

measurements inside the facility, but also measurements of nanomaterials in drains and in the 

exhausted air to help ensure protection of the environment. 

Finally, several companies are developing or have developed air monitoring devices for 

nanoparticle detection. The parameters that each device measures vary (Bennett, 2005, 193820; 

TRS Environmental, 2009, 196057; van den Brink, 2008, 196075). 

1.6.4. Summary of Analytic Methods 

Many techniques can be used to measure and characterize nanomaterials in the laboratory and 

manufacturing workplace, and some are available for detecting nanomaterials in the environment. 

However, no single instrument can characterize all of the physicochemical properties of interest. 

Technical difficulties still exist in certain aspects, such as measuring and characterizing 

nanomaterials in organisms, and distinguishing naturally-occurring nanomaterials from engineered 

nanomaterials in the environment. 
Chapter 2. Life Cycle Stages 

This chapter discusses the life cycle of nano-TiO2 as either a drinking water treatment agent or 

an ingredient in topical sunscreen. Each stage in the life cycles of the respective applications is 

considered from the standpoint of potential releases to the environment. 

2.1. Feedstocks 

Two ores, ilmenite (FeTiO3) and rutile (TiO2), predominate as feedstock materials for TiO2

production (nano and otherwise) (Haridasan et al., 2008, 155625). Ilmenite and rutile are often found 

together, but ilmenite is found and mined in far greater quantities (at a ratio of more than 10:1 by 

weight) (Gambogi, 2008, 155622) and supplies approximately 90% of Ti minerals worldwide. For 

the rutile-based manufacturing processes, the most common manufacturing pathway for producing 

TiO2 of all kinds is via the chloride route using titanium tetrachloride (TiCl4), a liquid used in 

approximately 60% of current manufacturing (Hext et al., 2005, 090567). Creating synthetic rutile 

from ilmenite is often more economical than eliminating impurities from natural rutile. 

World ilmenite production in 2007 was approximately 5.6 million metric tons (MT), and world 

rutile production was approximately 0.5 million MT. The nations that produce the greatest quantities 

of ilmenite are Australia, South Africa, Canada, China, Norway, India, the U.S., and Ukraine. 

Significant producers of rutile include Australia, Ukraine, South Africa, India, and the U.S. 

(Gambogi, 2008, 155622). An estimated 1 billion tons of TiO2 could be produced from existing 

world ilmenite resources, with another 230 million tons from rutile deposits (Mineral, 2009, 

195905). 

In the U.S., ilmenite and rutile are extracted by surface mining or reprocessing of mine tailings 

at two sites in Florida and Virginia. Combined ilmenite and rutile production is approximately 

0.3 million MT. Mine and mill employment at these sites was estimated at 229 persons in 2007, 

down from 344 in 2003 (Gambogi, 2008, 155622). 

Low levels of radioactive materials are present in ilmenite and natural rutile (Collier et al., 

2001, 155617; Haridasan et al., 2008, 155625). A study in India found that those who work with 

ilmenite could be exposed to an annual dose of 1 millisievert (mSv) of gamma radiation and another 

0.7 mSv of radioactivity via particle inhalation, mostly due to thorium. Thorium radioactivity in 

ilmenite was approximately 60% of the regulatory exemption limit established in the International 

Atomic Energy Agency (IAEA) Basic Safety Standards. Levels of radioactivity in natural rutile, 

ilmenite-derived synthetic rutile, and TiO2 pigment (produced by the chloride route, particle size not 

specified) are lower than ilmenite, while levels of radioactivity (from radium as well as thorium) in 

solid wastes and liquid effluent are elevated compared with ilmenite (Haridasan et al., 2008, 

155625). 

Another common feedstock is titanium sulfate solution, which can be hydrolyzed to form 

TiO2. The sulfate method begins with ground ilmenite or Ti slag. 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments. 

2-1 
2.2. Manufacturing 

Around 2005, annual global production of nano-TiO2 was estimated at 2,000 MT, with an 

overall market value of $70 million (Dransfield, 2005, 157809; Osterwalder et al., 2006, 157743). 

Approximately 65% of production at that time was thought to have gone to “personal care” 

applications such as topical sunscreens and cosmetics, with the remainder used in industrial 

applications such as plastics, catalysts, and ceramics. Commercial production of nano-TiO2 for years 

2006-2010 has been estimated at 5,000 MT/year, and more than 10,000 MT/year predicted for years 

2011-2014 (UNEP, 2007, 196074). In spite of some advantages of nano-TiO2 over conventional 

TiO2, nano-TiO2 cannot replace all conventional uses of TiO2. For instance, as a whitening agent, 

conventional TiO2, and not nano-TiO2, is needed to scatter visible light. From an economic point of 

view, the cheaper conventional TiO2 is likely to be used in applications that can use either TiO2 or 

nano-TiO2. Nonetheless, nano-TiO2 production based on a predicted trend of graduate and a 

theoretical upper bound of total replacement of conventional TiO2 was recently presented 

(Robichaud et al., 2009, 193617). The current and future worldwide production levels of nano-TiO2

was estimated to have an upper estimate of approximately 2.5 million MT by 2025 (Robichaud et al., 

2009, 193617). Thus far, nano-TiO2 production has represented a small fraction of overall TiO2

production, which commanded a market of 4.5 million MT and $9 billion (Dransfield, 2005, 157809; 

Osterwalder et al., 2006, 157743). 

Manufacturers and researchers report nano-TiO2 synthesis by various techniques, including 

chemical vapor deposition (CVD) and flame hydrolysis (Wahi et al., 2006, 090580). Further 

information on manufacturing of nano-TiO2 is provided in Appendix B. CVD, commonly used for 

production of both conventional and nanoscale TiO2, involves the conversion of a volatile compound 

to a nonvolatile solid that deposits on a substrate (Li et al., 2003, 090581; Nagaveni et al., 2004, 

090578). A variety of techniques are used to generate the vapor and collect the particles, including 

plasma, high temperatures, pressure, and injection, among others (Aitken et al., 2004, 090566). 

According to one industrial manufacturer of nanoscale titania, flame hydrolysis can generate 

high-purity nano-TiO2 using TiCl4 as a feedstock (Mulenweg, 2004, 090592). Like CVD, flame 

hydrolysis can be used to deposit a thin film on a surface, a process known as flame hydrolysis 

deposition (FHD). In FHD, an inert gas carries TiCl4 into a flame that produces hydrogen chloride 

(HCl) and a mixture of sizes of the metal oxide TiO2 (Tok et al., 2009, 196054). Flame hydrolysis is 

used for manufacturing P25 and yields agglomerated particles with a mean diameter of 

approximately 3.6 μm, with the smallest 4% of particles having an average diameter of 160 nm 

(Klaessig, personal communication, 2006, 196041). 

Anticipated by-products of this flame hydrolysis method of TiO2 production include those 

resulting from chlorine contamination of the TiO2 (from the TiCl4 precursor). Warheit et al. (2007, 

090594) have suggested that solutions of P25 in water are acidic (pH = 3.28) because of chloride ion 

artifacts on the particle surface. Manufacturer information, however, indicates that a steam washing 

step during the manufacturing process removes HCl adsorbed on the surface of P25 (Vormberg, 

2004, 157822). 

Another production method used to manufacture pigmentary-grade TiO2 is the sulfate process, 

although it can also be used to manufacture nano-TiO2 in certain commercial settings (Medley, 

personal communication, 2008, 196038). Details on this and other processes used in producing 

nano-TiO2 can be found in Appendix B. 

When photocatalytic or other applications require smaller particles, additional postmanufacturing processes of sufficient energy can be utilized to break apart the 

aggregates/agglomerates. Surfactants or solvents can be used to help keep the smaller particles from 

reaggregating after separation (Hewitt, 1996, 157936; Porter et al., 2008, 157508). Also, nanoscale 

particles might be sonicated to increase dispersion (Bihari et al., 2008, 157593). 

2-2 
2.2.1. Drinking Water Treatment 

No information was found on processes used in preparing or formulating nano-TiO2

specifically for use in drinking water treatment. P25 is used in a commercial water treatment system 

(Photo-Cat from Purifics) that can be used for drinking water, ground water, and wastewater 

treatment (NSF International, 2009, 196092; Pichat, 2003, 196037; Purifics, 2008, 196040). For this 

treatment system, P25 is neither specially prepared nor coated (Powell, personal communication, 

2009, 196056). 

2.2.2. Sunscreen 

Unlike drinking water treatment agents, information on the manufacture of topical sunscreens 

that incorporate nano-TiO2 is relatively abundant. Although specific details of manufacturing 

protocols are typically proprietary, general information on manufacturing processes and materials is 

readily available. The choice of a specific nano-TiO2 crystalline form is a key issue in manufacturing 

sunscreens because various forms differ in photostability. In particular, rutile is much more 

photostable than anatase (Chaudhuri and Majewski, 1998, 093308; Maynard, 2008, 157522). 

Although less photostable, anatase appears to be in common use. Barker and Branch (2008, 180141) 

studied five sunscreens containing nano-TiO2, purchased over the counter, and found that one was 

pure rutile, while the other four were anatase/rutile mixtures in which anatase predominated. 

To increase nano-TiO2 photostability, the particles are commonly given a surface coating such 

as silica, alumina, simethicone, or a variety of other compounds (see Appendix B for more 

information on coatings). Another technique for increasing photostability is by “doping” nano-TiO2

particles by embedding minute amounts of metals within them, such as manganese, vanadium, 

chromium, and iron (Park et al., 2006, 193593). 

Another important consideration in the manufacture of most topical sunscreens is the use of a 

liquid medium, or dispersion, to ensure that nano-TiO2 will be distributed evenly, thereby reducing 

aggregation and agglomeration. Aggregation and agglomeration can negatively impact UV scattering 

performance and transparency by increasing the effective particle size. Sunscreen manufacturers can 

purchase nano-TiO2 powder and formulate their own dispersion, or they can purchase ready-made 

“predispersions.” 

Surface coatings influence the interaction of nano-TiO2 with the dispersion medium, which 

can be water-based (aqueous), oil-based, or silicone-based. These and many other factors figure into 

the manufacture of sunscreens, including pH; emulsifiers; emollients; other physical UV blockers 

(e.g., ZnO, which can also be micronized); chemical UV filters; and various inert ingredients to 

achieve the desired viscosity/liquidity, spray-ability, color/transparency, water resistance, and 

spreadability. More detailed information on manufacturing processes is presented in Appendix B. 

2.3. Distribution and Storage 

Limited information is available regarding nano-TiO2 distribution and storage. P25 is shipped 

as a powder in 10-kg “multilayer ventilated paper bags, equipped with an additional polyethylene 

lining when required” (Degussa, 2007, 090576). P25 presumably could be stored as a powder in a 

chemical storage facility in the original 10-kg shipping bags. Degussa recommends storing it in 

closed containers under dry conditions (Degussa, 2007, 090576). Releases could occur if bags were 

damaged during shipping or storage, although such releases should be minimized by proper 

implementation of standard good management practices. 

Another brand of photocatalytic nano-TiO2 (KRONOS vlp 7000, 7001, and 7500) is also 

shipped in 10-kg paper bags (KRONOS International, 2006, 196046). Nano-TiO2 powders from 

2-3 
Sigma-Aldrich Corporation (Sigma), on the other hand, are shipped in amber glass bottles enclosed 

in foil or plastic bags, which are shrink-wrapped before being placed in cardboard boxes with 

shipping cushion peanuts. 

As a dispersion formulation, nano-TiO2 is shipped in pails, drums, or totes (Klaessig, personal 

communication, 2008, 196042). Sigma ships its nano-TiO2 dispersion in essentially the same way 

nano-TiO2 powders are shipped. Dispersion-formulated nano-TiO2 presumably would require 

protection from freezing in cold climates. Depending on where accidental releases of such 

dispersions occurred, nano-TiO2 could be released into water or soil during shipment or discharged 

into industrial or municipal wastewater treatment systems during storage. 

2.3.1. Drinking Water Treatment 

No information pertaining to the distribution and storage of nano-TiO2 used specifically for 

water treatment agents was identified. 

2.3.2. Sunscreen 

Topical sunscreen products are generally packaged in retail-sized bottles at the production 

facility and shipped in large containers to wholesalers, retailers, and direct marketers. Little 

information is available on methods of shipping or storage. Consumers generally handle only retailsized packages. 

Industry data from the 1990s, although perhaps out of date, shed light on the distribution chain 

of sunscreens. Sales in supermarkets, drugstores, and mass merchandise outlets accounted for 

approximately two-thirds of the total U.S. sun-care retail sales in 1992-1993, according to Davis 

(1993, 157949). The remaining one-third was attributed to sales in department stores and other 

“prestige” stores. Sun-care products are also sold by direct marketers (e.g., Avon, Amway, Mary 

Kay), discount stores, swimwear stores, and small variety stores (e.g., those near beaches and ski 

slopes) (Davis, 1993, 157949). 

At any point in the distribution-to-storage chain, accidental releases could occur. For example, 

a shipping accident, a dropped palette, or crushed retail-size container(s) could lead to releases. 

2.4. Use 

2.4.1. Drinking Water Treatment 

Nano-TiO2 could be used in various ways to treat drinking water, as discussed in Section 1.5.1. 

This discussion, however, is limited to nano-TiO2 that would be used to remove arsenic at drinking 

water treatment facilities. 

Roughly 54,000 community water systems in the U.S. serve more than 95% of the population 

(U.S. EPA, 2006, 091194). Most of these systems apply some form of treatment to remove or 

neutralize chemical or microbial contaminants. Those that do not apply treatment serve less than 5% 

of the U.S. population; these systems are generally small or medium sized (i.e., serving no more than 

10,000 people) and rely on ground water wells (U.S. EPA, 2002, 091192). Public water systems are 

required to keep arsenic concentrations in delivered water at or below a maximum contaminant level 

(MCL) of 0.01 mg/L (U.S. EPA, 2006, 091193). Approximately 5% of community water systems in 

the U.S. (i.e., approximately 3,000 systems serving 11 million people) have taken some action to be 

in compliance with the arsenic MCL (U.S. EPA, 2007, 091224). Likewise, approximately 5% of 

20,000 nontransient noncommunity water systems that serve at least 25 of the same people for more 

than 6 months of the year, such as schools, churches, nursing homes, and factories (i.e., 

2-4 
approximately 1,100 systems serving 2 million people) have also taken some action to comply with 

the arsenic MCL (U.S. EPA, 2007, 091224). Altogether, approximately 13 million people use water 

that is treated to remove arsenic. Although it is unknown to what extent nano-TiO2 might be used in 

any of these systems in the future, these numbers provide perspective on its potential usage for 

drinking water treatment. 

Depending on the type of drinking water treatment system, nano-TiO2 might be used as 

powder (e.g., in a slurry) or fixed on a supporting material as a component of adsorptive media. Each 

approach has its potential advantages and disadvantages. Powdered nano-TiO2 has a large surface 

area and offers highly efficient photocatalytic oxidation, but a means to filter out and/or recycle all 

of the photocatalyst is required (Dionysiou, personal communication, 2009, 193921; Pichat, 2003, 

196037). This suggests the possibility that some amount of nano-TiO2 suspended in water might pass 

through filters, including microfilters. Also, if nano-TiO2 builds up on the filter matrix (i.e., if it is 

not removed by filter backwashing and hydraulic cleaning of sand), it could saturate the filtration 

medium, and small quantities might be released with filtered water into subsequent steps of the 

treatment sequence. Fixed nano-TiO2 has a smaller surface area and thus is less efficient. Although 

the attachment to the supporting material should allow no leaching, a fixed photocatalyst might not 

require filters or recycling systems to remove nano-TiO2 from the final product (Dionysiou, personal 

communication, 2009, 193921). 

Zhang et al. (2008, 193735) investigated the removal of nano-TiO2 in a simulated conventional 

drinking water treatment procedure, which included coagulation, flocculation, sedimentation, 

filtration, and disinfection. Two types of nano-TiO2 (crystal form unspecified, primary particle sizes 

of 15 and 40 nm, and aggregates 200 and 500 nm, respectively) in 2-liter jars were subjected to the 

treatment procedure. Adding magnesium chloride (MgCl2) or alum (Al2(SO4)3·16H2O), followed by 

coagulation, flocculation, and sedimentation, still left more than 20% of an initial 10-mg/L 

concentration of nano-TiO2 in the settled water. Furthermore, the removal efficiency was lower in 

tap water than in buffered nanopure water (pH 5.6) due to the presence of organic matter in the tap 

water. Membrane filtration with a pore size of 0.45 μm (450 nm) after sedimentation removed 

nano-TiO2 aggregates larger than 500 nm, leaving only 1-8% of the initial TiO2 in the treated water. 

Although most, but not all, of the nano-TiO2 in the initial water was removed, this level of filtration 

is not typical in water treatment plants (Flummer, personal communication, 2008, 157573; Kline, 

personal communication, 2008, 157545), nor is it available in most whole-house filtration systems 

(Johnson, 2005, 157799). 

At least two commercially available water treatment systems can employ nano-TiO2, although 

to date they are not known to be routinely used in this manner. One system uses nano-TiO2 in a fixed 

membrane and the other uses nano-TiO2 in a slurry. A system from Matrix Photocatalytic Inc. uses a 

tube covered with fiberglass mesh in which nano-TiO2 is embedded; the tube contains water that 

circulates and UV lamps illuminate the outside (Dionysiou, personal communication, 2009, 193921; 

Pichat, 2003, 196037). In the Photo-Cat system by Purifics, nano-TiO2 (P25) circulates in a slurry 

inside a narrow annulus surrounded by a UV lamp (Pichat, 2003, 196037). A ceramic membrane 

filters out nano-TiO2 (Purifics, 2008, 196040). No empirical data are available on the life expectancy 

of either system or whether they can release nano-TiO2 into treated water. 

The Purifics system was pilot-tested for two months in a community drinking water treatment 

facility (Purifics, 2008, 196040). The ceramic membrane used to filter nano-TiO2 (particles as small 

as 12 nm) from the finished product was reported to require no servicing or cleaning during the 

2-month period because the nano-TiO2 particles collected in the membrane were removed by bursts 

of high-pressure air (Pichat, 2003, 196037; Purifics, 2008, 196040). Although the purpose of this 

pilot test was not to remove arsenic, several studies have bench-tested nano-TiO2 in slurry systems 

for removal of arsenic from water (Dutta et al., 2004, 157845; Ferguson et al., 2005, 090572; Lee 

and Choi, 2002, 193498; Li et al., 2003, 090581; Meridian, 2006, 090595). Higher arsenic oxidation 

rates occurred using a slurry that was continuously stirred (compared to immobilized nano-TiO2) (Li 

et al., 2003, 090581). In actual use, steps likely would be taken to keep nano-TiO2 dispersed during 

2-5 
treatment, which could affect solubility and particle agglomeration. Surface modification could 

affect dispersion and could also improve the material’s photocatalytic properties as described (Ryu 

and Choi, 2004, 193622). Additionally, numerous chemicals can be added for drinking water 

treatment (NSF International, 2009, 196092), any or some combination of which could affect the 

solubility, particle size, and behavior of the nano-TiO2. 

2.4.2. Sunscreen 

The estimated use of sunscreen can vary greatly among surveys, but it is clear that its use is 

significant (Kasparian et al., 2009, 193465; Keeney et al., 2009, 193466). Four U.S. studies that 

collected data in the years 1995-1999, with 1,000 to more than 10,000 participants in each survey, 

showed that approximately one in three people said they use sunscreen regularly (Cokkinides et al., 

2001, 193321; Geller et al., 2002, 193390; Santmyire et al., 2001, 193629; Weinstock et al., 2000, 

193716). In three studies, 31-45% of survey respondents said they routinely or often use sunscreen 

(Cokkinides et al., 2001, 193321; Geller et al., 2002, 193390; Weinstock et al., 2000, 193716). In 

another study, 30% of respondents said they were very likely to use sunscreen when they were 

outdoors (Santmyire et al., 2001, 193629). More recently, data from the 2005 Health Information 

National Trends Survey in the U.S. showed that among a total of 496 Latino participants, 15% 

reported that they always use sunscreen, 9% reported often use of sunscreen, and 20% reported that 

they sometimes use sunscreen (Andreeva et al., 2009, 193252). In the 2007 iVillage survey, the Skin 

Cancer Foundation (2008, 594955) found that 11% of respondents use sunscreen with a sunburn 

protection factor (SPF) of 15 or higher “every day,” and 59% of respondents use sunscreen at least 

occasionally (up from 39% in a 2003 survey), where SPF is defined by the U.S. FDA (2009, 196372) 

as a “measure of how much solar energy (UV radiation) is required to produce sunburn on protected 

skin (i.e., in the presence of sunscreen) relative to the amount of solar energy required to produce 

sunburn on unprotected skin.” Of those who wear sunscreen, 74% reapply it “at least every 4-6 hours 

or after swimming or sweating,” and 28% reapply it every 2 hours, the Skin Cancer Foundation’s 

recommended rate of reapplication (Skin Cancer Foundation, 2008, 594955). 

While the use of sunscreen may be lower in young adults and adolescents than adults 

(Kasparian et al., 2009, 193465) sunscreen use is likely to be higher in young children. Robinson 

et al. (2000, 193618) surveyed 503 people in the summer of 1997, and found that 54% of parents 

reported that their child always or usually used a sunscreen, but only 27% of parents used sunscreen 

themselves during the previous weekend. This is consistent with a survey of 254 parents in June-July 

of 1999 by Weinstein et al. (2001, 191128) in Chicago, in which parents reported more frequent use 

of sunscreen on their children than on themselves. 

The total amount of sunscreen, and more particularly the total amount of nano-TiO2 in 

sunscreen, used in the U.S. is unknown. Furthermore, the available survey data do not differentiate 

between sunscreen products with or without nano-TiO2, although the percentage of sunscreen with 

nano-TiO2 is thought to be substantial. In 2006, the Australian Therapeutic Goods Administration 

(TGA) estimated that 70% of sunscreens containing Ti and 30% of sunscreens containing zinc in 

Australia were formulated with nanoparticles (TGA, 2006, 089202). As noted in Section 2.2, annual 

global production of nano-TiO2 was estimated at 2,000 MT around 2005, with approximately 65%, 

or 1,300 MT, used in “personal care” products such as topical sunscreens and cosmetics (Dransfield, 

2005, 157809; Osterwalder et al., 2006, 157743). 

A poster presentation by Johnson et al. (2009, 644432) at SETAC Europe’s 19th Annual 

Meeting suggested that possible concentrations of nano-TiO2 in water, as a result of sunscreen use, 

are between 2,000 and 8,000 ng/L. This range is based on modeling assumed rates of sunscreen use 

over the course of a day, how much is expected to wash off, and how much will be removed by 

sewage treatment plants during various summer-time scenarios in the River Thames region of the 

UK. 

2-6 
2-7 

2.5. Disposal 

2.5.1. Drinking Water Treatment 

Most community water treatment filters, with regular backwashing, have an indefinite life 

span. Slow sand filters are generally cleaned not by backwashing, but by scraping and replacing the 

top layer of sand. Scraped sand is normally cleaned hydraulically and stockpiled for later reuse 

(Cleasby and Logsdon, 1999, 091181). This process creates wastewater, which might be recycled in 

the treatment train or discharged (e.g., to a municipal sewer). For processes in which nano-TiO2

would be introduced prior to or during the sand filtration process, the eventual disposal of the filter 

sand or other filter materials could result in nano-TiO2 entering landfills along with the filter. 

After nano-TiO2 is used in drinking water treatment, a sludge material (floc) containing 

nano-TiO2 would likely be created. In one scenario the sludge might be taken to a landfill; this is the 

case with approximately 30% of sludge generated from drinking water treatment (U.S. EPA, 2010, 

635678). Whether TiO2 could diffuse (and thus be released) from a solid matrix such as sludge is 

unknown. Some newly developed landfills are designed to collect and treat leachate, but leaks are 

still possible, and the ultimate fate of nano-TiO2 in the treatment process is unknown. In addition, 

some older landfills without leachate collection measures may still be in use. Nano-TiO2 and other 

contaminants such as residual arsenic could become suspended in leachate and enter ground water, 

or they could pass through a solid waste facility liner into the subsurface. 

Under a different scenario, the sludge could be used for land application (U.S. EPA, 2010, 

635678). This is the case with approximately 20% of sludge generated from drinking water 

treatment, which applied to land to improve soil conditions or to fertilize the soil. The sludge is 

plowed directly into the soil to limit water runoff and for sanitary reasons (U.S. EPA, 2010, 635678). 

Nano-TiO2 and other contaminants such as residual arsenic would then be present in these 

agricultural soils. 

If nano-TiO2 is present in finished drinking water that reaches the tap, it would eventually 

enter the ambient environment or be captured by a wastewater stream, after which it could enter 

sewage treatment facilities. 

2.5.2. Sunscreen 

Sunscreen containers likely would be disposed of primarily as municipal solid waste and thus 

end up in landfills or incinerators. The potential for leaching of nano-TiO2 from landfill disposal of 

containers would depend on many factors, including the integrity of liners and leachate collection 

systems, if present. Incineration of sunscreen containers raises the question of whether nano-TiO2

could enter the stack and be released to air, or become a trace contaminant in fly or bottom ash. 

Depending on the packaging, sunscreen containers might be recycled, suggesting the 

possibility that nano-TiO2 could be incorporated into recycled materials. Additional exposure 

pathways other than the specific handling of sunscreen containers are acknowledged as potentially 

important, and will be addressed as part of the fate and transport discussion in Chapter 3. 
Chapter 3. Fate and Transport 

Chapter 3 explores what might happen to nano-TiO2 after it is released to the environment at 

various stages of the product life cycles for water treatment agents or topical sunscreens. Nano-TiO2

could be released to air, water, or soil and then transported or transformed through chemical or 

biological processes. The lack of data on the fate and transport of nano-TiO2 by-products and waste 

produced during the manufacturing process also precludes a comprehensive discussion in this 

chapter. This chapter does, however, summarize what is known about the environmental pathways 

and transport and transformation processes of nano-TiO2 related to the various life-cycle stages 

described in Chapter 2. 

The preceding chapter discussed life cycle stages of nano-TiO2 with some considerations 

specific to its use in drinking water treatment for arsenic removal and in sunscreens. As this chapter 

focuses on the various pathways by which nano-TiO2 can potentially enter and propagate through 

environmental compartments, information related to wastewater treatment pathways and by-products 

will also be pertinent. Throughout this document, it is important to note the distinction between the 

two types of water treatment being discussed. The case study application of nano-TiO2 used in 

drinking water treatment for arsenic removal is distinct from the potential downstream appearance of 

nano-TiO2 in municipal wastewater treatment plants. The former scenario deals with the use of nanoTiO2, while the latter deals with its impacts after release to the environment. Because the processes 

for drinking water treatment and municipal wastewater treatment are different, they will lead to 

different scenarios for the fate and impacts of nano-TiO2. 

Although most studies cited in this chapter consider nano-TiO2 in aggregate or agglomerate

form (as discussed in Chapter 1), it is unclear whether all constituent primary particles remain in 

clusters if conditions change. Disagglomeration, for example, can occur at certain pHpzc levels. The 

pHpzc of a nanoparticle is defined as the pH at the “point of zero charge,” which occurs when the net 

electric charge at the particle surface is zero. At the pHpzc particles fail to electrostatically repel each 

other. In laboratory studies, the size range of aggregates and the presence of free nano-TiO2 particles 

(ranging from 5 to 50 nm in size) were found to be pH-dependent: when the solution pH differed 

from the pHpzc of the particles, the aggregates tended to be smaller (Dunphy Guzman et al., 2006, 

090584; Dunphy Guzman, personal communication, 2007, 091184). Sampled aggregates ranged up 

to 150 nm in size, and contained an estimated 8-4,000 nanoparticles (Dunphy Guzman et al., 2006, 

090584). The pHpzc depends in part on the crystal form of the nano-TiO2 particles. Finnegan et al. 

(2007, 193379) reported pHpzc values of approximately 5.9 for rutile and 6.3 for anatase. The degree 

of aggregation generally increases with increases in ionic strength (Domingos et al., 2009, 193347; 

French et al., 2009, 193384). The interaction between natural organic matter (NOM) and the 

aggregation state of nano-TiO2 is complex, and whether aggregation is enhanced or inhibited by the 

presence of these organic species can depend on factors such as concentration of NOM, 

concentration of nano-TiO2, pH, and the presence of divalent cations such as calcium (Kim et al., 

2009, 635778). 

Despite the presence, and sometimes the predominance, of large particles, several researchers 

investigating laboratory-synthesized and commercial nano-TiO2 products have found free particles 

or aggregates with diameters less than 100 nm in varying amounts, depending on synthesis method, 

temperature, solution pH, and the presence of buffers (Kormann et al., 1988, 090582; Li et al., 2003, 

090581; Nagaveni et al., 2004, 090578; Pena et al., 2006, 090573; Ryu and Choi, 2006, 090579; Sun 

et al., 2007, 193662; Wahi et al., 2006, 090580). Moreover, some preparations are specifically 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments. 

3-1 
designed to generate dispersed particles (e.g., Seok et al., 2006, 091198) to increase the efficacy of 

nano-TiO2 as a catalyst, increasing the potential for the presence of disagglomerated or even 

disaggregated nano-TiO2 to occur in the environment. However, a limited number of studies of 

nano-TiO2 agglomeration/disagglomeration behavior under “real-world” ambient environmental 

conditions, irrespective of medium, have been conducted (Kiser et al., (2009, 225305)Battin et al., 

2009, 201604). 

3.1. Water 

Although numerous studies characterize nano-TiO2 particles in aqueous solution under 

laboratory conditions, the fate and behavior of the particles in the environment have received less 

attention. One report indicated that nano-TiO2 was detected in river water in Montana, but the source 

(natural or engineered) and the concentration of nano-TiO2 were not determined (Wigginton et al., 

2007, 157415). 

Several physicochemical properties of nano-TiO2 can contribute directly to its environmental 

fate and transport in water. Long et al. (2006, 089584) reported that P25 rapidly aggregated in both 

Hank’s Basic Salt Solution (HBSS) and Dulbecco’s Modified Eagle’s Medium (DMEM) buffer 

solutions, both of which are high-osmolarity fluids that contain high concentrations of the 

monovalent cations Na+

 and K+ [160 millimolar (mM)] and the divalent cations Ca2+ and Mg2+

(2 mM). The ionic strengths of these two solutions are approximately 155 mM and 166 mM, 

respectively. After 1 minute of sonication, aggregation continued for 20-45 minutes until a steadystate, stable aggregate size formed. The steady-state aggregate sizes ranged from 826 to 2,368 nm 

and the concentration of P25 ranged from 2.5 to 120 parts per million (ppm). 

Ridley et al. (2006, 090599) found that results were reproducible for classical titration

procedures (with modification) to characterize the surface charging properties of a commercially 

available, uncoated anatase nano-TiO2 product (from Ishihara Techno Corporation, Osaka, Japan) in 

suspension. These findings showed that environmental pH can affect the surface charge properties. 

Schmidt and Vogelsberger (2006, 193634) studied the solubility of four types of nano-TiO2

(P25 from Degussa, DT51D and G5 from Millennium Chemicals, and an original formulation – 

presumably all uncoated particles) in various aqueous solutions, particularly focusing on the kinetics 

of the dissolution process. At the beginning of the process, solubility increased rapidly over time and 

then reached a steady-state value. The maximum solubility value (i.e., saturation concentration) was 

observed to depend on the morphology of the TiO2, the crystalline form of the nano-TiO2, and on the 

size of the nanoparticles exposed to dissolution. The saturation concentrations were higher in 

hydrolysis-generated nano-TiO2 than in precipitation-generated nano-TiO2, and higher in smaller 

particles than larger particles. However, since the equilibrium solubilities of the four types of nanoTiO2 ranged from micro-to nano moles per liter, while the saturated suspensions were in the range of 

milligrams per liter, dissolved Ti concentrations were negligible compared with the initial TiO2 input. 

Although many studies have demonstrated the potential to use the photocatalytic properties of 

nano-TiO2 in biocidal applications, including wastewater treatment (Chen and Ray, 2001, 193310; 

Han et al., 2009, 193407; Khataee et al., 2009, 193468; Rincon and Pulgarin, 2003, 157856; Wang et 

al., 2008, 193705; Watlington, 2005, 196080; Xu et al., 2009, 193726), data on the fate of nano-TiO2

in actual wastewater treatment facilities are scarce. The Water Environment Federation released a 

report including the behaviors and effects of nanomaterials in wastewater treatment, although very 

few studies were on nano-TiO2 (Effects of nanoparticles on the wastewater treatment industry 

(Report No, 2008, 195800). Kiser et al.(2009, 225305), however, have reported the occurrence of 

nano-TiO2 at full-scale wastewater treatment plants (in both raw and finished waters). The authors 

measured total Ti concentrations, which included some nano-scale particles, on the order of 10 μg/L 

in tertiary effluent from wastewater treatment. Another investigator studied the effects of nano-TiO2

3-2 
on aquatic microbial communities under environmental conditions, which has implications on both 

natural waters and on wastewater treatment environments (Battin et al., 2009, 201604). Several 

recent studies have used mass balance modeling to predict the accumulation of nanomaterials within 

various environmental compartments, including nano-TiO2 accumulation in wastewater treatment 

plants (Gottschalk et al., 2009, 633897; Mueller and Nowack, 2008, 157519). 

Other types of nanoparticles also have been studied in wastewater treatment plants. Limbach 

et al. (2008, 155628) studied the fate of cerium oxide nanoparticles (20- to 50-nm diameter) in a 

model wastewater treatment plant under a variety of conditions (e.g., with different surfactants to 

stabilize dispersions, and in media with different ionic strengths and pH values). They found that 

surfactants stabilized dispersions under a wide range of test pH values even at high ionic strength. 

The model sewage treatment plant consistently reduced the cerium oxide nanoparticle concentration 

in the wastewater from 100 ppm to 2-5 ppm. Most nanoparticles were removed via agglomeration 

with microorganisms in the sedimentation sludge. Comparing the physical properties and behavior of 

various oxides, the investigators speculated that TiO2 and other insoluble oxides would behave 

similarly to cerium oxide, while more soluble or reactive oxides like ZnO would be even more likely 

to aggregate and be more amenable to removal by sedimentation. The investigators cautioned, 

however, that the high nanoparticle concentration (100 ppm) used in the study favors aggregation, 

and that at more realistic initial concentrations, a greater percentage of nanoparticles are likely to 

break through. 

Kiser et al. (2010, 634458) investigated biosorption rates of eight nanoparticles, including 

TiO2, to wastewater treatment sludge. Investigators found that different nanoparticles biosorbed at 

different rates when placed in solutions with varying concentrations and types of biomass, which 

were designed to represent wastewater treatment sludge. For example, 23% of nanoscale TiO2 was 

removed via biosorption in biomass solution of 400 mg/L total suspended solids, compared to 88% 

of aqueous fullerenes in the same solution, 39% of functionalized Ag nanoparticles, and 13% of 

fullerol nanoparticles (Kiser et al., 2010, 634458). The authors noted that further research is needed 

to understand the specific mechanisms responsible for sorption. 

A limited number of studies are available on nano-TiO2 and its interactions with 

microorganisms and other NOM under “real-world” environmental conditions (Battin et al., 2009, 

201604; Kiser et al., 2009, 225305; Kiser et al., 2010, 634458). Battin et al. (2009, 201604) 

investigated damage to microorganisms from aggregated, agglomerated, and polydisperse nano-TiO2

under natural conditions in river microcosms. Their toxicity results correlated poorly with lab 

experiment results on monodisperse nano-TiO2 with monocultures, and contribute to the small but 

growing body of literature of nanoparticle toxicity in natural aquatic systems. It has long been 

recognized that anatase TiO2 can photogenerate fairly long-lived ROS such as hydrogen peroxide via 

photoinduced redox reactions or modification of the TiO2 surface in aqueous laboratory 

environments (Harbour et al., 1985, 090632). It is not clear how relevant results from such 

experiments would be for anticipating nano-TiO2’s behavior in wastewater or drinking water 

treatment plants. 

The interaction between nano-TiO2 and natural organic matter, which is ubiquitous in the 

environment, has been investigated in controlled conditions in the laboratory. Yang et al. (2009, 

190513) found that humic acid, a common type of natural organic matter, is easily adsorbed onto 

nano-TiO2 in aqueous media. Because humic acid adsorption decreased the zeta potential (i.e., 

increased electrostatic repulsion) of nano-TiO2 particles, humic acid-coated nano-TiO2 could be 

more easily dispersed and suspended and thus more stable in an aqueous medium than uncoated 

nano-TiO2 (Yang et al., 2009, 190513). 

Sediment, the solid fragments of inorganic or organic material that are carried by and settle to 

the bottom of natural waters, is another environmental matrix that could be affected by the release of 

nanomaterials. One study was identified on the transport and deposition of nano-TiO2 in natural 

streams and streambeds (Boncagni et al., 2009, 634454). Partitioning of nanomaterials, including 

3-3 
nano-TiO2, to sediments and other environmental compartments was modeled by Gottschalk et al. 

(2009, 633897). 

3.1.1. Drinking Water Treatment 

Although the processes for using nano-TiO2 for commercial water treatment are not yet well 

established and therefore a definitive understanding of nano-TiO2 fate is not possible, nano-TiO2 is 

not expected to be destroyed. The removal efficiencies of commercial nano-TiO2 in conventional 

water treatment processes (coagulation, flocculation, sedimentation, and filtration) have been 

reported in one study using jar testing (Zhang et al., 2008, 157462), although the condition was set 

for nano-TiO2 in source water and not as an agent in drinking water treatment. The study showed 

that more than 20% of initial 10 mg/L nano-TiO2 remained in the water after up to 24 hours of 

flocculation and 1 hour of sedimentation (in buffered nanopure water with MgCl2); more than 30% 

initial 10 mg/L nano-TiO2 remained in water after alum coagulation, flocculation, and sedimentation 

(in nanopure water); and filtration using a 0.45 μm membrane as a final process was able to leave 

only 1% to 8 % of the total TiO2 mass (in flocs smaller than 500 nm) in the water (Zhang et al., 

2008, 157462). It is expected that the actual removal efficiencies in drinking water treatment 

facilities would be different from these tested conditions due to the differences in process time, 

source water, and other factors. For instance, under the tested conditions (Zhang et al., 2008, 

157462), the most efficient nano-TiO2 removal was seen after 8- or 24-hours flocculation and 1-hour 

sedimentation. Flocculation is typically less than 1 hour in drinking water treatment plants, which 

may result in less removal than observed in the Zhang et al. study, while sedimentation is commonly 

several hours, which may result in more removal (AWWA Staff, 2003, 193818). In addition, the 

removal efficiencies of nanoparticles, not limited to nano-TiO2, were lower in tap water containing 

natural organic matter compared to nanopure water (Zhang et al., 2008, 157462). Since the removal 

of nano-TiO2 initially received as a suspension (200-nm aggregates) was less efficient than the 

removal of nano-TiO2 initially received as dry powders (500-nm aggregates), the authors speculated 

that the removal efficiencies would be lower for small aggregates than large aggregates at the same 

alum (coagulation agent) concentration. 

Several different waste streams that could contain nano-TiO2 could be generated from drinking 

water treatment facilities. For nano-TiO2 that settles with floc in the sedimentation step, nano-TiO2

presumably could become part of the sludge (AWWA Staff, 2003, 193818). The discarded sludge 

could be transported off-site for disposal or reuse, such as being buried in municipal solid waste 

landfills or directly applied to agricultural or recreational land. 

Theoretically, nano-TiO2 might become part of the filter matrix during the filtration step of 

water treatment. U.S. EPA’s Filter Backwash Recycling Rule (U.S. EPA, 2002, 644800) requires 

that, when the filter is backwashed, the water used must be recycled back into the coagulation 

process. This could reintroduce nano-TiO2 into the treatment process, but the implications for 

concentrations of nano-TiO2 in finished water are not clear. 

Various fate pathways could apply to nano-TiO2 used as a drinking water treatment agent. For 

example, if nano-TiO2 is not completely filtered out or otherwise removed from the final effluent, 

nano-TiO2 might remain in the water as aggregates/agglomerates and enter municipal water tanks or 

reservoirs. If some water were lost from the distribution system via leaks or spills, nano-TiO2 could 

end up in surface waters or in the subsurface environment. If nano-TiO2 were to enter ground water 

aquifers, nano-TiO2 would presumably persist, given that other inorganic compounds are not readily 

broken down in that environment and nano-TiO2 is poorly soluble; however, particle/agglomerate 

size and other characteristics could change. Conceivably, nano-TiO2 could contribute to the release 

of (or modify the bioavailability of) other water contaminants of concern. 

If nano-TiO2 were present in the final drinking water product that reaches the tap, it eventually 

might enter the ambient environment or be captured by a wastewater stream, after which it could 

reach a wastewater treatment facility. If the particular wastewater treatment method employed there 

3-4 
did not completely remove nano-TiO2, some level of nano-TiO2 would likely enter downstream 

water sources. 

3.1.2. Sunscreen 

The environmental fate of nano-TiO2 in topical sunscreens could be affected by the surface 

treatments and doping applied to nano-TiO2 particles, by the sunscreen vehicle, or by any number of 

other constituents in such products (Appendix B). Nano-TiO2 in emulsion, dispersion, and possibly 

powdered form could be present in wastewater (e.g., from equipment and site cleaning) and solid 

waste from sunscreen manufacturing facilities, depending on the trapping and filtration processes the 

facility uses. In the powdered form, nano-TiO2 could escape the facility through air venting and 

filtration systems. 

Nano-TiO2 could also be released to wastewater or to natural bodies of water through 

showering/bathing or through laundry water drainage following sunscreen use. Swimming after 

sunscreen use could result in an accumulation of sunscreen material in the swimming pool water and 

potentially be a point of release into the environment as untreated wastewater. If nano-TiO2 remained 

mobile in water, it could enter downstream water sources in a manner similar to that of the 

nano-TiO2 used for drinking water treatment. 

Auffan et al. (2010, 625063) investigated how nano-TiO2 particles formulated for use in 

sunscreens transform, or age, when placed in media that mimicked environmental conditions and 

conditions of product use. Their results showed that 90% wt of one coating constituent desorbed 

from the particle surfaces, that another constituent remained on the surface but was oxidized, and 

that the third constituent was chemically affected but remained sorbed at the surface. Though the 

remaining Al-based layer was still effective in protecting against the production of superoxide ions 

from the photoactive nano-TiO2 particle core under their experimental conditions, these changes in 

coating characteristics illustrate that transformations may occur once nano-TiO2 is released to the 

environment. 

The potential for release is suggested by recent studies that have detected topical sunscreen 

constituents in untreated wastewater, treated wastewater, surface water (lakes and rivers), fish from 

lakes and rivers, and biosolids (Balmer et al., 2005, 157817; Fent et al., 2008, 157574; Rodil and 

Moeder, 2008, 157503). The organic compounds detected in these studies were UV filter compounds 

such as 4-MBC (4-methylbenzylidene camphor) and octocrylene (OC), which generally biodegrade 

slowly and can bioaccumulate. Some evidence also indicates that nano-TiO2 can bioaccumulate 

(Zhang et al., 2006, 157722). Although nano-TiO2 is unlikely to behave exactly the same way as 

other components of sunscreen, the observed nano-TiO2 bioaccumulation in fish (Zhang et al., 2006, 

157722) suggests the possibility of persistent presence of nano-TiO2. However, no studies to date 

have documented the occurrence of nano-TiO2 specifically from sunscreens in wastewater or natural 

bodies of water. 

3.2. Soil 

Three studies addressed the fate and transport of nano-TiO2 in soil. Dunphy Guzman et al. 

(2006, 090584) studied the effect of pH on nano-TiO2 mobility in a model soil column. They found 

that both surface potential and aggregate size influence transport. In the pH range where electrostatic 

forces between nano-TiO2 aggregates and the experimental Pyrex surface should have been strong 

(pH 2.5-5.9), nano-TiO2 was highly mobile. The calculated interaction energy was expected to be 

greatest for the largest aggregates at pH 12, but these were the particles that most strongly attached 

to microchannel surfaces. At pH 3, where conditions were predicted to be favorable for 

negative/positive interaction, 84% of the particles were transported. The authors concluded that 

3-5 
current transport theory does not adequately predict transport of nanoparticles and aggregated 

nanoparticles. These results suggest that nano-TiO2 particles and aggregates of nanoparticles in a 

stable dispersion might be highly mobile in the subsurface over a wide range of conditions. This also 

raises the possibility that colloid transport mechanisms might be more relevant than particle 

transport. 

Lecoanet et al. (2004, 089258) showed that the mobility of aqueous anatase nano-TiO2

particles in a porous medium was comparable to that of other types of nanoparticles when compared 

on the basis of particle size. Primary particles of 40-nm diameter were found to be aggregated to a 

diameter of 198 nm. Approximately 55% was recovered after three pore volumes passed through the 

column, roughly twice the quantity of ferroxane particles with mean diameter of 303 nm and just 

more than half the quantity of silica particles with a diameter of 57 nm. After three pore volumes, 

approximately 95% of the 57-nm silica particles were recovered, compared with 60% of the 135-nm 

silica particles. Although the results were specific to the controlled experimental conditions, they 

suggest that particle size affects mobility of nanoparticles and that anatase might be mobile in 

ground water (Lecoanet et al., 2004, 089258). 

A recent study using soil samples from 11 different sites found that nano-TiO2 could remain 

suspended in soil suspensions for 10 days (Fang et al., 2009, 193371). Furthermore, the calculated 

maximum travel distance for some soil samples was more than 30 cm, which suggested that 

nano-TiO2 might be transferred to deeper soil layers or even to ground water. In general, large soil 

particles and low ionic strength conditions favor nano-TiO2 movement, while high clay content, 

dissolved organic carbon, and salinity conditions favor soil retention of nano-TiO2. 

If nano-TiO2 enters municipal sewage systems, liquid waste would be separated from solid 

waste and nano-TiO2 would likely be present in both waste streams. The solid waste, or sludge, could 

present a route by which nano-TiO2 could enter soil media, and could be dealt with in a number of 

ways. In one scenario, the sludge might be sent for land disposal. The ability of TiO2 to diffuse (and 

thus be released) from a solid matrix such as sludge is unknown. Nano-TiO2 and other contaminants 

such as residual arsenic could become suspended in leachate and enter ground water, or they could 

pass through a solid waste facility liner and reach the subsurface. 

Under a different scenario, the sludge could be used for land application. In this case, the 

sludge would undergo some type of treatment, generally to remove pathogenic organisms and 

regulated contaminants such as lead and arsenic (Ti is not regulated under U.S. EPA’s Biosolids 

Rule, Part 503) (see U.S. EPA, 1994, 090659). The treatment might include high temperature or 

strong alkaline pH processing, or both (U.S. EPA, 1994, 090659). The treated sludge could then be 

applied to land for agricultural use, reclamation sites, golf courses, public parks, and other areas 

where nutrient-rich organic matter is useful, including forests, parks, roadsides, and in some cases, 

residences (U.S. EPA, 1994, 090659). Roughly 50% of treated sewage sludge is applied to land, and 

treated sewage sludge is applied to less than 1% of all U.S. agricultural land (U.S. EPA, 2006, 

090658). 

Nano-TiO2 in sewage sludge could be broadly distributed to land used for crops or grazing, 

where it could enter the food chain, or to high-use areas such as parks, where people and pets could 

contact nano-TiO2 in soil or inhale wind-blown material. The nanomaterial could enter runoff and 

storm water during wet weather events, eventually returning to the aquatic medium. Ecological 

receptors also could also be exposed to nano-TiO2 in soil by direct contact with soils or via the food 

web, including uptake by vegetation. Because it is an inorganic compound, nano-TiO2 in soil could 

be expected to persist, in the same way that conventional TiO2 is very thermodynamically stable, and 

is unlikely to undergo significant transformation in the environment. Reactivity of nano-sized TiO2

might differ from conventional TiO2 due to nano-TiO2 particles’ greater surface area-to-volume ratio; 

the specifics of potential reactivity differences are largely unknown at this time. 

3-6 
3-7 

3.2.1. Drinking Water Treatment 

One scenario by which nano-TiO2 could enter soils would be through direct land application of 

sludge after specifically being used as an agent in drinking water treatment. In addition to the sewage 

sludge produced in wastewater treatment described above, a sludge material (floc) containing 

nano-TiO2 would likely be created in the process of using nano-TiO2 to treat drinking water. If 

nano-TiO2 settles with floc in the sedimentation step, it would likely become part of the sludge as 

well. Similarly, as described above, if nano-TiO2 were present in finished drinking water, it would 

eventually enter sewage treatment facilities where any remaining residual nano-TiO2 could also enter 

the sludge. The discarded sludge would be transported off-site and could be used as daily cover in a 

municipal solid waste landfill or used for direct land application. Either use would result in direct 

application of nano-TiO2-contaminated waste to soils. Alternatively, nano-TiO2 could enter soils if 

treated water were used to irrigate residential or agricultural vegetation. These scenarios could have 

implications for soil microbes and could also be noteworthy in relation to nutrient uptake by edible 

vegetation. 

3.2.2. Sunscreen 

As described above, nano-TiO2 in topical sunscreens could end up in the sludge produced at a 

wastewater treatment plant. The disposal of this sludge on land seems likely to represent the primary 

pathway by which nano-TiO2 in sunscreen could enter soil. 

3.3. Air 

Nano-TiO2 manufacturing facilities could emit such particles to the ambient atmosphere. An 

occupational exposure study by Berges et al. (2007, 157594) at a European nano-TiO2 manufacturing 

facility that supplies the nanomaterials for sunscreens and cosmetics found that “outside the plant,” 

the airborne TiO2 particle concentration was approximately 13,000 particles/cm3

, with nearly 94% of 

particles 100 nm or less in size, and approximately 52% at 40-60 nm (Berges, 2007, 157594; Berges, 

2008, 193274). The authors did not specify the duration or environmental conditions of the 

measurements. 

Some potential could exist for environmental or occupational atmospheric emissions and 

releases of nano-TiO2 if the transport or storage containers were to be compromised (e.g., due to a 

forklift error, train derailment, or truck accident). Direct land application of sludge, from either 

drinking water or wastewater treatment, might contribute nano-TiO2 to the atmosphere if dried 

material were to be re-entrained from wind turbulence. Nano-TiO2 is not expected to enter air via 

sunscreen application or from drinking water treatment processes. 

The large surface area of nano-TiO2 particles presents an opportunity for other co-occurring 

contaminants to adsorb onto their surface, potentially changing the physicochemistry of the particle 

and the behavior and effects of the other contaminant(s). Such interactions have been well 

documented for particulate matter and gases (U.S. EPA, 2004, 056905). When nano-TiO2 was 

dispersed for 0.5 hours in the air immediately next to thermal precipitators 1.5 m above the ground in 

various outdoor locations in the city of El Paso, Texas, USA, the collected nano-TiO2 particles were 

not only in agglomerate/aggregate form, but were also associated with other airborne nanoparticles, 

in particular, nanosilicate particulates (Murr et al., 2004, 196310). Environmental conditions at the 

study sites were not described, other than the investigators avoided collections in high-humidity 

environments. 
Chapter 4. Exposure–Dose Characterization 

This chapter examines the potential for biota and humans to be exposed to nano-TiO2 and 

associated pollutants through various environmental pathways tracing back to the life cycle of two 

types of applications of nano-TiO2, water treatment agents and topical sunscreens. Exposure is more 

than the occurrence of a substance in the environment; actual contact between the substance and an 

organism must occur. Exposure characterization entails much more than simply identifying the 

concentration of a substance in the environment. It also involves, for example, various temporal and 

spatial dimensions, including activity patterns and other complex variables. For nano-TiO2, even 

characterizing the primary material of interest, as discussed in Chapter 1, is not a simple matter. 

Further complications arise when considering the potential for aggregate exposure across multiple 

routes (e.g., inhalation, ingestion, dermal absorption) and for cumulative exposure to multiple 

contaminants that derive, either directly or indirectly, from the life cycle of the products in question. 

2 Dose refers to the amount of a substance that enters an organism by crossing a biological 

barrier such as the skin, the respiratory tract, the gastrointestinal tract, or the eyes. Dose can vary for 

individuals exposed to the same ambient concentration of a substance. For example, an adult and a 

child in a room breathing the same air containing a contaminant would both inhale the same 

contaminant concentration, but the inhaled contaminant quantity and absorbed dose would differ due 

to differences in physiology (e.g., respiration rates), morphology (e.g., lung volume and surface 

area), and other variables such as clearance. Dose can also reflect the integration of aggregate 

exposures across different routes of uptake. 

Organisms might be exposed to nano-TiO2 in the environment at any stage of the 

manufactured product’s life cycle. In the feedstock and manufacturing process, nano-TiO2 could be 

present in the air exhaust, waste-water effluent, and solid waste, if appropriate control technologies 

are not in use. Nano-TiO2 in the air can lead to inhalation exposure to organisms in the area. The 

material could agglomerate or attach to other pollutants and deposit on soil and water surfaces, as 

well as on animals, whose grooming habits could then result in ingestion of nanomaterials. 

Nano-TiO2 in soil could become airborne when the soil is dry and windblown, or leach into bodies of 

water when the soil is saturated with water. 

During the life cycle stages of distribution and storage, nano-TiO2 could be released 

accidentally into the environment, and cleaning the contaminated site with water could lead to 

nano-TiO2 exposure to both aquatic and terrestrial organisms. The use of nano-TiO2 in drinking 

water treatment could result in some level of nano-TiO2 in water, as described in Chapter 3, and thus 

potential exposure to human populations as well as biota. The use of sunscreens containing 

nano-TiO2 is expected to lead to nano-TiO2 presence in wastewater after users bathe or shower to 

remove residual sunscreen on the skin and launder clothes containing traces of sunscreen. Discharges 

of nano-TiO2 from wastewater treatment plants are not currently regulated, and are thus not designed 

or operated to remove nano-TiO2, although early research suggests that some removal can occur 

(Kiser et al., 2009, 225305). Therefore, nano-TiO2 might be present in the effluent and could lead to 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments. 

2

 The distinction between exposure and dose in this document is consistent with risk assessment usage (U.S. EPA, 1992, 041875). In 

toxicology, however, the term dose is often used to refer to the amount of a substance given to test subjects, as well as the amount that 

enters the subjects. Applied, external, and potential dose (e.g., on the skin, in the lung or digestive tract) in toxicology roughly equate to 

exposure in risk assessment; absorbed dose (amount entering the circulation) and target organ dose (amount taken up by a specific organ) 

in toxicology roughly equate to dose in risk assessment. 

4-1 
aquatic species exposure. In the life cycle disposal stage, wastes from factories and research facilities 

containing nanomaterials are often incinerated, possibly releasing nano-TiO2 into the air. Household 

waste containing consumer products made with nano-TiO2 might be incinerated or landfilled; 

landfilling might lead to nano-TiO2 leaching into ground water. 

Occupational exposure to nano-TiO2 and associated contaminants (e.g., waste by-products) 

could occur even with appropriate safety and protective practices (see Appendix C for a more 

thorough discussion of occupational exposure control measures). For instance, an accident or a 

mechanical failure might occur in spite of good safety practices. Such occupational exposures could 

differ from exposure to the general public in various ways. For example, workers could be exposed 

to free nano-TiO2, whereas the public might more commonly encounter nano-TiO2 embedded in a 

product. Exposure durations and concentration levels are likely to be higher in occupational settings. 

Likewise, target tissue dose levels could differ between workers and the general population or even 

between workers in different occupations at the same facility, depending on factors such as 

respiration rates in relation to sedentary or strenuous activity in the presence of airborne nano-TiO2. 

4.1. Biota 

Various scenarios and ways in which nano-TiO2 from water treatment agents and topical 

sunscreens could enter different environmental media were described in Chapters 2 and 3. Some of 

these scenarios will be further explored in this section, specifically examining various TiO2 exposure 

conditions and how they could affect aquatic and terrestrial organisms. The potential for 

bioaccumulation and entry of nano-TiO2 into the food web is discussed in Section 4.6. 

4.1.1. Aquatic Species 

Data on concentrations of nano-TiO2 in sediment, whether in a laboratory or a natural 

environment, are limited. Nano-TiO2 concentrations could be higher at the sediment surface than in 

the water (Handy et al., 2008, 157562). Settling of nano-TiO2 aggregates (with nano-TiO2 or with 

organic matter) would increase nano-TiO2 exposure to benthic and benthopelagic species, such as 

mussels, sea cucumbers, marine worms, flatfish, and other species that sometimes feed at the bottom 

of natural bodies of water. At the same time, settling decreases nano-TiO2 concentrations in the water 

column and would be expected to decrease exposure to suspension feeders (such as Daphnia) and 

animals that live in or drink the water. 

Nanoparticles can also deposit or aggregate on the surfaces of aquatic organisms. Surface 

aggregation can be caused by the slower flow near the interface between liquids and solids or by the 

viscous properties of the surface of an organism (Handy et al., 2008, 157562). Surface deposition or 

aggregation can result in a higher concentration of nano-TiO2 on the organism’s surface than in the 

water, and might cause toxicity even if the nano-TiO2 does not enter the cells (Handy et al., 2008, 

157562). Surface-acting metal toxicity of nano-TiO2 has been suggested as a cause of gill damage in 

rainbow trout where the Ti concentration in gill tissue was not increased (Federici et al., 2007, 

091222). 

Because water flow is also slower near the interface with air, higher concentrations of 

nanoparticles are also expected at the air-water interface (Handy et al., 2008, 157562). Consequently, 

organisms living at the water surface, such as zooplankton (microscopic invertebrates that float or 

swim in water), phytoplankton (primarily single-celled algae), and eggs of aquatic and amphibian 

species at the water surface, could be exposed to higher nanoparticle concentrations than organisms 

living throughout the water column. 

4-2 
4.1.2. Terrestrial Species 

Terrestrial organisms could be exposed to nano-TiO2 under various scenarios. For example, 

spillage during the life-cycle stages of shipping or storage, including breaching of containers by 

vermin, could result in contact by microbial, invertebrate, and vertebrate species. Plants could be 

exposed by taking up treated or wastewater containing nano-TiO2 or by growing in soil that contains 

nano-TiO2, for example, as a result of application of sludge from water treatment facilities. No 

empirical data on the potential for such exposures to terrestrial organisms have been located. 

4.2. Humans 

As noted at the beginning of this chapter, exposure is a complex function of not only the 

amount of a substance in the environment but also a function of various temporal and spatial 

dimensions of contact with the substance. At this early stage of investigation and understanding of 

human exposure to nano-TiO2, however, even basic information on the potential for and amount of 

human contact with this material is limited. Moreover, exposure characterization encompasses not 

just the primary material but the secondary waste and transformation products related to the entire 

life cycle of nano-TiO2 in various applications. These indirect and secondary aspects of exposure are 

even less well understood and therefore not discussed here. Their potential significance, however, 

should not be discounted. 

The potential for human exposure to nano-TiO2 depends first on the production and use of this 

material in the applications under consideration here. Generally, exposure related to life-cycle stages 

leading up to actual use appears more likely to occur in occupational situations, whereas exposure 

related to the use and disposal stages of the life cycle could occur in either occupational or 

nonoccupational settings. Although not absolute, this distinction provides a basis for discussing 

exposure with reference to either the general population or the occupational population, both of 

which are essential in examining the broad implications of nano-TiO2 use in drinking water 

treatment and in topical sunscreens. 

4.2.1. General Population 

4.2.1.1. Drinking Water Treatment 

Although the actual use of nano-TiO2 in water treatment facilities appears to be limited at 

present to pilot testing (Section 2.4), the potential for general population exposure to nano-TiO2 if it 

were to be used widely could involve sizeable numbers of people, given the number of U.S. 

community water suppliers that currently treat drinking water to reduce arsenic levels. As discussed 

in Section 2.4.1, such water suppliers serve roughly 13 million people in the U.S. alone. 

If nano-TiO2 were present in potable water, exposure could involve more than just ingesting 

the water. Such water could be used for bathing, including showering, which could imply exposure 

not only by dermal contact but by inhalation of water droplets and even contact through the eyes. 

Also, the general population includes infants and other individuals who could have relatively greater 

exposure to water and thus possible vulnerability if the water were contaminated. For example, on a 

body weight basis, 1- to 3-month-old infants consume far more water directly and indirectly than 

18- to 21-year olds. The 90th percentile consumption rate is 151 mL/kg-day for these infants versus 

17 mL/kg-day for the older age group (see Table 3-9 in U.S. EPA, 2008, 196062). Children also 

have a greater water intake while swimming, so they may be more vulnerable to contaminated water 

in that respect as well (U.S. EPA, 2008, 196062). 

4-3 
4.2.1.2. Sunscreen 

As discussed in Section 2.4.2, iVillage survey data from 2007 suggest that sunscreen might be 

used on a daily basis by 33 million people in the U.S. and on an occasional basis by another 

177 million. Moreover, sunscreen use appears to be increasing. According to the Skin Cancer 

Foundation (2008, 594955), the percentage of people who use sunscreen at least occasionally rose 

from 39% to 59% between 2003 and 2007. Sunscreen use is presumably greatest during the warmer 

months of the year, in warmer climates, or during outdoor recreational activities at various times 

during the year. No information was found regarding the proportion of use associated with water 

recreation and other specific venues or activities. 

Topical sunscreens are available as traditional lotions, in spray-on form, and as wipes (Jeffries, 

2007, 157682). Nano-TiO2 sunscreen powders are also available, according to the Project on 

Emerging Nanotechnologies at the Woodrow Wilson Center’s nanotechnology-based consumer 

product inventory (Woodrow Wilson International Center for Scholars, 2006, 196083). Another sun 

protection option available to consumers is “cosmeceuticals,” cosmetics that incorporate active 

sunscreen ingredients (Davis, 1994, 157946). In the mid-1990s, up to 30% of lipsticks and 20% of 

makeup were estimated to have SPF ratings, sunscreen claims, or both (Davis, 1994, 157946). Other 

products with active sunscreen ingredients include hair care products (e.g., hair spray, gel, mousse, 

and conditioner), alpha-hydroxy skin treatments, nail polish, and bath products. Sun-protective 

clothing is also available (Davis, 1994, 157946). 

For the general population, the principal exposure route to nano-TiO2 in sunscreen is through 

the skin. When sunscreen is applied by spray, inhalation presents another route, although it is not 

clear that the primary nanoparticles as such would be inhaled. Ingestion is also conceivable through 

hand-to-mouth contact and mucociliary clearance of inhaled nano-TiO2. 

Dermal Exposure 

Potential nano-TiO2 dermal exposure from sunscreen use can be estimated by the amount of 

applied sunscreen. Although the recommended sunscreen application rate is 2 mg/cm2

 of skin 

(roughly 1.5 ounces or 3 tablespoons for the entire body of an average adult), most consumers use 

0.5-1.5 mg/cm2

 skin (Srinivas et al., 2006, 157734). Assuming sunscreen is applied to all areas of 

skin exposed to sun on a day at the beach or exposed to water while swimming, an adult would use 

an estimated 10-46 g sunscreen/application, and a 3-year old would use an estimated 

3-15 g/application (Table 4-1). Assuming that a sunscreen contains 5% nano-TiO2 (the mass percent 

concentrations of nano-TiO2 in sunscreens range from 2% to 15%; see Table A-1 in Appendix A), the 

amounts of nano-TiO2 applied on the skin could range from 0.5 to 2.3 g/person/application for an 

adult, and 0.17 to 0.76 g/person/application for a 3-year old (Table 4-1). These exposure estimates 

are in line with estimates made by Hansen et al. (2008, 157560). Sunscreens, including the waterresistant or water-proof types, should be reapplied every 2 hours, regardless of the SPF values. 

Exposure to nano-TiO2 from sunscreen could range from 1.0 to 4.6 g for an adult and 0.33 to 1.5 g 

for a 3-year old for a half day at the beach (2 applications in 4 hours). As shown in Table 4-1, the 

ranges of applied nano-TiO2 would be 12 to 55 mg/kg of body weight/application for a 3-year old 

and 8.0-37 mg/kg of body weight/application for an adult. This relatively higher exposure in young 

children could be noteworthy in relation to indications that the skin of infants and young children 

might have less barrier function than matured skin (Hostynek, 2003, 193435), although this contrasts 

with another report indicating that human skin is mature both structurally and functionally at 

2-3 weeks of age (Makri et al., 2004, 193537). Although not everyone applies sunscreen at the 

recommended dose and frequency in real life, parents reported greater use of sunscreen on their 

children than on themselves (Weinstein et al., 2001, 191128). 

4-4 
Table 4-1. Estimated dermal exposure to nano-TiO2 from sunscreen containing 5% nano-TiO2 for 

adults and 3-year-old children 

Appliedb

nano-TiO2

(mg/person/ 

application) 

Applied sunscreen 

amount 

(mg/person/ 

application) 

Applied nano-TiO2

(mg/kg BW/ 

application) 

Applied sunscreen 

surface density 

(mg/cm2

) 

Surface areaa

 of 

skin (cm2

) Subject 

3-yr-old child, total body surface 

(50th percentile) 

6,640 0.5 3,320 166 12.0 

1.5 9,960 498 35.9 

2 13,280 664 47.9 

3-yr-old child, total body surface 

(95th percentile) 

7,640 0.5 3,820 191 13.8 

1.5 11,460 573 41.3 

2 15,280 764 55.1 

Adult, body surface area 

subjected to water contact in 

swimming (50th percentile) 

20,000 0.5 10,000 500 8.0 

1.5 30,000 1,500 24.0 

2 40,000 2,000 32.1 

Adult, body surface area 

subjected to water contact in 

swimming (95th percentile) 

23,000 0.5 11,500 575 9.2 

1.5 34,500 1,725 27.6 

2 46,000 2,300 36.9 

a

Body surface area values are based on Tables 6-6 and 6-16 of U.S. EPA (1997, 594981). 

b

Actual concentrations of nano-TiO2 in commercial sunscreen on the market vary, with the high at nearly 15%. (Table A-1 in Appendix A.) 

BW – Body weight. The body weights used in the calculation were 14 kg, the median for 36-month old females (2000, 157982), and 62 kg, the median for adults 18-74 years 

old; Table 7.5 of U.S. EPA (1997, 594981). 

Inhalation Exposure 

Consumers could inhale water aerosol while showering or from nebulizing room humidifiers. 

Spray sunscreen products also present an inhalation exposure scenario. For such products and for 

treated water containing nano-TiO2, the characteristics of the resulting aerosol have not been 

documented in the published literature. Section 4.2.2 discusses inhalation exposure from nano-TiO2

for several occupational scenarios. 

Oral Exposure 

Nano-TiO2 from sunscreen could be ingested by accident or as a result of routine hand-tomouth contact (from residual sunscreen on hands), particularly for young children. If nano-TiO2

were inhaled, mucociliary clearance could lead to uptake through the gastrointestinal tract. Although 

no estimates of this type of nano-TiO2 exposure are available, dietary intake of all sizes of TiO2 from 

all sources (food, pharmaceuticals, etc.) has been estimated. The estimation was based on 7-day food 

diaries and records of pharmaceutical, dietary supplement, and toothpaste use of 182 people in the 

United Kingdom. The amounts of TiO2 were calculated or estimated from product labels (the listing 

of food-additive TiO2 is required by British law in most foods), manufacturer reports, and laboratory 

testing. The total median dietary intake of nano-TiO2 and micro-TiO2 (0.1-3 μm) has been estimated 

between 2.5 and 5.4 mg/individual/day (Lomer, 2000, 635672; Lomer et al., 2004, 157382). Food 

was the main source of dietary TiO2, followed by pharmaceuticals, dietary supplements, and 

toothpaste. Individual TiO2 intake varied widely (0-112 mg/individual/day), and no particle size 

information was provided. 

4-5 
4.2.2. Occupational 

Nearly every stage of the life cycle for the applications considered here presents some 

potential for occupational exposure to nano-TiO2. Moreover, no exposure route can be ruled 

irrelevant to these workers. Thus, assessing occupational exposure is essential to completing a CEA 

of nano-TiO2 in either drinking water treatment agents or topical sunscreens. 

As a frame of reference, NIOSH (2005, 196072) proposed a draft occupational exposure limit 

of 1.5 mg/m3

 for fine TiO2 (primary particle <10 μm, see below for details) and 0.1 mg/m3 for 

ultrafine TiO2 (primary particle <0.1 μm, see below for details), as time-weighted average 

concentrations for up to 10 hours/day during a 40-hour work week. The “fine” particles in this 

NIOSH draft were defined as all particle sizes that are collected by respirable particle sampling (i.e., 

50% collection efficiency for particles of 4 μm, with some collection of particles up to 10 μm). 

“Ultrafine” particles were defined as the fraction of respirable particles with primary particle 

diameter <0.1 μm(2005, 196072). The NIOSH draft exposure limit was based on primary particle 

size, not the measured aggregate or agglomerate sizes. Agglomerates of ultrafine TiO2, which may 

by larger than 0.1 um, often exhibit biologically similar behavior to ultrafine TiO2 due to the surface 

area of the constituent particles, and therefore the recommended exposure limits for ultrafine TiO2

should apply (NIOSH, 2005, 196072). The draft recommended exposure limits were extrapolated 

from rat-based critical dose estimated to humans, using specific surface area measured by the BET 

method (6.68 m2

/g for fine TiO2 and 48 m2/g for ultrafine TiO2) to convert particle mass to surface 

area dose (NIOSH, 2005, 196072). Because the sizes and surface areas of fine and particularly 

ultrafine (nano) TiO2 vary, the risk estimates will vary for other particle sizes and surface areas 

(NIOSH, 2005, 196072), as well as other crystal forms of TiO2 and other conditions (Section 5.1). 

Most information on workplace TiO2 exposure relates to the production of conventional TiO2, 

not nano-TiO2 specifically. Additionally, given that nano-TiO2 tends to agglomerate or aggregate, 

occupational exposure conditions for nano-TiO2 could involve both nanoscale and larger than 

nanoscale TiO2 particles. The manufacturing stage of the life cycle comprises multiple processes that 

might vary in exposure characteristics. An epidemiologic study conducted in four TiO2

manufacturing facilities located in the U.S. indicated that occupational exposure to TiO2 is greatest 

during bagging, milling, micronizing, and internal recycling (shoveling spilled material from the 

floor into the processing bins) (Fryzek et al., 2003, 157864). However, it is possible that the levels of 

exposure can vary depending on the facility. 

The manufacturer of P25 has stated on its website that workplace inhalation exposures to TiO2

are typically less than 0.5 mg/m3

 (Degussa, 2007, 090576). If such exposures are sustained for less 

than 2 hours/day, they would not exceed the NIOSH proposed occupational exposure limit of 

0.1 mg/m3

, which is expressed as a 10-hour time-weighted average. The Web site also indicated that 

photocatalytic P25 production occurs in a closed reactor, which presumably limits exposure. The 

highest exposures the manufacturer reported were less than 0.5 mg/m3

 and occurred during the 

packaging step, which is also an enclosed process. This manufacturer is said to require the use of 

personal protective equipment during any repair work that could lead to dust exposure (Maier, 

personal communication, 2007, 091185). Such information suggests only limited potential for 

inhalation exposure during P25 manufacturing, but it does not address other routes such as dermal 

exposure or incidental ingestion from hand-to-mouth contact. 

Another manufacturer of nano-TiO2 products reported that air concentrations in particle 

manufacturing, packaging, and distribution areas for DuPontTM Light Stabilizer 210 and 220 (which 

protects plastic from UV damage) were less than 2 mg/m3

, and in most cases were lower than the 

detection limit of 0.3 mg/m3

 (size not specified) (DuPont, 2007, 157699). No worker exposure data 

were available for materials incorporation, packing, or product fabrication for nano-TiO2-containing 

polymer products. Although the potential for worker exposure was stated to be low (DuPont, 2007, 

157699), the detection limit (0.3 mg/m3

) is above the draft NIOSH recommended limit for ultrafine 

or nano-TiO2 of 0.1 mg/m3

 (NIOSH, 2005, 196072). 

4-6 
Preliminary estimates of workplace exposure in a factory that produces rutile nano-TiO2 for 

sunscreen and cosmetics were reported by Berges (2007, 157594; 2008, 193274). Measurements 

were made in 2006, and then in 2007, when improvements to local exhaust systems were in 

operation (Berges, 2008, 193274). In 2007, the TiO2 in the “inhalable” dust mass concentration at the 

bin filling station was 0.014 mg/m3

, and the TiO2 in the “respirable” dust mass concentration was 

0.004 mg/m3. (Inhalable refers to all particles that can enter the respiratory tract through the nose or 

mouth [e.g., up to approximately 100 μm]; respirable refers to particles that penetrate to the alveolar 

[pulmonary] region with a mass median aerosol diameter [MMAD] of approximately 4 μm1

 (CEN, 

1993, 078032)) In the bag filling area in 2007, the TiO2 inhalable fraction was 0.028 mg/m3

, and the 

respirable fraction was 0.022-0.042 mg/m3

. Personal sampling in 2007 over a 4.87-hour period 

measured 0.010 mg/m3

 TiO2 in the respirable fraction. It is not clear how applicable the results from 

this manufacturing facility are to occupational exposure in facilities where, for example, sunscreen 

products are formulated from the raw materials. 

Liao et al. (2009, 157456) further reported and analyzed the Berges (2007, 157594; 2008, 

193274) data, as well as data from several other sources to model the occupational exposure and 

characterize risk. In the bin filling area of the facility studied by Berges (2007, 157594; 2008, 

193274), the total airborne TiO2 particle number concentrations ranged from 15,000 to 

156,000 particles/cm3

, with a measured size range of 14 to 673 nm. More than 97% of the particles 

were 100 nm or less in size, and 60% were 20-30 nm. After a leak was sealed, the high-end 

concentration decreased to less than 29,000 particles/cm3

. Near the leak, the particle surface area 

concentrations reached 200 μm2

/cm3 for “alveolar deposited” particles and 50 μm2/cm3 for 

“tracheobronchial deposited” particles. Under normal operating conditions, the particle surface area 

concentrations were 50 μm2

/cm3 for the alveolar deposited particles and 13 μm2/cm3 for the 

tracheobronchial deposited particles. Outside the facility, the airborne TiO2 particle concentration 

was approximately 13,000 particles/cm3

. Their model indicated that the highest TiO2 burdens (in 

terms of lung surface area) of packers were 0.174 m2

 (anatase) and 0.122 m2 (rutile) for particles 

sized 10-20 nm. For particle sizes 80-300 nm, the burdens were 0.002 m2

 (anatase) and 0.0017 m2

(rutile). Employees classified as surface treatment workers (involved in drying, packing, and 

blending operations) had a higher TiO2 burden in their lung surface area. For particles 10-20 nm, the 

burdens were 0.40 m2

 (anatase) and 0.28 m2 (rutile). 

Using exposure data specific to particle size in the workplace from the Berges (2007, 157594; 

2008, 193274) reports as well as conventional TiO2 studies (Boffetta et al., 2004, 157849; Fryzek et 

al., 2003, 157864), Liao et al. (2009, 157456) used computer modeling to calculate that exposures to 

nano-TiO2 (expressed as particle surface area concentrations) were 0.1685 m2

 TiO2 per 300 m3 air 

(working space volume) for packers and 0.387 m2

 TiO2 per 300 m3 air for surface treatment workers. 

For nano-TiO2 in the 10- to 50-nm size range, the airborne concentrations (expressed as particle 

surface area concentrations) were higher in anatase nano-TiO2 than in rutile nano-TiO2 for both 

packers and surface treatment workers. The highest airborne concentration was anatase for surface 

treatment workers, followed in order by rutile for surface treatment workers, anatase for packers, and 

rutile for packers. 

Liao et al. (2009, 157456) also modeled the dose-response relationships from in vitro 

cytotoxicity studies of human dermal fibroblasts and inflammatory responses of human lung 

epithelial cells. They then compared exposure levels to the dose-response functions and concluded 

that packers and surface treatment workers at the studied location were “unlikely to [be at] 

substantial risk [of] lung inflammatory response, [but they] have significant risk [of] cytotoxicity 

response at relatively high airborne TiO2 anatase NP [nanoparticle] concentrations at size 10-30 nm” 

 

1

 The size for respirable particles cited here is the standard used by Berges (2007, 157594; 2008, 193274), and the size used in other 

studies may vary by standards set by different agencies or even laboratories. Some argue that approximate 50% of 5 μm particles are 

deposited in the alveolar region of humans who are engaged in activities requiring moderate ventilation levels, particularly associated with 

oronasal breathing. Thus, the respirable particulate size for the alveolar region in humans is closer to 7-8 μm (F.J. Miller, personal 

communication (2009, 625211)). 

4-7 
(Liao et al., 2009, 157456). Though these conclusions were not based on actual worker exposure, the 

combination of field data on relevant TiO2 size, laboratory lung cell studies and computer modeling 

generates data on the potential nano-TiO2 burden that could be faced by people handling these 

materials. 

In a presentation at a professional conference, Li et al. (2008, 196055) displayed photographs 

of a factory in Shanghai, China that mixed, but did not manufacture, nano-TiO2. The photographs 

appeared to show that nano-TiO2 was stored in shipping bags piled on pallets. White powder was 

visible on the facility floor, but its composition was uncertain as the factory also handled 

conventional “pigmentary grade” and “food grade” TiO2 (Ichihara, personal communication, 2009, 

196034). Li et al. (2008, 196055) reported that workers had been given masks and shirt-like 

protective clothing but that the masks were not always worn. The authors also noted that shirt-like 

protective clothing provided no protection for the forearms and legs of the workers, many of whom 

wore short-sleeved tops and shorts. The authors noted that this factory exhibited particularly poor 

conditions compared to others with similar processes, but even so their investigation can be used to 

illustrate how inhalation and dermal exposure might occur during the manufacturing or mixing 

process. 

As noted in Section 2.3, nano-TiO2 is routinely shipped in paper bags, which could be a source 

of exposure if they were to be ruptured, punctured, or otherwise compromised during distribution or 

storage. Nano-TiO2 in dispersion form shipped in pails, drums or totes (Klaessig, personal 

communication, 2008, 196042) could be subject to accidents resulting from forklift errors, train 

derailments, and truck accidents, but no empirical data on such incidents specifically related to 

nano-TiO2 were available. 

The above information suggests that inhalation and dermal exposure could occur during 

manufacturing, packaging, shipping, and storage of nano-TiO2. To fully characterize potential risk, 

toxicity data at conditions comparable to be the reported exposure conditions would be useful, 

although extrapolation from higher concentrations can be used as well. 

4.3. Aggregate Exposure to Nano-TiO2 from Multiple 

Sources and Pathways 

Nano-TiO2 is used in various manufactured products, raising the possibility that biota and 

humans could be exposed to nano-TiO2 from more than one source. Such sources might include 

drinking water treatment agents, topical sunscreens, cosmeceuticals (traditional cosmetics such as 

moisturizers and color cosmetics that incorporate active sunscreen ingredients containing 

nano-TiO2), sun-protective clothing, cleaning agents, air purifiers, coatings, and food packaging, 

among many others (The Project on Emerging Nanotechnologies, 2009, 196052). It should, however, 

be recognized that nano-TiO2 particles from these different sources may have different properties 

such as size distribution, crystalline phase, and surface treatment. Kaegi et al. (2008, 193457), for 

example, reported nano-TiO2 in water runoff from both new and naturally aged building façades 

painted with paint containing nano-TiO2. Hsu and Chein (2007, 193437) found that nano-TiO2

powder-coated materials (wood, polymer, and tiles) under various conditions emitted nanoparticles 

to the air. Of course, merely the presence of nano-TiO2 in a product does not mean that exposure will 

occur. For example, if nano-TiO2 is firmly embedded in a product and the product remains intact, 

little or no exposure to nano-TiO2 might actually occur. 

A hypothetical scenario for aggregate exposure to nano-TiO2 in both treated water and 

sunscreen could involve a person’s ingesting the water (oral route), bathing (dermal) or showering 

(dermal and inhalation), applying sunscreen lotion to the skin (dermal), ingestion of sunscreen 

through hand-to-mouth contact (oral), or uptake from hand-to-eye (ocular) contact. The latter two 

exposures pathways are particularly relevant for young children. Biota also could be subject to 

4-8 
aggregate exposures. A fish, for example, could take up nano-TiO2 that originated from a wastewater 

treatment facility and could also ingest prey whose contamination originated from ambient water, 

sediment, or other prey or plants that already contained sunscreen constituents. The seemingly 

widespread occurrence of nanoparticles of various types in aquatic media reported by Wigginton 

et al. (2007, 157415) lends plausibility to these scenarios. 

4.4. Cumulative Exposure to Nano-TiO2 and Other 

Contaminants 

Nano-TiO2 is not the only substance relevant to the life cycle of products containing 

nano-TiO2 to which biota and humans could be exposed. As noted in Chapter 2, releases of other 

contaminants might also occur during various stages of the product life cycle, particularly waste 

materials during feedstock processing and during manufacturing of the primary product. Such waste 

materials are not necessarily nanoscale in size. As described in Chapter 3, if wastes were released 

into the environment, they could undergo transformation, potentially resulting in even more types of 

contaminants; they might also be transported to other locations, e.g., downstream or downwind. 

The creation of secondary contaminants through transformation processes in various 

environmental media also raises the possibility of exposure to substances indirectly related to 

nano-TiO2. Many nanoparticles, including nano-TiO2, tend to bind transitional metals and organic 

chemical pollutants (Nagaveni et al., 2004, 090578; Pena et al., 2006, 090573). With a tendency to 

adsorb other pollutants and an ability to absorb into the body and cells (Sections 4.6.1, 4.6.3, and 

4.6.4), the possibility cannot be ruled out that nano-TiO2 could carry toxic pollutants to target sites 

where the pollutants would not normally go (Moore, 2006, 089839). Such activity could result in 

increased uptake of other pollutants or interactive effects that would otherwise not occur if these 

substances were only present individually. 

4.5. Models to Estimate Exposure 

The EPA uses various models to estimate exposures for chemical assessments, some of which 

are described on the websites for the Council for Regulatory Environmental Modeling (U.S. EPA, 

2009, 196065) and the Center for Exposure Assessment Modeling (U.S. EPA, 2009, 196064). For 

example, the Exposure and Fate Assessment Screening Tool Version 2.0 (E-FAST V2.0) is a publicly 

available program EPA uses for screening-level assessments of conventional industrial chemicals. 

The tool provides estimates of aquatic exposure, general population exposure, and consumer 

exposure based on release data (U.S. EPA, 2007, 196060). Other fate and transport models also 

might be relevant, for example, the Particle Tracking Model (PTM) that the Army Corps of 

Engineers developed (Demirbilek et al., 2005, 193887); the Eulerian model that treats particles as a 

liquid (Kollias, 2009, 624994); Publicly Owned Treatment Works (POTW) models for evaluating 

fate in wastewater treatment plants (to sludge, effluent, and air); receiving water models for 

evaluating environmental transport and fate, and bioaccumulation models (Minerva, 2009, 625210); 

and over 20 models of aquatic fate and transport (for comparison, see a review by Paquin et al., 

2003, 196867). However, these models were not developed for nanomaterials and have not been 

tested for their ability to estimate nanomaterial exposures, although they perhaps could be used or 

adapted for qualitative exposure estimation in lieu of quantitative release data. 

Although empirical data on nano-TiO2 concentrations in the environment are currently 

lacking, a recent study used computer modeling to predict nano-TiO2 concentrations in different 

environmental media. Using limited data from published literature and various assumptions, 

4-9 
researchers in Switzerland developed models to estimate predicted environmental concentrations 

(PEC) and predicted no-effect concentrations (PNEC) (Mueller and Nowack, 2008, 157519). PEC 

values were calculated for “realistic exposure scenarios” (based on nano-TiO2 use, estimated as 

25 MT per year in Switzerland) and for “high exposure scenarios” (based on 500 MT per year). The 

authors estimated that more than 60% of nano-TiO2 is used in cosmetics, including sunscreen, and 

that most of it is discharged into wastewater. To estimate PNEC, the lowest no-observed-effect 

concentration (based on a published study on acute toxicity to Daphnia, Hund-Rinke and Simon, 

2006, 090607), was divided by an assessment factor of 1,000, in accordance with the Technical 

Guidance Document on Risk Assessment published by the European Chemicals Bureau, because, as 

the authors noted, the “accuracy of the data was low” (European Chemicals Bureau, 2003, 196375; 

Mueller and Nowack, 2008, 157519). The PEC of nano-TiO2 in water was 0.7 μg/L (“realistic 

scenario”) or 16 μg/L (“high scenario”), compared to a PNEC of <1 μg/L (for Daphnia). The authors 

(Mueller and Nowack, 2008, 157519) stated that, given that the PEC was close to or greater than the 

PNEC, European Union authorities would consider the substance “of concern” and call for more data 

to validate the result (Umweltbundesamt, 2009, 196071). Gottschalk et al. (2009, 633897) followed 

up on this work using similar modeling procedures to predict environmental concentrations of 

several nanomaterials across a variety of compartments and regions. The team also investigated the 

overall applicability of probabilistic modeling to predict environmental exposure to nanomaterials 

(Gottschalk et al., 2010, 635674). 

Based on available information about the applied concentration of nanoparticles in cosmetics, 

personal care products and paints, Boxall et al. (2007, 196111) used a series of algorithms to 

estimate the PECs of nanoparticles in soil and water. Although anticipating that 10% market 

penetration probably provides a conservative estimate (with the exception of sunscreens), the 

researchers calculated the PEC for three scenarios assuming that 10%, 50% and 100% of the 

products on the market contained nanoparticles. The total predicted concentrations in water were 

found to be 24.5-245 μg/L. 

4.6. Dose 

Dose is defined as the amount of a substance that actually enters an organism by crossing a 

biological barrier. Uptake of nano-TiO2 by different routes has been investigated in various species. 

It is important to note that upon entering an organism, a substance may still be transported and 

undergo changes as it moves throughout the organism. For this reason, understanding dose includes 

understanding additional fate and transport considerations specific to the media encountered by a 

substance once it is taken up; several investigations have been identified in this area. Sager et al. 

(2007, 090633) attempted to disperse nano-TiO2, and other types of nano-sized particles in several 

suspension media, including phosphate-buffered saline (PBS), rat and mouse bronchoalveolar lavage

fluid (BALF), and dipalmitoyl phosphatidylcholine (DPPC). Although PBS was not a satisfactory 

medium, BALF was an excellent medium for dispersing the particles. The dispersion was also 

unsatisfactory in saline containing albumin alone or DPPC alone at concentrations found in BALF. 

Combinations of protein and DPPC were satisfactory, but slightly less effective, substitutes for 

BALF. These findings demonstrate the importance of the suspension media in determining the 

behavior of nano-TiO2 within a given system. 

Exposure to nano-TiO2 in aquatic organisms has been studied mostly by measuring tissue 

concentrations in fish exposed to it in water. However, information related to exposure of substances 

other than nano-TiO2 is often also mentioned, appropriately reflecting the multiple substances to 

which aquatic organisms may be exposed in the natural environment. For terrestrial organisms, 

including laboratory animals used for toxicological studies and used as models for human health 

effects, the route of exposure is important in determining the dose that actually enters the body, 

4-10 
hence information on uptake of nano-TiO2 is presented here according to the route of uptake, i.e., 

inhalation, dermal, or ingestion. While differences in animal models, such as the differences in 

human and rodent nasal pathways leading to the olfactory bulb, are known to underlie some 

differences in toxicological results between species, studies across a biological continuum are drawn 

upon herein to collect a spectrum of potentially informative data. 

Additionally, this section discusses special biological barriers (blood brain barrier [BBB] and 

placenta), and issues related to dose-metrics for nano-TiO2. Again, because internal transport of the 

materials will influence the ultimate dose to the organism, it should be noted that multiple routes of 

exposure will be considered, even though all routes may not be equally significant. Some routes 

(e.g., i.v., i.p., and i.m. injections) could have relevance when internal transport is considered. 

4.6.1. Uptake in Aquatic Species 

4.6.1.1. Bioaccumulation 

Zhang et al. (2006, 157722) found that nano-TiO2 can accumulate internally in carp 

(Table 4-2). The authors exposed carp to photocatalytic nano-TiO2 (P25) for up to 25 days. Before 

dissection and TiO2 analysis, carp were rinsed and wiped. The nominal concentrations of nano-TiO2

in the water were 3 and 10 mg/L (based on the amount of stock nano-TiO2 suspension added to the 

fish tank), and the authors reported that nano-TiO2 concentrations were 2 and 7 mg/L after 24 hours, 

with most of the decreases occurring within 4 hours after the addition of stock solution. The TiO2

concentration in carp tissue increased rapidly over the first 10 days and then more gradually between 

day 10 and day 25. TiO2 concentrations were highest in visceral organs, distantly followed by gills, 

and then closely followed by skin and scales (one sample), and muscle. The bioconcentration factors 

in the visceral organs were approximately 2,100 at 3 mg/L, and approximately 1,400 at 10 mg/L. 

In contrast to the finding of bioaccumulation of nano-TiO2 in carp that Zhang et al. (2006, 

157722) reported, Federici et al. (2007, 091222) detected no accumulation in rainbow trout exposed 

to up to 1 mg/L nano-TiO2 for 14 days. Although the findings appear contradictory, each study might 

simply reflect the results of the specific test conditions. For instance, the rainbow trout were exposed 

to lower concentrations of nano-TiO2 than were the carp. The Federici et al. (2007, 091222) study 

used photocatalytic nano-TiO2 (also P25), but 80% of the water in the fish tank was changed every 

12 hours. Similar to Zhang et al. (2006, 157722), Federici et al. (2007, 091222) reported that more 

than 85% of the initial nano-TiO2 concentrations in the tank water remained after 12 hours. Other 

environmental factors, such as water temperature at 14oC for rainbow trout and at 23oC for carp, 

could influence the behavior or effects of nano-TiO2 and contribute to the difference between these 

two studies. Furthermore, carp feeding behavior mainly consists of grubbing in sediments, and 

therefore carp could have a higher exposure to settled nano-TiO2 aggregates than rainbow trout. 

Studies on kinetics of uptake and clearance, which could be valuable in understanding nano-TiO2

bioaccumulation and relevant factors, were not available. 

Although nano-TiO2 may bioaccumulate in fish, the uptake mechanism is not clear. Substances 

in water can enter fish through waterborne exposure (through gills and then into blood through 

absorption), dietary uptake, or cutaneous absorption. Handy et al. (2008, 157563) suggested that the 

absorption of nano-TiO2 on the gill surface into the blood might be slow or uncertain, but that 

nano-TiO2 on the gut surface might be taken into cells by endocytosis. Although intact fish skin is 

unlikely to be permeable to nano-TiO2, these authors proposed that cutaneous uptake of nano-TiO2

might be possible if the skin is infected or inflamed (Handy et al., 2008, 157563). Handy et al. 

(2008, 157563) did not provide experimental data to support nano-TiO2 uptake through endocytosis, 

but a recent in vitro study indicated that an endocytosis inhibitor, Nystatin, decreased the mutation 

frequencies induced by exposures to 5-nm and 40-nm nano-TiO2, but not 325-nm TiO2, in mouse 

embryo fibroblasts, implying that endocytosis is involved in modulating cellular response to 

4-11 
nano-TiO2 exposure (Xu et al., 2009, 157452). The concentration of nano-TiO2 or Ti in cells was not 

measured (Xu et al., 2009, 157452). 

4.6.1.2. Food Web 

Nano-TiO2 could enter the food web at various levels, depending on the point and extent of its 

release to the environment. If nano-TiO2 were dispersed in water, for example, it could be taken up 

by algae, which are primary producers of chemical energy needed to fuel ecosystems. Many 

invertebrates, which are primary consumers of chemical energy in aquatic ecosystems, eat algae and 

in turn are consumed by larger animals such as fish. A common aquatic invertebrate is the water flea 

(genus Daphnia), which is a small crustacean filter feeder (also known as suspension feeder). 

Daphnids use their legs to generate water flow and use the comb-like setae on their thoracic limbs to 

strain or catch smaller organisms (such as algae) for consumption. Because daphnids have been 

reported to filter up to 120-160 mL each per day (Vanoverbeke, 2008, 157477), they could be 

exposed to quite high numbers of nanoparticles in water (Griffitt et al., 2008, 157565). Even if 

nano-TiO2 were not absorbed into tissues, nano-TiO2 in the digestive tract of daphnids could still 

contribute to bioaccumulation in the food web. Although nano-TiO2 has not been tested for trophic 

transfer in the food web, one study found evidence of transfer of carboxylated and biotinylated 

CdSe-based quantum dots to higher trophic organisms (rotifers) through eating ciliated protozoans 

exposed to quantum dots (Holbrook et al., 2008, 192383). Since quantum dot uptake in this study 

was inferred from quantitation of Cd2+ and assumed no dissolution of the nanoparticles, it is unclear 

to what extent these results are applicable to poorly soluble nano-TiO2. Biomagnification was not 

observed at the top predator level (rotifers), because biomagnifications factors (BMF) values for the 

quantum dots ranged from between 0.29 and 0.62 (Holbrook et al., 2008, 192383). 

4.6.1.3. Cumulative Dose of Nano-TiO2 and Other Pollutants 

Increased uptake of other pollutants in the presence of nano-TiO2 has been reported by Sun 

et al. (2007, 193662) and Zhang et al. (2006, 157722; 2007, 090114) (Table 4-2). Sun et al. (2007, 

193662) demonstrated that arsenic as arsenate [As(V)] strongly binds to Aeroxide® P25 (P25) in 

water and that fish (carp) exposed to water containing 10 mg/L of this photocatalytic nano-TiO2 and 

200 μg/L arsenate accumulated more arsenic than fish exposed to either nano-TiO2 or arsenic alone. 

The bioconcentration factor of arsenic1

 was more than twice as high when nano-TiO2 was present 

than when it was not (Sun et al., 2007, 193662). The tested arsenate concentration, 200 μg/L, is 

environmentally relevant, given that higher total arsenic concentrations (mainly inorganic arsenic in 

the forms of arsenite and arsenate) in drinking water have been reported in many countries, including 

Bangladesh, China, Chile, and India (Basu et al., 2004, 087896; Feng et al., 2001, 193374; Moore et 

al., 1997, 193553; Tian et al., 2001, 193679). Although data on nano-TiO2 concentrations in the 

environment2

 are lacking, the tested nano-TiO2 concentration (10 mg/L) may be higher than the 

likely environmental concentrations, with the exceptions of spills or accidents. The presence of 

nano-TiO2 did not alter the distribution of arsenic within fish tissues. Over various time intervals, 

arsenic and TiO2 accumulated significantly in the intestine, stomach, and gills, and to a lesser degree 

in liver, skin, and scales; the least accumulation occurred in muscle. Because the accumulation of 

arsenic was much greater in the presence of nano-TiO2, Sun et al. (2007, 193662) concluded that 

adsorption to nano-TiO2 facilitated arsenic transport and uptake. 

 

1

 The bioconcentration factor of arsenic = 1,000 × arsenic concentration in fish (μg/g dry weight)/arsenic concentration in water (μg/L). 

2

 Limited information is available on nano-TiO2 in the ambient air within or surrounding nano-TiO2 production facilities, or from the 

runoff from structures painted with paints containing TiO2. In the various compartments of the environment, however, nano-TiO2

concentrations are unknown, and current technologies have not been able to distinguish man-made nano-TiO2 from naturally-occurring 

nano-TiO2. 

4-12 
Table 4-2. Tissue concentrations of various pollutants in fish after exposures to nano-TiO2 in water 

Protocol 

(no UV illumination, 

unless specified) 

Test Species Material Study Outcome Reference 

Fish (carp, Cyprinus 

carpio) 

21-nm primary 

particle, 50- to 

200-nm aggregates 

in water (P25) 

(photocatalytic) 

Up to 25-day exposure to 3 and 

10 mg/L nano-TiO2 (water changed 

daily, TiO2 concentrations in water ~2 

and ~7 mg/L, respectively, after the first 

few hr) 

TiO2 accumulated in internal organs > gills 

> skin and scales > muscle 

Zhang et al. (2006, 

157722) 

Bioconcentration factors were higher at 

3 mg/L than at 10 mg/L 

Fish (carp, Cyprinus Sun et al. (2007, 

carpio) 

21-nm primary 

particle, 40- to 

500-nm aggregates 

in water (P25) 

(photocatalytic) 

Up to 25-day exposure to 10 mg/L 

nano-TiO2 with and without 200 μg/L 

arsenate 

Arsenate adsorbed onto nano-TiO2 

193662 Higher arsenic concentrations in tissues 

(skin and scales; muscle; gills; liver; 

stomach; intestine) with arsenate plus 

nano-TiO2 exposure, compared to arsenate 

exposure alone 

) 

Fish (carp, Cyprinus Zhang et al. (2007, 

carpio) 

21-nm primary 

particle, BET 50 m2

/g 

(P25) (photocatalytic) 

Up to 25-day exposure to ~97 μg/L Cadmium adsorbed onto nano-TiO2 

cadmium alone, cadmium with 10 mg/L 

nano-TiO2, or cadmium with 10 mg/L 

natural sediment particles 

090114 Higher cadmium concentrations in tissues 

( skin and scale; muscle; gills; viscera; 

whole body) with cadmium plus nano-TiO2

exposure, compared to cadmium exposure 

alone, or cadmium plus natural sediment 

particles 

) 

Fish (rainbow trout, Federici et al. (2007, 

Oncorhynchus 

mykiss) 

21-nm, 75% rutile: 

25% anatase, 

sonicated (P25) 

(photocatalytic) 

0-, 7-, or 14-day exposure to 0, 0.1, 

0.5, or 1.0 mg/L nano-TiO2

No clear treatment or time-dependent 

effects on Ti levels in gill, liver, or muscle. In 

brain, a transient but statistically significant 

decrease in Ti concentrations compared to 

control fish on day 0, but no exposure 

concentration-effect. 

091222) 

Respiratory distress, organ pathologies, and 

oxidative stress at concentrations as low as 

0.1 mg/L. 

BET – Brunauer, Emmett, Teller method of calculating surface area 

P25 – Aeroxide® P25 

Zhang et al. (2007, 090114) showed that fish (carp) exposed to cadmium in water 

(approximately 97 μg/L) along with 10 mg/L photocatalytic nano-TiO2 accumulated more cadmium 

than fish exposed to either nano-TiO2 or cadmium alone (Table 4-2). After 20 days of exposure, the 

bioconcentration factor for whole-body cadmium was 64.4 in carp exposed to cadmium alone, but 

reached 606 in carp exposed to both cadmium and nano-TiO2. After 25 days of exposure, cadmium 

concentration in the whole fish was 9.07 μg/g in the cadmium-only group and 22.3 μg/g in the 

cadmium-plus-nano-TiO2 group, indicating a 146% increase in the cadmium bioconcentration factor 

in the presence of nano-TiO2. When carp were analyzed after 20 days of exposure, cadmium 

concentrations in all groups were higher in internal organs than in gills, muscle, and skin and scale 

(Zhang et al., 2007, 090114). Unlike nano-TiO2, natural sediment particles (19 μm) (at equivalent 

concentrations) did not affect cadmium bioaccumulation. Both nano-TiO2 and sediment particles 

adsorb cadmium and reach equilibrium within 30 minutes, but nano-TiO2 adsorbed more than 

5 times as much cadmium as the sediment particles. Based on the facts that nano-TiO2 can adsorb 

cadmium and that tissue concentrations of cadmium and nano-TiO2 (measured as Ti) are positively 

correlated, the authors suggested that increased cadmium uptake in the presence of nano-TiO2 may 

have been due to accumulation of cadmium adsorbed on nano-TiO2 (i.e., facilitated transport). The 

transport routes could be from water onto the gill surfaces or from consumed food into internal 

organs. Toxicity was not measured in this study. 

The fact that organic disinfection by-products can be formed by the photocatalytic oxidation of 

drinking water treatment with conventional TiO2 (Richardson et al., 1996, 193612) suggests the 

possibility that nano-TiO2 could have the same effect. Richardson et al. (1996, 193612) compared 

4-13 
the organic disinfection by-products detected after using: (1) chlorine as the sole disinfectant; and (2) 

TiO2/UV light treatment followed by chlorination. The authors reported detecting an additional byproduct (tentatively identified as dihydro-4,5-dichloro-2(3H)furanone) after the combined TiO2/UV 

and chlorine treatment compared to chlorine treatment alone. Overall, however, the numbers and 

concentrations of chlorinated disinfection by-products were lower after combined TiO2/UV and 

chlorine treatment than after chlorination alone. 

Cumulative exposure to nanomaterials could also occur. Some consumer products contain 

more than one type of nanomaterial; e.g., nano-TiO2 and nano-silver (nano-Ag) have been used 

together in multiple products (The Project on Emerging Nanotechnologies, 2009, 196052). 

4.6.2. Respiratory (Inhalation and Instillation) 

Instillation can be performed in various ways, but essentially involves the direct administration 

of a substance to the respiratory tract. Animal studies have shown that inhaled or instilled nano-TiO2

can translocate into the interstitium of the lung, lymph nodes (Ma-Hock et al., 2009, 193534; 

Oberdörster et al., 1992, 045110; Oberdörster et al., 1994, 046203), blood (Geiser et al., 2005, 

087362), and the brain (Wang et al., 2005, 193703; Wang et al., 2007, 090290; Wang et al., 2008, 

157473). 

Particles in the nasal cavity may enter the brain through: (1) the olfactory nerve (Elder et al., 

2006, 089253; Oberdörster et al., 1994, 046203) [upper particle size limit: 200 nm (Elder et al., 

2006, 089253)]; (2) the circulating blood and then crossing the blood-brain barrier (Oberdörster et 

al., 2004, 055639); and (3) the olfactory mucosa and through the ethmoid bone into cerebrospinal 

fluid (Illum, 2000, 157897). One of the most visually convincing demonstrations of olfactory nerve 

transport, as mentioned in Oberdörster et al. (2004, 055639), is a study by DeLorenzo (1970, 

156391). DeLorenzo showed sequential TEM images of intranasally instilled gold nanoparticles in 

the olfactory mucosa, uptake into the olfactory rods, retrograde translocation within the olfactory 

dendrites, anterograde translocation in the axons of the olfactory nerve, and appearance in the 

olfactory bulbs. For more discussion of nanoparticle translocation from the nasal cavity to the brain, 

see Oberdörster et al. (2004, 055639). 

Intranasal instillation of three sizes of nano-TiO2 particles (approximately 20, 70, and 155 nm) 

at approximate 0.05 g/kg BW every other day for 30 days resulted in increased Ti concentrations in 

the olfactory bulb of mice (Wang et al., 2005, 193703). Also, two forms of nano-TiO2 particles (80-

nm rutile and 155-nm anatase) were found to increase Ti concentrations in the hippocampus, central 

cortex, and cerebrum, in addition to olfactory bulb, in mice after repeated intranasal instillation at 

approximate 24 mg/kg BW every other day for 30 days (Wang et al., 2008, 157473). The authors 

noted that the fact that brain tissue Ti concentrations were higher than lung tissue concentrations 

suggested that the olfactory nerve was the route of transport to the brain in this study. 

For respiratory exposure, the deposition pattern and concentration of particles in the 

respiratory tract can influence the health effects of these particles. Particles of various sizes can have 

different mechanisms of deposition (Gebhart, 1992, 157951; Heyder et al., 1985, 006919; 

Oberdörster et al., 2005, 087559). For nanoparticles, diffusive deposition, also known as 

thermodynamic deposition or diffusion (due to Brownian motion), predominates, whereas for 

particles larger than 1 μm, aerodynamic deposition predominates. Between 0.1 and 1 μm, the 

combined effects of aerodynamic and diffusive deposition are important. 

Oberdörster et al. (2005, 087559) summarized the principles and models of respiratory tract 

nanoparticle deposition and retention in the lung. Modeling of humans who are resting and breathing 

through the nose indicated that for 1-nm particles, approximately 90% will be deposited in the nasal, 

pharyngeal, and laryngeal region; approximately 10% in the tracheobronchial region; and almost 

none in the alveolar region. These results contrast with the modeling of a 5-nm particle, which is 

deposited roughly equally in the three regions. Approximately 50% of larger, 20-nm particles are 

deposited in the alveolar region, with approximately 15% deposition in each of the other two 

4-14 
regions. Since these simulations are based on the International Commission on Radiological 

Protection (ICRP) model, which was designed for larger particles (supplement of Oberdörster et al., 

2005, 087559), the performance of this model regarding particles at the lower end of the size 

distribution is unclear. 

In contrast to these results, a Multiple Path Particle Dosimetry (MPPD) model that 

incorporated convective flow, axial diffusion , and convective mixing (dispersion) predicted that 

very few small nanoparticles would deposit in the alveolar area (Asgharian and Price, 2007, 

093119). Nanoparticles less than 10 nm in diameter were predicted to deposit mainly in the 

tracheobronchial airway, and very few nanoparticles smaller than 5 nm were predicted to reach the 

alveolar region (Asgharian and Price, 2007, 093119). Depending on particle size, consideration of 

axial diffusion and dispersion could result in increased predicted deposition in the alveolar region of 

up to 10%. This modified MPPD model for nanoparticles found good agreement between predicted 

depositions of nanoparticles with measurements reported in the literature. 

Inhaled nano-TiO2 persisted in the lung longer than fine TiO2 in rats (Oberdörster et al., 1994, 

046203). After 12 weeks of inhalation (6 hours/day, 5 days/week) of approximately equivalent mass 

concentrations of fine TiO2 (22.3 ± 4.2 mg/m3

) and nano-TiO2 (23.5 ± 2.9 mg/m3), the total retained 

lung burdens were 6.62 ± 1.22 mg for fine TiO2 and 5.22 ± 0.75 mg for nano-TiO2. The estimated 

retention half-times were 174 days for fine TiO2 and 501 days for nano-TiO2 (Oberdörster et al., 

1994, 046203). 

In animal studies of nano-TiO2 disposition (Table 4-3), 13 weeks of inhalation exposure to 

nano-TiO2 increased TiO2 burden in lymph nodes in rats (2 and 10 mg/m3

), mice (10 mg/m3), but not 

in hamsters (at up to 10 mg/m3

) (Bermudez et al., 2004, 056707). 

4-15 
Table 4-3. Nano-TiO2 disposition in animals after inhalation or intratracheal instillation 

Species/Strain Aerosol Study Protocol Observations Reference 

Bermudez et al. 

(2004, 

Fischer 344 rats, 

females (6 wk) 

TiO2: 1.29-1.44 μm 

MMAD 

Animals exposed via inhalation 

6 hr/day, 5 days/wk, for 13 wk to 

0.5, 2, and 10 mg/m3

. 

TiO2 pulmonary retention half-times for the low-, mid-, 

and high-exposure groups, respectively: 63, 132, and 

365 days in rats; 48, 40, and 319 days in mice; and 

33, 37, and 39 days in hamsters. Burden of TiO2 in 

lymph nodes increase with time postexposure in midand high-dosed rats, and in high-dosed mice, but was 

unaffected in hamsters at any time or in any dosage 

group. In high-exposure groups of mice, epithelial 

permeability remained elevated (~2 × control groups) 

out to 52 wk without signs of recovery. Epithelial 

permeability was 3 to 4 × control in high exposure 

group rats through 4 wk postexposure, but 

approached control by 13 wk. Epithelial permeability 

was unaffected in all groups of hamsters. 

056707) 

B3C3F1 mice, 

females (6 wk) 

(σg = 2.46-3.65), 

21-nm primary particles Control animals exposed to filtered 

air. Hamsters, 

females (6 wk) Animals sacrificed at 0, 4, 13, 26, 

and 56 days (49 for hamsters) 

postexposure. 

Groups of 25 animals per species 

and time point. 

Distributions of particles among lung compartments Geiser et al. (2005, 

(airspace, epithelium/endothelium, connective tissue, 

capillary lumen) were directly related to the volume 

fractions of compartments and did not differ 

significantly between 1- and 24 hr postexposure. On 

average, 79.3 ± 7.6% of particles were on the luminal 

side of the airway surfaces, 4.6 ± 2.6% in epithelial or 

endothelial cells, 4.8 ± 4.5% in connective tissues, 

and 11.3 ± 3.9% within capillaries. Particles within 

cells were not membrane-bound. 

TiO2 (22-nm CMD, 

σg = 1.7) 

Rats exposed 1 hr via endotracheal 

tube while anesthetized and 

ventilated at constant rate 

Wistar rats, 

20 adult males, 

250 ± 10 g 

087362) 

Spark generated 

Lungs fixed at 1 or 24 hr 

postexposure 0.11 mg/m3

7.3 × 106 particles/cm3

(SD 0.5 × 106 

particles/cm3

) 

Re-evaluation of the data from Geiser et al. (2005, Muhlfeld (2007, 

087362) with a new statistical method in which a 

relative deposition index (RDI) was calculated for 

each compartment. When RDI=1, the particle number 

is the same as one would expect for the size of the 

compartment, if the particle distribution is random. 

RDI>1 suggests a preferential distribution. The new 

analysis suggested that at 1 hr postexposure, 

connective tissue was the preferential target for the 

nano-TiO2, while capillary lumen was the preferential 

target at 24 hr postexposure. This study suggested 

pulmonary clearance via microvasculature, and does 

not exclude clearance through exhalation or 

mucociliary escalator. 

091106) 

WKY/NCrl 

(Charles River) 

rats, 

5 young adult 

males, 

250 ± 10 g 

TiO2 (22-nm CMD, 

σg = 1.7) 

Rats exposed 1 hr via endotracheal 

tube while anesthetized and 

ventilated at constant rate 

Of particles in tissues, 72% were aggregates of 2 or Kapp et al. (2004, 

more particles; 93% of aggregates were round or 

oval; 7% were needle-like. The size distribution of 

particles in lung tissues (29 nm CMD, σg = 1.7) was 

remarkably similar to the aerosol; the small 

discrepancy could have been due to differences in 

sizing techniques. A large 350-nm aggregate was 

found in a type II pneumocyte, a 37-nm particle in a 

capillary close to the endothelial cells, and a 106-nm 

particle within the surface-lining layer close to the 

alveolar epithelium 

156624) 

Spark generated 

Lungs fixed immediately 

postexposure 

CMD – Count median diameter; MMAD – Mass median aerosol diameter; σg – Geometric standard deviation 

Source: U.S. EPA (2009, 196063) 

4.6.3. Dermal 

Because sunscreen is used on the skin, human skin penetration of nano-TiO2 (as particles in a 

solubility vehicle or in sunscreens) has been discussed in several reports and reviews (NanoDerm, 

2007, 157660; Nohynek et al., 2007, 090619; TGA, 2006, 089202). Most dermal exposure studies 

reviewed used human skin and pig skin (Sadrieh et al., 2010, 594511); several were in vivo studies 

in humans. Compared to other routes of exposure, dermal exposure may be more directly relevant in 

assessing potential health effects associated with its use in sunscreens, at least for unflexed skin from 

healthy adults. 

4-16 
Because of the relatively noninvasive nature of skin penetration testing, several laboratory 

studies have focused on skin absorption in humans, rather than animals. Human skin regulates the 

penetration of contaminants primarily through the stratum corneum layer, which contains keratinized 

cells and has no blood vessels. The thickness of the layer varies, ranging from approximately 60 μm 

to greater thickness on the plantar and palmar surfaces (Monteiro-Riviere et al., 1990, 625073). 

Other aspects of skin may also vary in different parts of the body (e.g., face versus forearm). 

Although published studies indicate the anatomy of stratum corneum of full-term infants and babies 

is comparable to that of adults (Fairley and Rasmussen, 1983, 193370), the physiology is not. Both 

the anatomy and physiology of pre-term infants’ skin are not comparable to that of adults (Kalia et 

al., 1998, 196039). Skin studies include a range of experimental conditions, including in vivo and 

ex vivo/in vitro. With few exceptions discussed below (Kertesz et al., 2005, 180334; Menzel et al., 

2004, 180361; Sadrieh et al., 2008, 157500), some of which were attributed to artifacts from sample 

preparation, most of these human and animal studies (Table 4-4) found clear evidence that nano-TiO2

does not penetrate beyond the stratum corneum or hair follicles, and does not penetrate into living 

cells of healthy skin (Figure 4-1). 

In healthy human skin, topically applied nano-TiO2 penetrates only into the upper layers of the 

stratum corneum (Table 4-4). The pathways of skin penetration can include intracellular penetration, 

intercellular penetration, and penetration through hair follicles (Figure 4-1) (Nohynek et al., 2007, 

090619). Penetration through sweat glands has not been reported, according to one source 

(NanoDerm, 2007, 157660). Although increased skin penetration of other nanomaterials has been 

reported in flexed porcine skin (Rouse et al., 2007, 157644) and flexed or abraded rat skin (Zhang 

and Monteiro-Riviere, 2008, 193735) and in UV-exposed murine skin in vivo (Mortensen et al., 

2008, 155612), studies of skin penetration in healthy flexed human skin or damaged skin have not 

been identified for nano-TiO2. Similarly, studies developing transcutaneous vaccine delivery 

detected the presence of nanoparticles in immune cells after topical application of nanoparticles on 

tape stripped skin (Mahe et al., 2009, 225307), but nano-TiO2 has not been tested in these conditions. 

Nano-TiO2 was observed in some hair follicles (Lekki et al., 2007, 180280), but did not reach 

the living follicle cells (with the exception of one study in hairless mice (Wu et al., 2009, 193721), 

see below). The presence of nano-TiO2 in hair follicles is most likely due to mechanical force, such 

as the movement of the hair during sunscreen application. Nano-TiO2 in hair follicles might 

contribute to increased Ti levels in the dermis (Sadrieh et al., 2008, 157500) because the shaft of the 

hair is exposed to the surface but the hair follicles are in the dermis. Nanoparticle loss from hair 

follicles is expected to be slow because the elimination occurs only by its flowing out with sebum or 

by its being pushed out with sebum. In a study using a hydrogel formulation containing 

fluorescence-labeled nanoparticles [Resomer RG 50.50 H, poly(lactide-co-glycolide)] on human skin 

(Lademann et al., 2007, 157678), approximately 15% of total nanoparticles detected in hair follicles 

30 minutes after application remained in the hair follicle for 10 days, which is at least 10 times 

longer than particles remain in the stratum corneum (Lademann et al., 2006, 157758). 

A recent in vitro and in vivo study using pig and hairless mice suggested that repeated in vivo 

dermal exposure may lead to nano-TiO2 penetration into the living cells of epidermis and possibly 

systemic distribution (Wu et al., 2009, 193721). Similar to other studies, Wu and colleagues’(2009, 

193721) 24 hour exposure in vitro of porcine skin to nano-TiO2 did not show penetration beyond the 

stratum corneum. Thirty days of in vivo exposures to 4 nm nano-TiO2, but not larger nano-TiO2, on 

the ear skin of pigs, however, resulted in penetration deep into the basal cell layer of the epidermis. 

No nano-TiO2 was observed in the dermis. After 60 days of in vivo dermal exposure to 10-60 nm 

nano-TiO2, hairless mice showed increased Ti concentrations in multiple organs, including skin, 

subcutaneous muscle, heart, liver, spleen, as well as pathological changes in skin, liver, spleen and 

lung. The various tested sizes of nano-TiO2 do not behave the same. For instance, in the heart, the 

increases in Ti concentration were similar in all nano-TiO2 treatments, but the pathological changes 

were only seen in the 10 nm nano-TiO2 group. Given that hairless mouse skin has a much thinner 

stratum corneum than human skin (Haigh and Smith, 1994, 625322) and other differences, it is 

4-17 
unclear to what extent the observed systemic distribution of nano-TiO2 after repeated dermal 

exposure in the hairless mouse may occur in humans. 

In human skin that is diseased, nano-TiO2 might penetrate more deeply. The only available 

study of nano-TiO2 on skin with dermal lesions was completed on psoriatic skin. Psoriatic skin is a 

symptom of a chronic, and possibly immune-mediated or genetic, disease called psoriasis. Unlike 

normal skin cells, which mature and are shed in 28-30 days, psoriatic skin cells mature in 3-4 days, 

accumulate on the skin surface (instead of shedding, because new skin develops faster than dead skin 

sheds), and develop into patches of dead skin (National Psoriasis Foundation, 2006, 157748; 

Pinheiro et al., 2007, 180160). Psoriatic skin has a looser corneocyte organization than healthy skin 

due to the loss of stratum corneum cohesion (Pinheiro et al., 2007, 180160). In the Pinheiro et al. 

(2007, 180160) study, nano-TiO2 in a sunscreen formulation penetrated into deeper areas of the 

stratum corneum in psoriatic skin than in healthy skin, but not into living cells in either psoriatic or 

healthy skin (Table 4-4). 

4-18 
Source: Adapted from and used with permission from CRC Press, Monteiro-Riviere (1991, 157957; 2004, 157834); Used with permission from Informa 

Healthcare, Riviere and Monteiro-Riviere (1991, 625197); Used with permission from Informa Healthcare, Nohynek et al. (2007, 090619) 

Figure 4-1. Possible pathways of nano-TiO2 skin penetration. 

Top Graphic: Nanoparticles may penetrate into skin by passing 

through: (1) the intercellular space between cells; (2) transcellular 

space; (3) opening of hair follicles; or (4) opening of sweat glands. 

Nano-TiO2 has been seen in the stratum corneum and inside hair 

follicles, but not in sweat glands. Bottom Graphic: Skin surface (from 

stratum corneum to stratum basal) at a high magnification showing 

simplified paths of nanoparticles passing: (1) between cells; and (2) 

through cells. Nanoparticles are not drawn to scale in either graphic. 

4-19 
Table 4-4. Overview of TiO2 skin absorption/penetration studies

Skin Modela

(Sampling Technique) Test Material Results Reference 

Sunscreen Formulations Containing Nano-TiO2

Lademann et al. 

(1999, 

Nano-TiO2 in a 

sunscreen formulation 

Primary particle 17 nm 

(Kemira, 2000, 

Human forearm, repeated 

application for 4 days (tape 

stripping, biopsy) 

Most particles on and in the upper layers of 

stratum corneum. In the lower half of the 

horny layer, only in the openings of hair 

follicles and sebaceous glands. In deeper 

tissue, exclusively in the follicle channels. 

157896), rutile, 

Al2O3/stearic acid coated, 

aggregates 150-170 nm (UVTitan M 160) in an oil-in-water 

emulsion, provided by L’Oréal 

(Clichy, France) 

090591) 

No penetration into living skin. 

Pinheiro et al. 

(2007, 

Sunscreen that 

contains nano-TiO2

Deeper nano-TiO2 penetration in psoriatic 

skin than in healthy skin. 

Not specified Human skin (healthy and 

psoriatic), in vivo, 2 hr (biopsy) 180160) 

No penetration beyond stratum corneum in 

both psoriatic and healthy skin. 

Nano-TiO2 in a 

sunscreen formulation 

20-nm nano-TiO2, coated with 

silicone 

Human skin, in vitro, and human 

skin, in vivo (skin stripping) 

Penetration limited to upper layers of stratum 

corneum. Nanoparticles in skin furrows or 

follicular opening could be mistaken to be in 

the epidermal compartment. 

Mavon et al. 

(2007, 090587) 

Kertész et al. 

(2005, 

Sunscreen that 

contains nano-TiO2

A commercially available 

sunscreen, hydrophobic 

emulsion containing nano-TiO2

(Anthelios XL SPF 60, La 

Roche Posay, France) 

Human foreskin grafts 

transplanted onto SCIDb

 mice; 

TiO2 emulsion on the graft in 

occlusion for 1, 24, or 48 hr 

TiO2 in the corneocyte layers of stratum 

corneum. 180334) 

In two cases, penetration through the stratum 

corneum, to the stratum granulosum was 

observed. 

Sunscreen that 

contains nano-TiO2

A commercially available 

sunscreen, hydrophobic 

emulsion containing nano-TiO2

(Anthelios XL SPF 60, La 

Roche Posay, France) 

Human foreskin grafts 

transplanted onto SCID mice; 

TiO2 emulsion on the graft at 

2 mg/cm2

 in occlusion for 24 hr 

TiO2 in stratum corneum, not in deeper layers 

of the skin. 

Kiss et al. (2008, 

157547) 

Nano-TiO2 in 

sunscreen 

formulation/sunscreen 

that contains 

nano-TiO2

50-100 nm, mixture of anatase 

and rutile, no coating 

information 

Human abdominal skin, in vitro Penetration limited to upper layers of stratum 

corneum. Dussert and Gooris (1997, 

193359) 

Female Yucatan minipigs (in 

vivo), 2-mg emulsion/cm2

 skin, 

5 days/wk for 4 wk (necropsy) 

Sadrieh et al. 

(2010, 

Various TiO2 in 

sunscreen 

formulations 

Sunscreen base formulation 

containing no TiO2 or 5% of 

one of three types TiO2: 

Increased Ti levels in epidermis in all TiO2-

treated groups. 594511) 

No penetration of TiO2 particles into the 

Micro-sized TiO2 dermis. 

No increases in Ti levels in lymph nodes or 

liver of any treated animals. 

Nano-TiO2, uncoated 

Nano-TiO2, coated with 

aluminum hydroxide and 

dimethicone/methicone 

copolymer 

Photostable 

nano-TiO2 in various 

formulations 

Photostable nano-TiO2, 

needle-like shape, 45-150 nm 

× 17-35 nm, coated with 

alumina and silica (Lodén et 

al., 2006, 

Pig skin, in vitro Particles on/in the stratum corneum; minimal 

penetration into stratum granulosum. 

Menzel et al. 

(2004, 180361

157757), in the 

following formulations: (1) 

Eucerin® Micropigment 

Crème 15: commercial 

sunscreen, 5% TiO2

concentration (Beiersdorf 

company); (2) a liposome 

dispersion: 18% TiO2, 

containing Phospholipon 90 G 

and Tioveil AQ-N (Tioxide 

Specialties Ltd., Billingham, 

UK); (3) formula SG110: 4.5% 

TiO2, containing Tioveil AQ-N; 

and(4) pure predispersion 

Tioveil AQ-N: 40% TiO2

) 

No penetration into living skin. 

4-20 
Skin Modela

(Sampling Technique) Test Material Results Reference 

Photostable 

nano-TiO2 in sunscreen 

formulations 

(1) T-Lite SF-S: rutile, coated 

with SiO2 and methicone; and 

(2) T-Lite SF: rutile, coated 

with methicone 

Pig skin, in vitro, up to 24 hr 

(tape stripping) 

No penetration beyond stratum corneum. Gamer et al. 

(2006, 090588

Both primary particles are 

needle-like: 30-60 nm × 

10 nm. Aggregates and 

agglomerates in water phase, 

mostly up to 200 nm 

Both are oil/water emulsions 

containing 10% TiO2

Receptor solution recoveries of 0.8-1.4% of 

applied dose. 

) 

Other Nano-TiO2 Formulations 

T 805 Degussa Coated with 

trimethyloctylsilane; ~20 nm in 

diameter 

Pig, in vivo TiO2 found exclusively in the outermost 

stratum corneum layer. Traces of TiO2 were 

found in the upper part of the follicle, with no 

evidence of uptake into the follicular 

epithelium. 

Pflucker et al. 

(1999, 644132) 

Human forearm, in vivo, 6 hr 

(biopsy) 

Penetration of particles into the upper layers 

of stratum corneum. 

Pflücker et al. 

(2001, 

Various nano-TiO2 in 

oil-in-water emulsions 

Emulsions contained 4% 

nano-TiO2, only differed in 

nano-TiO2 types: (1) 20-nm 

cubic primary particle, coated 

with trimethyl octylsilane, 

hydrophobic surface (T805, 

Degussa); (2) 10-15 nm 

primary particle, aggregated 

into ~100-nm needles, coated 

with Al2O3 and SiO2, 

amphiphilic surface (Eusolex 

T-2000, Merck); and (3) 100-

nm needles, coated with 

alumina and silica, hydrophilic 

surface (Tioveil AQ-10P, in 

dispersion, Solaveil) 

157887) 

and Schulz et al. 

(2002, No penetration into living skin. 157872) 

10-100 nm, coated with SiO2

–

, 

Al2O3

–

, Al2O3,/SiO2

Nano-TiO2 Human, in vivo (biopsy) Particles on or in the outmost surface of the 

stratum corneum. 

Schulz et al. 

(2002, 157872) 

No penetration into living skin. 

Various TiO2 and 

nano-TiO2

14 nm-200 μm, anatase and 

rutile, coated and uncoated 

materials 

Pig and human skin, in vivo and 

in vitro (skin stripping or biopsy) 

No penetration beyond the stratum corneum 

in any study. 

SCCNFP (2000, 

092740) 

4-21 
Skin Modela

(Sampling Technique) Test Material Results Reference 

(A) No penetration beyond the stratum 

corneum 

Various nano-TiO2 100% anatase, uncoated, 

nano-TiO2 (Zhejiang Wanjin 

Material Technology Co., Ltd.): 

(A) Porcine skin, in vitro, 

isolated pig ear skin (without 

and with tape stripping) on a 

modified Franz equipment, 

nano-TiO2 suspension (4, 10, 

25, 60, or 90 nm) on the skin for 

up to 24 hr 

Wu et al. (2009, 

193721) 

(B) After 30 days, nano-TiO2 was detected in 

all layers of epidermis (stratum corneum, 

stratum granulosum, prickle cell layer, and 

basal cell layers), but not in the dermis of 

porcine skin. Only 4 nm nano-TiO2 penetrated 

into the deeper layer of the epidermis (basal 

cell layer). 

(C) After 60 days, hairless mice had 

increased Ti in the skin, subcutaneous 

muscle, liver, heart, and spleen, but not in the 

blood or subcutaneous saccus lymphaticus in 

10-, 21-, 25-, and 60-nm groups. Almost 

negligible changes in the brain and kidney, 

with the exception of increased Ti in the brain 

after 21 nm nano-TiO2 exposure. Increased Ti 

in the lung may be significant in the 21- and 

60-nm groups. 

4 nm, hydrophobic surface, 

measured particle size 

5 ± 1 nm, surface area 

200 m2

/g 

(B) Porcine skin, in vivo, shaved 

pig ear starting at age of 4 wk, 

approximately 24 mg of test 

formulation containing 5% 

nano-TiO2 (4 or 60 nm) and 

Tween 80 was topically applied 

in the marked test area on the 

right ear skin for 30 

consecutive days. Punch 

biopsies collected at 24 hr after 

the last treatment for TEM 

10 nm, hydrophobic surface, 

measured particle size 

10 ± 1 nm, surface area 

160 m2

/g 

75% anatase/25% rutile, 

uncoated nano-TiO2 (P25 from 

Degussa, Germany): 

21 nm, hydrophilic surface, 

surface area 50 m2

/g (C) BALB/c hairless mice skin, 

in vivo, starting at age of 7-8 wk. 

Test formulation containing 5 % 

nano-TiO2 (10 nm, 21, 25, 60, or 

90 nm), carbopol 940, and 

triethanolamine was applied on 

the dorsal skin for 60 

consecutive days at 8 mg 

emulation (or 400 μm 

nano-TiO2) per cm2

 skin. 3 hr 

after application, the dressing 

was removed and residual 

nanomaterials were removed 

from the skin with lukewarm 

water and the skin was dried. 

100% rutile, uncoated, 

nano-TiO2 (Zhejiang 

Hongsheng Material 

Technology Co., Ltd.): 

25 nm, hydrophilic surface, 

measured particle size 

25 ± 5 nm, surface area 

80 m2

/g 

60 nm, hydrophobic surface, 

measured particle size 

60 ± 10 nm, surface area 

40 m2

/g 

90 nm, hydrophobic surface, 

measured particle size 

90 ± 10 nm, surface area 

40 m2

/g 

Lansdown and 

Taylor (1997, 

TiO2 Mixed particle sizes, mostly 

less than 10 μm in aqueous 

solution (range from <2 μm to 

>20 μm), no coating 

information, 20% TiO2 in 

water, castor oil, or 

polyethylene glycol 

Rabbit skin, in vivo, 4 hr for 

1 day or 2 hr daily for 3 days 

Penetration of particles into stratum corneum 

and outer hair follicles. 

157928 No penetration into living skin. ) 

Uptake of TiO2 affected by the vehicle: in 

caster oil>in water>in polyethylene glycol. 

Nano-TiO2 in various Lekki et al. (2007, 

gels 

For ion microscopy study: 20-

nm × 100-nm primary 

particles, coated (photostable 

UV-filter) (Eusolex® T-2000, 

Merck). Four formulations: 

hydrophobic basis gel, 

isopropyl myristate gel, 

microemulsion gel, and 

polyacrylate gel, each 

containing 5%-weight 

nano-TiO2 particles 

After wash with water, nano-TiO2 remains on 

skin, with most in stratum corneum and some 

in hair follicles. 

Porcine and human skins, for 

30 min to 48 hr (biopsy) 180280) 

Nano-TiO2 observed seen in hair follicles as 

deep as 400 μm, but not in living cells 

surrounding the follicles. 

For autoradiography study: 

proton-irradiated 20-nm TiO2, 

rutile (R-HD2, Huntsman), 

coated with alumina 

(Huntsman, 2008, 157555) 

4-22 
Skin Modela

(Sampling Technique) Test Material Results Reference 

TiO2/Nano-TiO2 Particles of Unknown Size 

Sunscreen that 

contains TiO2

Not specified Human (tape stripping) Particles on or in the outmost layers of the 

stratum corneum. No penetration into living 

skin. 

Gottbath and 

Mueller-Goymann 

(2003, 193401) 

TiO2 Not specified Mouse, pig, and human skin, 

in vitro 

TiO2 detected in the intercellular spaced 

between corneocytes of the outermost layers 

of the stratum corneum. No penetration into 

living skin. 

Gontier et al. 

(2004, 193398) 

Sunscreen that Tan et al. (1996, 

contains TiO2

Sunscreen containing 8% 

microfine TiO2 (size, crystal 

form, and coating were not 

specified) 

Human skin (13 patients, 

59-82 yr old), in vivo, applied 

TiO2 sunscreen daily for 

9-31 days until 2 days prior to 

surgical removal of the skin 

(tape stripping) 

Ti concentration in the dermis of patients 

exposed to sunscreen was higher than 

concentration in cadavers (controls), after 

exclusion of one control outlier. 

157933) 

No correlation between the duration of 

sunscreen application and Ti concentration. 

Various “microfine” 

TiO2

Commercial microfine TiO2

dispersions in octyl palmitate 

and in water (Tioxide 

Specialties Ltd, Billingham, 

U.K.) 

Human abdominal skin, in vitro, 

and skin equivalents 

(keratinocytes and fibroblasts of 

native human origin), in vivo 

Microfine TiO2 penetrates into the human 

stratum corneum probably via sebum lipids of 

the hair follicles. 

Bennat and 

Muller-Goymann 

(2000, 157403) 

a

Topical application unless specified. 

b

SCID = Severe combined immune deficiency. 

Mortensen et al. (2008, 155612), working with quantum dots rather than TiO2, reported greater 

skin penetration following UV exposure and suggested that even mildly sunburned skin might be 

more susceptible to penetration by nanoparticles of similar size and chemistry to the quantum dots 

used in their study. Though the size and chemical composition of the quantum dots differ from the 

nano-TiO2 used in sunscreens, this increased susceptibility to penetration is of note. The authors 

qualified their results by noting that under no circumstances was there evidence for massive quantum 

dot penetration, and that quantum dots collected preferentially in the folds and defects in the stratum 

corneum and in hair follicles. 

Using “microfine” TiO2, Tan et al. (1996, 157933) compared uptake in skin samples from 

13 elderly persons (age 59-82 years) with samples from 6 control cadavers (used to determine 

background exposure). The authors reported some dermal uptake, although they suggested caution 

when interpreting their results, citing the advanced age of their participants, the fact that skin 

samples were taken from different locations, and the fact that TiO2 concentrations were close to 

analytical detection limits. Kertész et al. (2005, 180334) reported penetration of nano-TiO2 into the 

stratum granulosum of grafted human foreskin in two samples (of an unknown total number). 

Sadrieh et al. (2010, 594511) found elevated levels of Ti in the dermis and epidermis skin of 

minipigs exposed to coated and uncoated nanosize TiO2. Investigators found no evidence of Ti 

penetration through expected routes such as the hair follicles, and concluded that the very few 

randomized Ti particles detected in living cells of the dermis were accounted for by contamination 

during sample preparation, possibly by small pieces of epidermis which contained 300- to 500-fold 

higher Ti concentrations than the dermis (Sadrieh et al., 2010, 594511). Several other studies that 

evaluated absorption using pig skin suggest little or no absorption beyond the stratum corneum. In a 

study using nano-TiO2 in four formulations on pig skin (Menzel et al., 2004, 180361), the authors 

stated that nano-TiO2 penetrated through the stratum corneum into the underlying stratum 

granulosum (but not into stratum spinosum) via intercellular space. The presence of Ti in the dermis, 

however, was deemed to be an artifact of the preparation process. Other studies using pig skin did 

not find nano-TiO2 penetration beyond the stratum corneum (Gamer et al., 2006, 090588; Lekki et 

al., 2007, 180280; Pflücker et al., 2001, 157887). 

Some nanomaterials have been shown to penetrate deeper in damaged skin than in intact skin 

[quantum dots in UV-exposed murine skin (in vivo) (Mortensen et al., 2008, 155612) and abraded rat 

4-23 
skin (in vitro) (Zhang and Monteiro-Riviere, 2008, 193735); nano-silver coated with 

polyvinylpyrrolidone in abraded human skin-skin (ex vivo) (Larese et al., 2009, 193493)], but no 

experimental data on nano-TiO2 dermal penetration in damaged skin were found. Preliminary data 

showed that two types of coated nano-TiO2 topically applied on either dermabraded or intact skin of 

SKH-1 hairless mice did not increase Ti concentrations in blood, lymph nodes, liver, spleen, or 

kidney (Gopee et al., 2009, 193399; Gopee et al., 2009, 667592). The depth of nano-TiO2

penetration in either damaged or intact skin was not reported. Hairless mice data, however, do not 

exclude the possibility that nano-TiO2 might penetrate deeper into damaged human skin than intact 

human skin because relative penetration of chemicals between hairless mice and humans varies and 

could be chemical specific (Benavides et al., 2009, 193270; Simon and Maibach, 1998, 193647). 

4.6.4. Ingestion 

Currently only three toxicological studies of nano-TiO2 through oral exposure have been 

reported (Section 5.3.1.2), and of these, only one (Wang et al., 2007, 090290) reported tissue 

concentrations of nano-TiO2. In the Wang et al. (2007, 090290) study, male and female mice 

received a single oral gavage of a fixed large dose of 5,000 mg/kg TiO2 as 25-nm rutile spindles, 

80-nm rutile spindles, or 155-nm anatase octahedrons (10 male and 10 female mice for each type of 

TiO2, and negative controls) (Table 4-5). The organs with elevated TiO2 concentrations (measured 

only in female mice) were liver, spleen, kidney, lung, and brain. Although the liver is expected to 

receive most of the TiO2 absorbed from the gastrointestinal tract through the portal vein, elevated 

TiO2 levels in the liver were observed only in the 80-nm group. The reason for this size-specific 

elevation in hepatic TiO2 concentration is unknown. 

4.6.5. Blood Brain Barrier and Placental Transfer 

The general potential for nanoparticles to cross the BBB has been investigated and developed 

primarily in relation to drug delivery systems (Beduneau et al., 2007, 193266; Emerich and Thanos, 

2007, 193365). In addition to size (Sonavane et al., 2008, 193652), the surface properties of 

nanoparticles influence the potential for a nanomaterial to penetrate the BBB (Singh and Lillard, 

2009, 193650). Nanoparticles developed for drug delivery often have ligands conjugated on the 

surface or other surface modifications to facilitate cellular uptake (Beduneau et al., 2007, 193266). 

4-24 
Table 4-5. Animal studies that measured Ti concentrations in brain after nano-TiO2 exposures 

through injection or oral gavage 

Nano-TiO2 Study design Findings in the brain Reference 

Ti concentrations in brain were increased in 

all three TiO2 treatment groups compared to 

negative controls. The increase was smaller 

in the 25-nm group than the 155-nm group, 

while the 80-nm group had the same 

increase as the 155-nm group. 

Nano-TiO2, 25 nm and 80 nm, 

rutile, uncoated (from Hangzhou 

Dayang Nanotechnology Co. Ltd. 

Fine TiO2, 155 ± 33 nm TiO2, 

anatase, uncoated, >10 wt% at 

<100 nm (from Zhonglina 

Chemical Medicine Co. (Chen, 

personal communication, 2008, 

Single oral gavage at 5,000 mg/kg to male and Wang et al. (2007, 

female CD-1(ICR) mice 090290) 

Ti content was measured 2 wk after gavage by 

ICP-MS with a detection limit of 0.074 ng/mL 

Vacuoles in the neuron of hippocampus, 

suggesting fatty degeneration, observed in 

the 80-nm (but not typical) and 155-nm 

(frequently) groups, but not in the 25-nm 

group. 

157588) 

TiO2 was not detected in the brain at any 

tested time points. 

Single i.v. injection at 5 mg/kg BW through the tail 

vein of male Wistar rats 

Nano-TiO2, 20-30 nm, 17% 

anatase, 30% rutile, uncoated, 

BET surface area 48.6 m2

/g 

Fabian et al. (2008, 

157576) 

TiO2 concentrations in the brain were measured 

on days 1, 14, and 28 by ICP-AES with a Thermo 

Jarrell Ash “IRIS 1” spectrometer with a detection 

limit of 0.5 μm/organ 

Nano-TiO2, 15 nm, rutile, coated 

with silica (27.5 wt%) 

Single i.v. injection at approximately 60 mg/kg BW 

through the tail vein of male ddY mice 

No increase of Ti in the brain of treated mice 

was observed compared to negative controls 

at any tested time points. 

Sugibayashi et al. 

(2008, 157489) 

Ti concentrations in brain were measured at 

5 min, 72 hr, and 1 mo after injection by ICP-MS 

with an unspecified detection limit 

Ti concentrations in the brain increased with 

increasing nano-TiO2 doses. All TiO2 

treatments increased Ti concentration in the 

brain, as compared to negative controls. At 

150 mg/kg, brain Ti concentration was higher 

in the nano-TiO2 group than in the 

conventional TiO2 group. 

Multiple i.p. injection to female CD-1 (ICR) mice 

once per day for 14 days with nano-TiO2 at 5, 10, 

50, 100, and 150 mg/kg BW or conventional TiO2

at 150 mg/kg BW 

Nano-TiO2, 5 nm, anatase Liu et al. (2009, 

193516 Conventional TiO2 ) 

Both types of TiO2 were made 

from controlled hydrolysis of 

titanium tetrabutoxide. Ti concentration was measured 14 days after the 

treatment began by ICP-MS with a detection limit 

of 0.076 ng/mL 

Nano-TiO2 particles were seen in the brain 

(olfactory bulb and the cerebral cortex – 

frontal and temporal lobes) of the 6-wk-old 

mice from nano-TiO2-exposed dams. (Results 

from 4-day-old mice were not reported.) 

Nano-TiO2, 25-70 nm, anatase, Takeda et al. (2009, 

surface area 20-25 m2

/g, purity 

99.9% (from Sigma-Aldrich) 

s.c. injections of 100 μL of 1 mg/mL nano-TiO2

(i.e., 0.1 mg nano-TiO2) each time per pregnant 

Slc:ICP mice once per day at 3, 7, 10 and 14 days 

post-mating. 

193667) 

Presence of nano-TiO2 in the brain was assessed 

in the male offspring at age of 4 days and 6 wk by 

FE-SEM/ EDS 

Markers of apoptosis (activation of caspase-3 

and crescent-shaped cells), occlusion of 

small vessels, and perivascular edema 

observed in the brain of 6-wk-old mice from 

nano-TiO2-exposed dams. 

BET – Brunauer, Emmett, Teller method of calculating surface area ICP-MS – Inductively coupled plasma-mass spectrometry 

BW – Body weight i.p. – Intraperitoneal 

FE-SEM/EDS – Field emission-type scanning electron microscopy/energy dispersive Xray spectrometry i.v. – Intravenous 

s.c. – Subcutaneous 

ICP-AES – Inductively coupled plasma atomic emission spectrometry 

Increased Ti concentrations in the brain were observed in mice 2 weeks after they were 

exposed to fine and nano-TiO2 through a single dosage by oral gavage (Wang et al., 2007, 090290), 

and in mice following a 14-day exposure period of once-daily i.v. injections of nano-TiO2 (Liu et al., 

2009, 193516) (Table 4-5). No increase in Ti concentration in the brain was observed in rats or mice 

exposed to nano-TiO2 via a single i.v. injection (Fabian et al., 2008, 157576; Sugibayashi et al., 

2008, 157489). Due to the variations in nano-TiO2 treatment regimens, and other experimental 

design elements, no specific characteristic of nano-TiO2 or its administration has been identified as 

determining factors for BBB penetration. 

A recent study demonstrated presence of TiO2 particles, and pathological changes, in the brain 

of 6-week-old mice born to nano-TiO2 exposed dams (Takeda et al., 2009, 193667) (Table 4-5), 

suggesting that nano-TiO2 might be passed through undeveloped or developing BBB in embryos or 

4-25 
young mice. Because the dams were exposed to nano-TiO2 during pregnancy and the offspring were 

tested at 4 days and 6 weeks of age, the nano-TiO2 exposure to the offspring could have been in 

utero (i.e., nano-TiO2 could penetrate the placental barrier) or through milk, which was not tested in 

this study. In addition to the brain, nano-TiO2 particles and pathological changes were also observed 

in the reproductive system of male offspring of nano-TiO2-exposed dams (female offspring were not 

studied) (Takeda et al., 2009, 193667). Although no evidence was identified from human studies for 

nano-TiO2 passing through the placental barrier, an ex vivo study using perfused human placentas 

showed that nano-gold (PEGylated gold nanoparticles at 15 and 30 nm) did not cross the placenta 

into the fetal circulation at the tested condition (Myllynen et al., 2008, 187028). Nano-gold might 

behave differently from nano-TiO2, given that uncoated nano-gold does not penetrate either the BBB 

or placental barrier in mice (Sadauskas et al., 2007, 091407), whereas nano-TiO2 does pass through 

the BBB in mice (Liu et al., 2009, 193516; Wang et al., 2007, 090290). 

4.6.6. Dose Metrics 

Quantitative risk assessment relies on dose-response relationships. Selecting a measurable 

characteristic of dosage that would be appropriate for predicting nanoparticle toxicity has drawn 

attention from both researchers and risk assessors. No single metric is recommended in this 

document, but supporting evidence for various selections of a dose metric is noted. The criterion for 

selecting a “good” dose metric is often based on generating a consistent dose-response relationship. 

However, an appropriate dose metric need not constitute measurement of only one physicochemical 

property (such as surface area, mass, or number of particles). Although dose metrics based on one 

property, such as mass concentration, have been used successfully in toxicology, a combination of 

measurements of two or more physicochemical properties also might be appropriate for use in 

assessing nanomaterial toxicity. Recently, the OECD developed a guidance document on sample 

preparation methods and dosimetry for safety testing with nanomaterials (OECD, 2010, 644192). 

Total particle surface area, which is closely related to primary particle size, has been suggested 

as a suitable dose metric for inhalation and instillation studies (Faux et al., 2003, 625074; Liao et al., 

2009, 157456; Oberdörster et al., 2005, 087559). Although two distinctive dose-response curves for 

fine TiO2 and nano-TiO2 can be drawn based on mass concentration, certain observed respiratory 

effects of fine TiO2 and nano-TiO2 have been shown to fit well with a single linear dose-response 

curve based on primary particle surface area, even where both types of particles agglomerated to 

approximately 0.7 μm in diameter (Oberdörster et al., 1994, 046203). Hext et al. (2005, 090567) 

found that, compared to gravimetric lung burden (particle mass per lung mass), administered primary 

particle surface area correlated better with lung burdens, clearance half-lives, and certain biological 

responses in rats, mice, and hamsters. However, the evidence on this issue is somewhat mixed. For 

instance, biological responses after exposure to similarly-sized agglomerates of fine TiO2 and 

nano-TiO2 were similar in severity according to Warheit et al. (2006, 088436; 2007, 091305); by 

contrast, Sager and Castranova (2009, 193625) found that well-dispersed nano-TiO2 yielded greater 

effects than well-dispersed fine TiO2. 

As mentioned previously, any one or more of various characteristics, including particle 

number, size (including agglomerations or aggregations), shape, crystalline form, mass, surface area, 

and surface modifications, could play a role in nano-TiO2 toxicity. Including one or more of these 

factors in the dose metric could be a better choice than surface area alone. For instance, based on 

administered primary particle surface area, the data used in the Hext et al. study (2005, 090567) – 

the increases in the numbers of pulmonary polymorphonuclear neutrophil (PMN) due to exposure to 

anatase fine and nano-TiO2 (Oberdörster et al., 1994, 046203) and rutile fine TiO2 (Cullen et al., 

1999, 157905) – would better fit two dose-response curves (one each for anatase TiO2 and rutile 

TiO2), instead of one dose-response curve. Similarly, a recent study of pulmonary effects of 

intratracheal instilled rutile fine TiO2 and 80% anatase/20% rutile nano-TiO2 (Sager et al., 2008, 

157499) showed that when dose was normalized to surface area of the particles administered, the 

4-26 
4-27 

dose-response curves for inflammogenic responses were not statistically different between fine and 

nano-TiO2, but the anatase-rutile nano-TiO2 always yielded greater (1.3- to 2-fold) responses than the 

rutile fine TiO2. 

Due to limited toxicological data from oral or dermal exposure to nano-TiO2, the choice of 

dose metric for these exposure routes has not been widely discussed. For in vitro studies, 

nanoparticle concentration (mass or surface area) is often used to express dose. In vitro cytotoxicity, 

however, has been reported to be affected by both the concentration and the total mass (or total 

number or total surface area, since these three are closely related) of nanoparticles (Lison et al., 

2008, 157530). In the Lison et al. study (2008, 157530), when cells were cultured in various volumes 

of a medium containing the same amount of nano-silica (same mass/number/surface area), higher 

toxicity occurred in a lower volume of medium, that is, in higher nano-silica concentrations. When 

the medium contained the same concentrations of nano-silica, higher toxicity occurred in cells 

cultured with a higher volume of medium than lower volume of medium. 
Chapter 5. Characterization of Effects 

The preceding chapters have laid a foundation for the present chapter by providing an 

exposure context for characterizing the effects of nano-TiO2 used for drinking water treatment and in 

topical sunscreens. This chapter provides information on the factors that influence nano-TiO2

ecological and health effects (Section 5.1), the ecological effects of nano-TiO2 (Section 5.2), and the 

toxicological and human health effects of nano-TiO2 (Section 5.3). Whether there are specific byproducts (e.g., waste and transformation products) or interactions with other substances that should 

or can be evaluated has not yet been determined. For this reason, the focus of this chapter is on 

nano-TiO2. 

Although literature exists on the effects of conventional TiO2 on humans and laboratory 

animals (NIOSH, 2005, 196072), comparatively less information is available on the effects of 

nano-TiO2. Consistent with studies of other nanomaterials (Ostrowski et al., 2009, 193592), most 

nano-TiO2 studies have investigated the ecological or health effects of nano-TiO2 itself, and 

relatively few have investigated the ecological or health effects of end-use products containing 

nano-TiO2 or their life-cycle by-products. 

The physicochemical characteristics of nano-TiO2 could be important to the biological effects 

of these materials (Section 5.1), yet those characteristics frequently are not evaluated or reported as 

part of studies of such effects. This observation should serve as a caveat in examining and 

interpreting the results described throughout this chapter. 

The following sections are not meant to be an exhaustive review of the ecological and human 

health effects literature for nano-TiO2. Instead, this chapter is intended to highlight recent work on 

the effects of nano-TiO2 and to identify current knowledge status and gaps in information needed for 

assessing potential risks of nano-TiO2 in water treatment and sunscreen. 

5.1. Factors that Influence Ecological and Health Effects 

of Nano-TiO2

The large number of variables associated with nano-TiO2 material itself and its ecological and 

health effects makes it extremely difficult to identify the primary characteristic(s) of nano-TiO2 

contributing to an effect or to compare the importance of different characteristics to such effects. A 

common statement from early studies is the announcement of size effects (or the lack of size effects) 

from nano-TiO2 of different crystalline forms or anatase/rutile ratios. That size alone does not 

account for the effects of nano-TiO2, however, is now generally accepted; other factors, such as 

shape, surface chemistry, photoreactivity, and other characteristics, could also play a role in these 

effects (Gonzalez et al., 2008, 157569; Hassellöv et al., 2008, 157559; Powers et al., 2006, 088783). 

With the advance of nanoparticle synthesis, the influence of different physicochemical 

characteristics of nano-TiO2 has been investigated using well-characterized nano-TiO2 and better 

control of variables in recent studies (Jiang et al., 2008, 156609). 

Three categories of factors (nano-TiO2 physicochemical characteristics, experimental 

conditions, and environmental conditions) that could influence the ecological and toxicological or 

health effects of nano-TiO2 are discussed here in Section 5.1. These are not the only factors of 

potential importance. As noted previously, exposure route can play a major role in the effects of 

nano-TiO2, and the importance of this is reflected in the fact that much of the information in this 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments. 

 5-1 
chapter is organized around environmental matrices and routes of exposure. Host effects, particularly 

species differences, can also play an important role in the effects of nano-TiO2. For example, skin 

penetration is greatest in rabbits, followed by rats, pigs, monkeys, and humans (Nohynek et al., 

2007, 090619). However, little information is available on these species differences or on differences 

in susceptibility of different cell types to nano-TiO2 effects (Kiss et al., 2008, 157547). The 

phenomenon of pulmonary particle clearance “overload” and subsequent effects in rats and mice are 

much more understood and are discussed in Section 5.3.1.2. In the following sections, the order in 

which factors are presented does not imply relative importance. This section focuses on factors that 

have been shown to be germane to nano-TiO2, but findings related to other types of nanomaterials 

are noted where relevant. 

5.1.1. Nano-TiO2 Physicochemical Characteristics 

Size, crystal structure, and surface chemistry (such as coating) are among the factors that 

influence nano-TiO2 effects. Other physicochemical properties, such as shape (Warheit et al., 2006, 

088436; Yamamoto et al., 2004, 157820), manufacturing process, doping, and purity (or impurities) 

could also play a role in nano-TiO2 toxicity, but such information is usually not reported in 

ecological and toxicological studies. Contributing to this lack of reported characteristics are 

limitations in the availability of analytic methods for characterizing such nanomaterials. Databases 

describing detailed nanoparticle properties and health effects are being developed (Miller et al., 

2007, 092297). 

The need for characterization of nanomaterials used in toxicity studies has been noted in 

reports and journal articles, with possible attributes for minimal characterization including chemical 

composition, size and size distribution (for primary particles and agglomerates), shape, specific 

surface area, and number of particles per unit mass (Department for, 2007, 195461; Powers et al., 

2006, 088783; Powers et al., 2007, 090679; Warheit et al., 2007, 091075). For more information on 

nanomaterial physiochemical characteristics that could affect ecological and toxicological effects, 

readers are referred to reports listing recommended information to be included in nanomaterial 

studies (OECD, 2008, 157512; Attachment 5 to Appendix D of Taylor, 2008, 157487; Warheit et al., 

2007, 091305). A compilation of characterization recommendations from a multi-stakeholder group 

can also be accessed at http://characterizationmatters.org. 

5.1.1.1. Size 

Size is a main determining factor for the distribution of (inhaled or instilled) nano-TiO2 in and 

outside of the respiratory tract (Oberdörster et al., 2004, 055639). For particles with a diameter less 

than 100 nm, the smaller the particles are, the more total particle deposition in the respiratory tract 

and deposition in nasopharyngolaryngeal regions (Oberdörster, 2000, 036303). Smaller sizes, 

however, do not always result in more deposition in other regions of the respiratory tract. For 

example, the highest percentages of alveolar deposition have been observed in nanoparticles of 

approximately 20 nm in size, and the highest percentages of tracheobronchial deposition were 

observed in nanoparticles 1-10 nm in size (Oberdörster, 2000, 036303). Furthermore, particles less 

than 200 nm in size can be transported from olfactory mucosa to the olfactory bulb of the brain via 

the olfactory nerve (Elder et al., 2006, 089253). Exposures to nano-TiO2 (with mean diameters of 

21.05 ± 5.08 nm, 71.43 ± 23.53 nm, and 154.98 ± 32.98 nm) through intranasal instillation increased 

Ti concentrations in the olfactory bulb in mice (Wang et al., 2005, 193703; Wang et al., 2007, 

090290), and two types of nano-TiO2 particles (80-nm rutile and 155-nm anatase) were found to 

increase Ti concentrations in hippocampus, central cortex, and cerebrum, in addition to olfactory 

bulb, in mice after repeated intranasal instillation (Wang et al., 2008, 157473). 

Jiang et al. (2008, 156609) investigated the size effects of nano-TiO2 on the generation of ROS 

per unit of particle surface area. Using nine different sizes (4-195 nm) of anatase nano-TiO2, the 

 5-2 
investigators found that the highest levels of ROS generation per unit surface area were generated by 

30 nm and larger particles. For nano-TiO2 less than 30 nm, the ROS generation per surface area 

decreased with decreasing particle diameter down to 10 nm, below which it was constant (Jiang et 

al., 2008, 156609). 

5.1.1.2. Crystallinity 

TiO2 crystalline forms also influence TiO2 and nano-TiO2 photoreactivity, reactive species 

generation, and toxicity. Nano-TiO2 containing more anatase tends to generate more free radicals and 

induce more toxicity (e.g., cytotoxicity, inflammatory response) than nano-TiO2 containing ore rutile 

(Hidaka et al., 2005, 157804; Sayes et al., 2006, 090569; Uchino et al., 2002, 090568). The influence 

of crystal forms of nano-TiO2 on ROS generation was investigated using a fixed total surface area by 

Jiang et al. (2008, 156609), who tested 13 nano-TiO2 particles of varying crystallinity, all within the 

size range of 42 to 102 nm. The researchers found that the ROS generation per unit surface area was 

highest in amorphous nano-TiO2, followed by anatase and then nano-TiO2 containing both anatase 

and rutile, and was lowest in rutile nano-TiO2 (Jiang et al., 2008, 156609). This finding is consistent 

with those of a study investigating unusually fast weathering (loss of gloss) or degradation of surface 

coating on steel roofing, associated with sunscreens left by workers during installation (Barker and 

Branch, 2008, 180141). Nano-TiO2 in the coating-damaging sunscreens was an anatase/rutile 

mixture, whereas nano-TiO2 in the one sunscreen that did not accelerate loss of gloss was pure rutile 

(Barker and Branch, 2008, 180141). 

The cytotoxicity of anatase and anatase-mixtures was further increased by UV illumination. 

Anatase nano-TiO2 can be 100 times more cytotoxic under UV than rutile of similar size (Sayes et 

al., 2006, 090569). The hydroxyl (·OH) radical production by nano-TiO2 in cultured cells was found 

to depend on the crystalline form and size, but differences in OH radical production were not 

explained by the differences in UV-A absorption between anatase and rutile (Uchino et al., 2002, 

090568). Smaller particles that contain more anatase, however, are not always more toxic either 

in vitro (Sayes et al., 2006, 090569) or in vivo (Warheit et al., 2006, 088436) than larger particles 

containing more rutile. 

5.1.1.3. Surface Chemistry 

Although coatings have been used to decrease the photoreactivity of nano-TiO2 intended for 

sunscreen (Section 2.2.2), coatings affect more than photoreactivity. In particular, the presence of a 

surface coating changes the nature of the interface between the nano-TiO2 particle and the 

environment or an organism, and it is not clear whether the surface coating or the core material 

dominates particle-environment and particle-organism interactions. Coatings for nano-TiO2 particles 

can be designed to reduce agglomeration/aggregation, which in turn affects the behavior of the 

particles in various matrices, including sedimentation behavior. This also affects the exposure 

conditions of organisms living in different parts of bodies of water or feeding on different sized 

particles. Particle surface modifications can also change the effects of nano-TiO2 on living cells, 

tissues, or organisms. Using in vitro methods, Serpone et al. (2006, 157736) reported that a 

“thermally assisted” modification of the TiO2 particle surface reduced photocatalytic activity, which 

in turn decreased (if not eliminated) toxicity to DNA plasmid, human cells, and yeast. In rats 

intratracheally instilled with two types of nano-TiO2 having the same core material, the nano-TiO2

with a hydrophobic surface (Aeroxide® T805, silanized) caused a slightly lower bioactivity than 

 5-3 
1 hydrophilic P25, although the authors concluded that silanization did not “lead to remarkable 

differences in lung reaction” (Rehn et al., 2003, 090613). 

5.1.1.4. Recommended Characterization of Nanomaterial for Ecological and 

Toxicological Studies 

As noted in Chapter 1, nanomaterials, and nano-TiO2 in particular, can be characterized in 

several ways in terms of physicochemical properties (Table 1-1). Given that the relationship between 

such properties and the behavior and effects of nanomaterials, including nano-TiO2, remains to be 

fully understood, it might seem desirable for researchers to characterize every possible property of 

the material they are investigating. In practice, this is not feasible. Consequently, recommendations 

for characterization of nanomaterials have periodically been made. 

For in vitro studies, Murdock et al. (2008, 193563) recommended characterizing nanomaterial 

dispersion in solution for (in no specific order) particle size and size distribution; particle 

morphology; particle composition; surface area; surface chemistry; particle reactivity; 

agglomeration; zeta potential; and impact of sonication. For human and environmental testing, a 

roundtable discussion at the 2005 Society of Toxicology Annual Meeting considered the following as 

essential parameters in nanomaterial physicochemical properties: size distribution, agglomeration 

state, crystalline structure, chemical composition, and shape (Holsapple and Lehman-McKeeman, 

2005, 088087). For “hazard studies with nanoparticle-types,” Warheit (2008, 193706) prioritized the 

characterization needs as (highest priority first): (1) particle size and size distribution (wet state) and 

surface area (dry state) in the relevant media; (2) crystal structure/crystallinity; (3) aggregation status 

in the relevant media; (4) composition and surface coatings; (5) surface reactivity; (6) method of 

nanomaterial synthesis and/or preparation; and (7) purity of sample. 

An expert working group convened by the International Life Sciences Institute (ILSI) 

Research Foundation/Risk Science Institute recommended both off-line (i.e., not using time-resolved 

continuous techniques) and on-line (continuous) measurement of mass, size distribution, surface 

area, and particle number for exposure characterization in inhalation studies (Table 5-1), and 17 offline measurements/aspects for nanomaterial characterization for toxicological studies (Table 5-2) 

(Oberdörster et al., 2005, 090087). 

 

1

 Silanization is the covering of a surface that has hydroxyl (OH) groups with molecules that contain only silicon and hydrogen (silane), 

such as SiH4. Silanization is one type of surface modification applicable to particles, such as metal oxides, and can render the particle 

surface chemically inert. 

 5-4 
Table 5-1. Published recommendations for measuring nanomaterial parameters for exposure 

during characterization inhalation studies 

Recommendation 

Metric Measurement 

Off-line (Discrete)a On-line (Continuous)b

Mass E (coupled with on-line) E 

Size distribution E E/D 

Surface area O O 

Particle Number N E 

a

Off-line: Collected and analyzed later. 

b

On-line: Real-time collection and analysis during the process. 

E – These measurements are considered to be essential. 

D – These measurements are considered to provide valuable information, but are not recommended as essential due to constraints associated with complexity, cost and 

availability. 

O – These measurements are considered to provide valuable but nonessential exposure information. 

N – These measurements are not considered to be of significant value to inhalation studies. 

Source: Modified with permission from BioMed Central, Oberdörster et al. (2005, 090087). 

 5-5 
Table 5-2. Published recommendations for off-line nanomaterial characterization using 

noncontinuous techniques for toxicological studies 

Toxicity Screening Studies 

Characterization Human Exposure Supplied Material Material as 

Administered 

Material after 

Administration 

(in vivo/in vitro) 

E (combine with 

agglomeration state) Size distribution (primary particles) E D D 

Shape E E O O 

Surface area D E D O 

Composition E E O O 

Surface chemistry D E D D/O 

Surface contamination D N D N 

Surface charge – suspension/solution O E E O 

Surface charge – powder (use bio fluid surrogate) O E N O 

Crystal structure O E O O 

Particle physicochemical structure E E D D 

Agglomeration statea E N E D 

Porosity D D N N 

Method of production E E -- -- 

Preparation process -- -- E -- 

Heterogeneityb D E E D 

Prior storagec of material E E E -- 

Concentration E -- E D 

E – These characterizations are considered to be essential. 

D – These characterizations are considered to provide valuable information, but are not recommended as essential due to constraints associated with 

complexity, cost and availability. 

O – These characterizations are considered to provide valuable but nonessential information. 

N – These characterizations are not considered to be of significant value to screening studies. 

a

As primary particle, secondary particle (primary particle agglomerates and self-assembled structures) and tertiary structure (assemblies of secondary 

structures). When possible, material agglomeration or de-agglomeration in different liquid media should also be characterized. b

Time and conditions, including temperature, humidity, exposure to light and atmosphere composition. c

Ratios of different components. 

Source: Reprinted with permission from BioMed Central, Oberdörster et al. (2005, 090087). 

Three factors figured into these recommendations: “the context within which a material is 

being evaluated, the importance of measuring a specific parameter within that context, and the 

feasibility of measuring the parameter within a specific context” (Oberdörster et al., 2005, 090087). 

5.1.2. Experimental Conditions 

Experimental conditions, particularly the choice of medium/vehicle in which to disperse 

nano-TiO2, preparation of testing solutions or suspensions, and the formation of aggregates, can 

influence the behavior and effects of nano-TiO2 and other nanomaterials. For example, instability of 

nanoparticle suspensions may cause particle settling during the experiment, leading to the effective 

concentration being either higher or lower than the initial added concentration. This can result in 

 5-6 
improper interpretation of the dose-response relationship. The advantages and disadvantages of 

various dispersion preparation methods are compared in a recent publication of nanomaterial 

ecotoxicity test methods (Crane et al., 2008, 157583). 

5.1.2.1. Medium/Vehicle 

Nano-TiO2 in an oily dispersion penetrates deeper into skin than nano-TiO2 in an aqueous 

dispersion, as shown in an ex vivo study using healthy adult skin (intact samples of tissue removed 

from the body, and manipulated in vitro) (Bennat and Muller-Goymann, 2000, 157403). Nano-TiO2

did not penetrate into living cells of the skin in either aqueous dispersion or oily dispersion. When 

the dispersal of nano-TiO2 was made in the aqueous phase of an oil-in-water emulsion, nano-TiO2

did not penetrate into skin, but the emulsion was not stable. Although the stability could be improved 

by encapsuling the nano-TiO2 into liposomes, liposome formulation increases nano-TiO2 skin 

penetration (Bennat and Muller-Goymann, 2000, 157403). In another relevant phenomenon, the 

nanoparticles by themselves can act as a dispersant, forming so called Pickering emulsions and 

essentially acting as the surfactant that helps make liposomes. An in vivo study (Lansdown and 

Taylor, 1997, 157928) in rabbits also demonstrated that uptake of TiO2 particles in sizes ranging 

from 2 to 20 μm was affected by the vehicle: uptake was greatest in castor oil, followed by water, 

and then polyethylene glycol. 

Different levels of free radical production were observed in cultured cells exposed to similar 

nano-TiO2 but within different formulae of suspensions (Uchino et al., 2002, 090568). Although 

nano-TiO2 F-1R (a formula containing nano-TiO2 that is 3% anatase and 97% rutile, with an average 

size of 93 nm and a surface area of 17 m2

/g) produced OH radicals after UV-A exposure, no OH 

radical production was detected after UV-A exposure in nano-TiO2 in a different formula, St-C 

(sunscreen standard C from the Japan Cosmetic Industry Association containing nano-TiO2 that is 

2% anatase, 98% rutile, with an average size of 85 nm and a surface area of 19 m2

/g). Most rutile 

nano-TiO2 is relatively inefficient in free radical production, and the F-1R used in this study 

produced more OH radicals than all four other, mainly rutile nano-TiO2 forms and one of the anatase 

forms tested (Uchino et al., 2002, 090568). Although nano-TiO2 has been reported to generate ROS 

in cell-free conditions but not in cells (a murine macrophage cell line, RAW 264.7) (Xia et al., 2006, 

089620), whether nano-TiO2 in different formulae also causes different levels of ROS production in 

cells has not been verified. 

The purity of water affects the degree of aggregation, which in turn may affect exposure-dose 

and toxicity. The degree of aggregation generally increases with an increase in ionic strength, and the 

extent of aggregation can also be affected by the presence of NOM and other constituents such as 

divalent cations (Domingos et al., 2009, 193347; French et al., 2009, 193384; Kim et al., 2009, 

635778). Aggregation was more severe in tap water than in nanopure water (Zhang et al., 2008, 

157462), and is likely to be more severe in fish tank water or pond water than in tap water. Because 

nano-TiO2 in the environment is more likely to be present in aggregated form, results from 

nano-TiO2 suspensions with aggregates can be informative, and as noted earlier, might even be more 

relevant than results from a perfectly dispersed suspension with nano-TiO2 in primary particle form. 

The lack of accurate measurement of nano-TiO2 (e.g., size distribution, mass concentrations, 

numbers, and surface area) and a generally-agreed-upon choice of dose metrics, however, impede the 

establishment of a reliable dose-response relationship. 

In respiratory exposure studies, intratracheal instillation exposure typically uses saline as a 

vehicle for TiO2 delivery while inhalation exposure uses air. The behavior of nano-TiO2 (such as 

agglomeration) is expected to be different in air than in solution. Furthermore, the vehicle alone can 

affect respiratory system responses, at least for a short time. Transient inflammation in the 

respiratory tract occurs in rats given saline alone through instillation (Driscoll et al., 1990, 087145; 

Henderson et al., 1995, 002744). Sager et al. (2007, 091214) tried to disperse several types of nanosized particles, including TiO2, in several suspension media, including: PBS; rat and mouse BAL

 5-7 
fluid; and PBS containing DPPC or mouse serum albumin or both. Although the dispersion in PBS 

was not satisfactory, BAL fluid was an excellent vehicle for dispersing the particles. The dispersion 

was also unsatisfactory in saline containing albumin alone or DPPC alone, in concentrations found in 

BAL fluid. Adding protein plus DPPC in PBS, however, produced satisfactory, albeit slightly less 

effective, substitutes for BAL fluid. The Sager et al. (2007, 091214) experiment demonstrates the 

importance of the suspension medium and suggests that the immediate milieu (such as the BAL fluid 

and protein and DPPC in lung) affects not only the agglomeration of nano-TiO2, but also the 

consequent effects on nano-TiO2 behavior and effects. 

5.1.2.2. Dispersion Preparation 

The potential importance of dispersion preparation for nanomaterial ecotoxicity is illustrated 

by fullerene (C60) studies. C60 toxicity in daphnids and fishes was higher when the C60 suspension 

was prepared with the organic solvent tetrahydrofuran (THF) than when the suspension was prepared 

by stirring and sonication (Henry et al., 2007, 157684; Zhu et al., 2006, 157721). Entrapped or 

residual THF in the C60 and THF degradation products were suspected to have contributed to toxicity 

(Henry et al., 2007, 157684). Nevertheless, no difference in toxicity to daphnids was observed 

between nano-TiO2 suspensions prepared with and without THF (Klaper, personal communication, 

2008, 157546; Lovern and Klaper, 2006, 088040). Regardless of dispersion method, aggregation of 

nano-TiO2 might be unavoidable. Several studies reported that nano-TiO2 formed aggregates or 

agglomerates in water, and that these aggregates/agglomerates could not be separated into primary 

particles by ultrasound or chemical dispersants (Griffitt et al., 2008, 157565; Jeng and Swanson, 

2006, 090085; Zhang et al., 2008, 157462). Furthermore, an unfiltered nano-TiO2 suspension with 

aggregates/agglomerates has been reported to be less toxic to daphnia than a filtered nano-TiO2

suspension that mostly contained primary particles (Lovern and Klaper, 2006, 088040). In contrast to 

Lovern and Klaper’s (2006, 088040) reported difficulty of disagglomerating particles by sonication 

or chemical dispersants, Federici et al. (2007, 091222) reported effective dispersion of P25 by 

sonication in ultrapure water at final working concentrations up to 1 mg/L, although they did not 

evaluate potential agglomeration in test tank water at these concentrations. 

In addition to the medium itself, the dispersion method can affect not only the nanoparticle 

agglomeration or aggregation (such as the degree and size of agglomerates) but also the effects of 

nanoparticles (Bihari et al., 2008, 157593). For example, sonication with ultrasound has been used to 

decrease nano-TiO2 agglomeration (Bihari et al., 2008, 157593) and has been shown to generate 

particles or agglomerates in the nanoparticle range (Maier et al., 2006, 090451). However, sonication 

alone could increase the size of nano-TiO2 agglomerates, as reported by Porter et al. (2008, 157508) 

who found that the mean agglomerate size of P25 in PBS increased from 1,930 nm before sonication 

to 2,849 nm immediately after sonication, while the same sonication procedure decreased the sizes 

of agglomerates of P25 dispersed in BAL fluid and in a mimic BAL fluid that contained Ca2+- 

and Mg2+-free PBS, serum albumin, and DPPC. No explanation was provided. Furthermore, 

ultrasound sonication has been reported to increase nano-TiO2 catalytic activity in breaking down an 

organic dye (acid red B) (Wang et al., 2009, 157453), but also to decrease changes in enzyme 

activity caused by ingested nano-TiO2 in isopods (Jemec et al., 2008, 157552). Post-preparation 

analysis of particle size is important when comparing laboratory studies and formulations with 

sunscreen preparations. Although studies of nano-TiO2 particle and agglomerate sizes are available 

(Delrieu et al., 2007, 157449), very few health effects studies have characterized nano-TiO2 in 

sunscreen formulations and only a few studies characterized nano-TiO2 in other experimental 

mediums. Most health effects studies have reported characteristics of only dry nano-TiO2 primary 

particles, which are important but not representative of all exposure scenarios. 

Finally, without a special hydrophilic coating, nano-TiO2 forms a suspension in water (rather 

than a solution). Standard ecotoxicological test methods are intended for soluble or poorly soluble 

substances, and not designed for testing suspensions (BAuA, 2007, 157694). 

 5-8 
5.1.3. Environmental Conditions 

Once nano-TiO2 is released into the environment, its fate depends on abiotic and biotic 

conditions, which are likely to be more complex and diverse than standard ecological testing 

conditions. Of the many environmental factors that might be relevant to nano-TiO2 ecotoxicity, UV 

light exposure, purity of water (Zhang et al., 2008, 157462), and presence of organic matter 

(Domingos et al., 2009, 193347) have been investigated. Factors that affect nano-TiO2 aggregation, 

such as pH value, ionic strength, and cation valence (Domingos et al., 2009, 193347; 

Dunphy Guzman et al., 2006, 090584; French et al., 2009, 193384), would influence not only 

nano-TiO2 fate and transport (Chapter 3), but also potential exposure and possibly ecological effects. 

Only environmental factors that have been shown to affect toxicity in organisms used for ecological 

effects testing are discussed here. 

UV light is well known to increase the cytotoxicity of nano-TiO2, particularly photocatalytic 

nano-TiO2 such as anatase or anatase/rutile mix, to cultured mammalian cells (Sayes et al., 2006, 

090569) and fish cells (Reeves et al., 2008, 157506; Vevers and Jha, 2008, 157475) as well as 

microorganisms (Adams et al., 2006, 157782). Genotoxicity (Nakagawa et al., 1997, 157927) and 

clastogenicity (Nakagawa et al., 1997, 157927) of nano-TiO2 to cultured mammalians cells were also 

increased by UV exposure. This UV-increased toxicity is at least partially due to the greater number 

of hydroxyl radicals (·OH) generated by anatase than by rutile under UV exposure (Sayes et al., 

2006, 090569; Uchino et al., 2002, 090568). UV exposure may influence the effects of nano-TiO2 in 

sunscreen indirectly by causing sunburn, which can make skin more permeable (Mortensen et al., 

2008, 155612). In addition to UV, visible light was shown to increase the cytotoxicity of nano-TiO2

(carbon-doped TiO2 and TiO2 modified with platinum [IV] chloride complexes) in bacteria and fungi 

(Mitoraj et al., 2007, 157665). 

Nano-TiO2 was found to form aggregates more in pond water than in pure water (MilliQ 

water), although no nano-TiO2 toxicity to soil bacteria, green algae, or water fleas was detected in 

either pond water or pure water at up to 100 mg/L (Velzeboer et al., 2008, 157476). The adsorption 

of nano-TiO2 onto certified reference material sediment did not increase the toxicity of the sediment 

(Blaise et al., 2008, 157592). 

Additional environmental factors that might indirectly influence the effects of TiO2

nanoparticles in sunscreen include moisture; pH and water chemistry; and temperature. High 

humidity in the environment could increase the hydration level of the stratum corneum, and could 

lead to increases in skin permeability and penetration of both hydrophilic and lipophilic components 

(Benson, 2005, 193273; Zimmerer et al., 1986, 193744). For example, the level of penetration of 

nano-TiO2 on soaked skin, which is likely to occur after swimming or other water activities, has not 

been investigated. Similar to medium and vehicle effects on nano-TiO2, the pH and chemistry of the 

water in which sunscreen may be mixed, e.g., in a swimming pool versus a lake or an ocean, might 

also modulate nano-TiO2 effects. Finally, sunscreen is often used at much higher temperatures than 

typical ambient laboratory temperatures. Although nano-TiO2 itself is not expected to change in the 

temperature range tolerable for human beings, increased body temperature and sweat may affect 

nano-TiO2 dermal penetration and thus its effects (Lu et al., 2008, 157526). 

The influence of the immediate milieu on nano-TiO2 behavior and effects is also evident when 

nano-TiO2 is inside an organism. For instance, in vitro studies showed that in rat BAL, nano-TiO2

formed smaller aggregates and the aggregates remained small longer than nano-TiO2 in PBS (Porter 

et al., 2008, 157508; Sager et al., 2007, 091214; Sager et al., 2007, 090633). Because pH affects the 

electrostatic charge of nano-TiO2, it is plausible that nano-TiO2 would behave differently in tissues 

and cellular organelles with different pH values, such as very acidic pH values in the stomach and in 

lysosomes. 

 5-9 
5.1.4. Summary 

Nano-TiO2 physicochemical properties, experimental conditions, and the immediate 

environment or milieu, all can influence nano-TiO2 ecological and health effects. For example, 

nano-TiO2 size, crystalline form, and surface characteristics all influence nano-TiO2 behavior, 

including distribution, exposure potential, and effects. Although the influences of media, vehicles, 

and dispersion methods on particle aggregation and distribution have been reported, information on 

these influences on health effects is very scarce (Jemec et al., 2008, 157552). The presence of UV 

and visible light often increase photocatalytic nano-TiO2 activity and toxicity; other environmental 

factors, such as pH, ionic strength, and presence of organic matter of the aquatic environment, could 

also affect nano-TiO2 behavior and effects. 

5.2. Ecological Effects 

The ecological effects of nanomaterials have been gaining attention from the research and 

regulatory communities, and several review articles (Baun et al., 2008, 157598; Christian et al., 

2008, 157586; Hassellöv et al., 2008, 157559; Navarro et al., 2008, 157517; Nowack and Bucheli, 

2007, 092294) and conferences (such as the annual International Conference on the Environmental 

Effects of Nanoparticles and Nanomaterials) have addressed this topic. Although new information on 

nanomaterial ecotoxicity seems to emerge almost daily, available data thus far have been insufficient 

for a quantitative risk assessment of any particular nanomaterial. A thorough discussion of methods 

for ecotoxicity testing and characterization of nanomaterials (including in environmental media) is 

beyond the scope of these case studies, and has been reviewed elsewhere in peer-reviewed articles 

(Christian et al., 2008, 157586; Crane et al., 2008, 157583; Handy et al., 2008, 157562; Hassellöv et 

al., 2008, 157559) and in several public databases, such as those sponsored by the OECD 

(International Council on Nanotechnology, 2010, 644440; OECD, 2009, 644433; 

Project on Emerging Nanotechnologies, 2010, 644439). Nonetheless, a brief review of ecological 

effects testing and the importance of the tests are presented at the beginning of each of the following 

sections for the readers’ reference. 

Section 5.2.1 features a review of the ecological effects of nano-TiO2 exposure. Effects on 

bacteria and fungi are discussed in Section 5.2.1.1, effects on aquatic organisms are discussed in 

Section 5.2.1.2, effects on terrestrial organisms are discussed in Section 5.2.1.3, and indirect and 

interactive toxicity are discussed in Section 5.2.1.4. Section 5.2.1.5 summarizes the available 

ecological toxicity information. 

5.2.1. Ecological Effects of Nano-TiO2 Exposure 

Most of the nano-TiO2 ecological effect studies surveyed in this report (Table 5-3) used 

photocatalytic nano-TiO2, some of which could be suitable for water treatment purposes. Two of the 

studies used photostable nano-TiO2 intended for topical sunscreen (Wiench et al., 2007, 090635) and 

for protecting plastic from UV degradation (Warheit et al., 2007, 091075). Current FDA regulation 

of TiO2 in topical sunscreen does not specify crystalline form and does not require proof of 

photostability (or lack of photoreactivity). Pure anatase nano-TiO2 is much more photoreactive than 

pure rutile nano-TiO2, but it is possible to have photostable anatase or an anatase/rutile mix of 

nano-TiO2 by using doping or surface treatments, such as coating with silica. The coating of 

photostable nano-TiO2 is designed to endure the manufacturing process and consumer use 

(Lademann et al., 2000, 157895), but the long-term stability of coated TiO2 in sunscreen remains 

unclear. Once nano-TiO2 is released into the environment, various environmental factors, such as 

high ionic strength in sea water and high acidity in landfill leachate, could compromise some 

 5-10 
nano-TiO2 coatings. In a study presented in a professional conference, nano-TiO2 from a 

commercially available sunscreen has an outer hydrophobic polydimethylsiloxane coating and an 

intermediate AlOOH coating, and the coating was gradually degraded in water, as evidenced in one 

study by altered dispersion and release of silicon and Al from the coated nano-TiO2 product (Botta et 

al., 2009, 625076). Additionally, the by-products penetrated into aquatic organisms and were toxic at 

high concentrations (Botta et al., 2009, 625076). Therefore, the ecological effects of photocatalytic 

nano-TiO2 might be relevant not only for nano-TiO2 used in drinking water treatment but also for 

nano-TiO2 in sunscreen, because photoreactive nano-TiO2 can be used as the core material of 

photostable nano-TiO2 in sunscreen. For example, the core of Aeroxide® T805 is P25, a 

photocatalyst, and has been used as a UV filter in some sunscreens (Barker and Branch, 2008, 

180141; Evonik, 2007, 157696). 

Because mass concentration is reported for all studies reviewed, this dose metric is presented 

in Table 5-3 and in all subsequent discussion referring to the literature. Whenever information on 

surface area of the particles (to calculate particle surface area concentration) or the measured 

nano-TiO2 concentration (versus calculated based on added mass) present in the final test suspension 

was available, it is also provided in Table 5-3. The environmental relevance of the tested 

concentrations is unclear due to limited information on nano-TiO2 concentrations in the environment, 

although even the use of elevated concentrations in laboratory studies is informative in 

demonstrating the potential for an effect. It should be noted that several studies reported visible 

turbidity in nano-TiO2 stock suspension (Velzeboer et al., 2008, 157476; Zhang et al., 2006, 157722; 

Zhang et al., 2008, 157462). Because turbidity is likely caused by large aggregates of nano-TiO2, 

which can settle out of the liquid phase by gravity, actual concentrations of nano-TiO2 in the liquid 

phase might be lower than concentrations calculated based on mass of nano-TiO2 added. 

Table 5-3. Summary of nano-TiO2 ecological effects

Protocol 

(No UV illumination, unless 

specified) 

Test Species 

(Reference) Material Study Outcome 

Acute Exposure to Microorganisms 

Bacteria (Escherichia 

coli and Bacillus subtilis) 

(Adams et al., 2006, 

In dark, similar growth inhibition 

for both bacteria 

66-nm powder, ~35% rutile:65% anatase, 

average 330-nm in water (Sigma product 

634662) (Lyon, personal communication, 2008, 

6-hr exposure to: (1) 50, 100, 500, 1,000, 

2,000, 5,000 ppm in mediuma, in direct 

sunlight; or (2) 1,000 ppm in mediuma

, in 

dark In light, B. subtilis: 0%, 75%, and 

99% growth inhibition at 500, 

1,000, and 2,000 ppm, 

respectively 

157782) 157524) 

E. coli: 0%, 15%, 44%, and 46% 

inhibition at 100, 500, 1,000, and 

2,000 ppm, respectively 

Bacterium (Vibrio 

fischeri) 

(Blaise et al., 2008, 

<100-nm powder (Sigma product 634662, IC25 >100 mg/L 

Canada or France) 

15-min exposure, measure the reduction of 

light output from bioluminescent marine 

bacterium, Vibrio fischeri (Microtox® toxicity 

test) as an indicator of growth inhibition, 

tested concentrations not specified 

157592) 

Mix in a 1:1 ratio with certified reference 

material sediment, measure light output 

(Microtox® toxicity test) (indirect 

toxicity/interaction) 

Nano-TiO2 did not affect the 

toxicity of certified reference 

material sediment 

Bacterium (Vibrio 

fischeri) 

(Heinlaan et al., 2008, 

25- to 70-nm powder mixture of anatase and 

rutile, ratio not disclosed (Sigma product 13463-

67-7, Estonia) (Heinlaan, personal 

communication, 2008, 

30-min exposure for up to 20,000 mg/L 

nano-TiO2 and conventional TiO2, 8-hr 

exposure to 20,000 mg/L conventional TiO2

. 

Measure the reduction of light output from 

Vibrio fischeri (Flash assay) as an indicator 

of growth inhibition 

The highest concentration tested: 

20,000 mg/L nano-TiO2 (30-min 

exposure) did not decrease 

193414) 157558) bacterial growth 

Conventional TiO2: size and crystal form not 

disclosed (Sigma product 14027, Estonia; a 

former Riedel-de Haën product) (Heinlaan, 

personal communication, 2008, 

The highest concentration tested: 

20,000 mg/L conventional TiO2

(30-min and 8-hr exposure) did 

157558) not decrease bacterial growth 

 5-11 
Protocol 

(No UV illumination, unless 

specified) 

Test Species 

(Reference) Material Study Outcome 

EC50 >100 mg/Lb Bacterium (Vibrio 

fischeri) 

(Velzeboer et al., 2008, 

<75-nm (primary particle) nano-TiO2 in water 

suspension (Sigma product 643017, the 

Netherlands), mixture of rutile and anatase, ratio 

not reported (Velzeboer et al., 2008, 

15 min, 1, 10, 100 mg/L, measure reduction 

of light output from bioluminescent bacteria 

(Microtox® method, which could be 

affected by turbidity of 100 mg/L TiO2

suspension)b 157476) 157476) 

EC50 >100 mg/Lb Bacteria (from a soil 

sample, species not 

identified) 

(Velzeboer et al., 2008, 

<75-nm (primary particle) nano-TiO2 in water 

suspension (Sigma product 643017, the 

Netherlands), mixture of rutile and anatase, ratio 

not reported (Velzeboer et al., 2008, 

7 days (Biolog® test, gram positive), 

100 mg/L b 

157476

157476) 

) 

Bacteria and yeast 

(proprietary information) 

(Blaise et al., 2008, 

<100-nm powder (Sigma product 634662, 

France), characteristics in water not reported 

18-hr growth inhibition of 10 bacteria and 1 

baking yeast (microbial array for risk 

assessment [MARA] assay), tested 

concentrations not specified 

MTC >100 mg/L 

157592; Dando, 

personal 

communication, 2008, 18-hr exposure to the filtered elutriate from 

certified reference material sediment with 

and without nano-TiO2 mixed in a 1:1 ratio 

(MARA assay) (indirect toxicity/interaction), 

tested concentrations not specified 

Nano-TiO2 did not affect the 

toxicity of the elutriate of certified 

reference material sediment 

157582) 

Acute Exposure to Aquatic Organisms 

Alga (green alga, 

Desmodesmus 

subpicatus) 

(Hund-Rinke and 

Simon, 2006, 

72-hr growth inhibition, following the 

guidelines for EU standard algal assay (DIN 

38412-33, 1991, 

EC50 and effects of additional 

particle cleaning: 

25-nm primary particle, 20% rutile:80% anatase 

(Degussa P25) (Baun et al., 2008, 157598

090607) 

) 

(photocatalytic)(Product 1) 667415; ISA 8692, 2004, Product 1 (P25): EC50 was not 

different between nano-TiO2

washed once as manufacturer 

recommendation (32 mg/L) and 

nano-TiO2 with an additional 

wash (44 mg/L), suggesting 

toxicity was not from 

contaminants 

667212; OECD 201, 2006, 199838) with 

modifications to include pre-illumination of 

nano-TiO2 dispersion with simulated 

sunlight (wavelength 300-800 nm) at 250 

watts for 30 min; illumination alone did not 

affect D. subpicatus growth 

100-nm primary particle, 100% anatase; 

(Hombikat UV100) (Baun et al., 2008, 157598); 

photocatalytic (Mehrvar et al., 2002, 193541) 

(Product 2) 

Algal growth (without pre-illumination): 0, 

3.1, 6.2, 12.5, 25, 50 mg/L (products 1 

[P25] and 2 [UV100]) Product 2 (UV100): EC50

>50 mg/L, both nano-TiO2 with 

and without the additional wash 

(at up to 50 mg/L) caused less 

than 40% decrease in growth 

Shading effect: 0, 12.5, 25, 50 mg/L 

Algal growth (with pre-illumination): 12.5, 

25, 50 mg/L (Product 1[P25]) 

No shading effect: when 

nano-TiO2 dispersion (at up to 

50 mg/L) was between algae and 

light source (but not in contact 

with algae) for 72 hr, no effects 

on algal growth, suggesting 

nano-TiO2 effects was not due to 

lowered light intensity, but due to 

a toxicity of nano-TiO2

Pre-illumination of nano-TiO2

(Product 1 [P25]) did affect 

nano-TiO2 effects on algal growth 

EC50 >100 mg/Lb Alga (green alga, Pseudokirchneriella 

subcapitata) 

(Velzeboer et al., 2008, 

<75nm (primary particle) nano-TiO2 in water 

suspension (Sigma product 643017, the 

Netherlands), mixture of rutile and anatase, ratio 

not reported (Velzeboer et al., 2008, 

4.5 hr, in light, 100 mg/L 

Photosynthesis efficiency was measured as 

a pulse amplitude modulation (PAM) 

fluorescence test, which could be affected 

by turbidity of 100-mg/L TiO2 suspensionb

157476

157476) 

) 

EC50 21 mg/L (based on 

decreases in healthy cell 

number) 

Alga (green alga, 

Pseudokirchneriella 

subcapitata) 

(Warheit et al., 2007, 

199838

091075) 

140 nm in water, 79% rutile: 21% anatase, 

coated (90-wt % TiO2, 7% alumina, and 1% 

amorphous silica) (DuPont uf-C TiO2) (photopassivative/ photo-stable) (Warheit, 2008, 

OECD 201 (2006, ) (72-hr growth), 

with lighta

0.01, 0.1, 1, 10, and 100 mg/L (uf-C TiO2

and fine TiO2) EC50 87 mg/L (based on inhibition 

of growth rate) 157470) 

Fine TiO2: 380 nm in water, rutile, coated (~99% 

TiO2 and ~1% alumina) 

EC50 16 mg/L (based on 

decreases in healthy cell 

number) 

EC50 61 mg/L (based on inhibition 

of growth rate) 

 5-12 
Protocol 

(No UV illumination, unless 

specified) 

Test Species 

(Reference) Material Study Outcome 

Alga (green alga, 

Pseudokirchneriella 

subcapatitata) 

(Huang et al., 2005, 

Photocatalytic nano-TiO2: Deguassa P25 (75% 

anatase, 25% rutile, 30 nm) 

Short-term chronic toxicity – Adsorbsion 

onto surface of algae; concentrations not 

specified. 

Algae carried 2.3 times their own 

weight in TiO2 particles on their 

surface 

Cellular weight increased by 

157801) >130% 

Ala (green alga, ) OECD 201 (2006, 

(Aruoja et al., 2009, 

Nano-TiO2 199838

193254) Conventional (Bulk) TiO2

) (algal growth 

inhibition test) Algal cell culture; formation 

of agglomerates. 

Nano-TiO2 formed large 

agglomerates with almost all 

algal cells entrapped. 

EC50 = 5.83 mg/L 

NOEC = 0.98 mg/L 

Conventional TiO2 formed small 

agglomerates with some algal 

cells entrapped and some 

remained free. 

EC50 = 35.9 mg/L 

NOEC = 10.1 mg/L 

Invertebrate (water flea, 

Daphnia magna) (HundRinke and Simon, 2006, 

667232

090607) 

25-nm primary particle, 20% rutile:80% anatase 

(Degussa P25) (2008, 

ISO 6341 (1996, 

157598) (photocatalytic); 

ultrasonic dispersion 

), OECD 202 

(2004, 

Pre-illumination increased toxicity 

compared to the same 

concentration 

667207) and DIN 38412-30 (1989, 

667416) (48-hr immobility), exposure to up 

to 3 mg/L, 16:8 hr light:dark cycles, 

compare the effects of pre-illuminated and 

nonilluminated nano-TiO2

No dose-response relationship 

with either pre-illuminated or 

nonilluminated nano-TiO2

0, 1, 1.5, 2, 2.5, 3 mg/L 

100-nm primary particle, 100% anatase; 

(Hombikat UV100) (2008, 

Pre-illumination showed a trend 

157598); of increasing toxicity 

photocatalytic (Mehrvar et al., 2002, 193541); 

ultrasonic dispersion No dose-response relationship 

with either pre-illuminated or 

nonilluminated nano-TiO2

Invertebrate (water flea, LC50 5.5 mg/L 

Daphnia magna) 

(Lovern and Klaper, 

2006, 

Primary particle <25 nm (smallest 5-nm), 

anatase, uncoated (photocatalytic) (Klaper, 

personal communication, 2008, 

EPA 48-hr tox test (U.S. EPA standard 

operating procedure 2024, 1991, 667211

088040) 

157546); filtered 

through a 0.22-μm nylaflo filter, secondary 

particle 20-30 nm in deionized water 

) 

(mortality) LOEC 2.0 mg/L 

NOEC 1.0 mg/L Filtered nano-TiO2: 0.2, 1, 2, 5, 6, 8, and 

10 ppm 

Primary particle <25 nm (smallest 5 nm), 

anatase, uncoated (photocatalytic) (Klaper, 

personal communication, 2008, 

Sonicated, unfiltered nano-TiO2: 50, 200, 

250, 300, 400, and 500 ppm 

LC50 >500 mg/L 

157546) 

sonicated, unfiltered, secondary particle 

100-500 nm in deionized water 

Invertebrate (water flea,

Daphnia magna) 

(Wiench et al., 2007, 

OECD 202 (2004, 667207

090635) 

20-30 nm, 80% anatase, 20% rutile, no surface 

coating, BET surface area 48.6 m2

/g 

), part 1 (48-hr 

immobility), tested concentrations: 0 

(untreated control), 0.01, 0.1, 1.0, 10.0 and 

100.0 mg/L 

EC50 >100 mg/L 

50 nm × 10 nm, rutile, surface coating aluminum 

hydroxide, dimethicone/methicone copolymer, 

BET 100 m2

/g (T-LiteTM SF) (photostable UV 

filter) 

EC50 >100 mg/L 

50 nm × 10 nm, rutile, surface coating aluminum 

hydroxide, hydrated silica, 

dimethicone/methicone copolymer, BET 100 

m2

/g (T-LiteTM SF-S) (photostable UV filter) 

EC50 >100 mg/L 

50 nm × 10 nm, rutile, surface coating aluminum 

hydroxide, hydrated silica, 

dimethoxydiphenylsilane/ triethoxycaprylsilane 

crosspolymer, BET 100 m2

/g (T-LiteTM MAX) 

(photostable UV filter) 

EC50 >100 mg/L 

~300 nm, BET surface area 6 m2/g (pigment 

grade) 

EC50 >100 mg/L 

Invertebrate (water flea, 

Daphnia magna) 

(Lovern et al., 2007, 

30 nm, anatase 1-hr exposure to 2.0 mg/L No changes in heart rate or 

behaviors 

091069) 

 5-13 
Protocol 

(No UV illumination, unless 

specified) 

Test Species 

(Reference) Material Study Outcome 

Invertebrate (water flea, EC50 >100 mg/L 

Daphnia magna) 

(Warheit et al., 2007, 

667207

091075) 

140 nm in water, 79% rutile:21% anatase, 

coated (90-wt % TiO2, 7% alumina, and 1% 

amorphous silica) (DuPont uf-C TiO2) (photopassivative/ photo-stable) (Warheit, 2008, 

OECD 202 (2004, ) (48-hr 

immobility) (10% immobility at 100 mg/L) 

0.1, 1.0, 10, and 100 mg/L (uf-C and fine 

TiO2) 157470) 

Fine TiO2: ~380-nm in water (buffered), rutile, 

BET surface area 5.8 m2

/g, coated with alumina 

(~99% TiO2 and ~1% alumina) 

EC50 >100 mg/L 

(10% immobility at 10 mg/L, 0% 

immobility at 100 mg/L) 

LC50 >10 mg/L for both D. pulex

and C. dubia

Invertebrates (water 

flea, Daphnia pulex and 

Ceriodaphnia dubia) 

(Griffitt et al., 2008, 

48-hr mortality, 14:10 hr light:dark cycle, for 

D. pulex adults and C. dubia neonates 

(<24-hr old) 

20.5-nm primary particle, mainly 220.8 or 

687.5 nm in moderately hard water, 20% 

rutile:80% anatase, BET surface area 45 m2

/g; 

sonicated (Degussa P25) (photocatalytic) Gradient of concentrations up to 10 mg/L 

(The estimated median lethal concentration 

(LC50) from range-finder tests, and 0.6-, 

0.36-, 1.67-, and 2.78-fold the estimated 

LC50. However, the estimated LC50 was not 

specified.) 

157565) 

EC50 >100 mg/L b Invertebrate (water flea, 

Chydorus sphaericus) 

(Velzeboer et al., 2008, 

157476

<75 nm (primary particle) nano-TiO2 in water 

suspension (Sigma product 643017, the 

Netherlands), mixture of rutile and anatase, ratio 

not reported (Velzeboer et al., 2008, 

48-hr mortality, 17:7 hr light:dark cycle 

(Chydotox test) b

) 157476) 

NOEC >20,000 mg/L for T. 

platyurus; not tested in D. magna

Invertebrates (water 

flea, Daphnia magna; 

fairy shrimp, 

Thamnocephalus 

platyurus) 

(Heinlaan et al., 2008, 

25- to 70-nm powder mixture of anatase and 48-hr mortality for D. magna 

rutile, ratio not disclosed (Sigma product 13463-

67-7, Estonia) (Heinlaan, personal 

communication, 2008, 

24-hr immobilization for T. platyurus 

157558) 

Conventional TiO2: size and crystal form not 

disclosed (Sigma product 14027, Estonia; a 

former Riedel-de Haën product) (Heinlaan, 

personal communication, 2008, 

Up to 20,000 mg/L for both nano- and 

conventional TiO2

NOEC >20,000 mg/L for T. 

platyurus; 60% mortality at 

20,000 mg/L for D. magna

193414) 

157558) 

Invertebrate (fairy 

shrimp, 

Thamnocephalus 

platyurus) 

(Blaise et al., 2008, 

<100-nm powder (Sigma product 634662, LC50 >100 mg/L 

France), characteristics in water not reported 

24-hr lethality (ThamnoToxkit assay), tested 

concentrations not specified 

157592) 

Invertebrate (freshwater 

hydra, Hydra attenuata) 

(Blaise et al., 2008, 

<100-nm powder (Sigma product 634662, EC50 in 10 to 100 mg/L range 

France), characteristics in water not reported 

96-hr morphological changes, tested 

concentrations not specified 

157592) 

Fish cell (trout primary 

hepatocytes) 

(Blaise et al., 2008, 

<100-nm powder (Sigma product 634662, 

France), characteristics in water not reported 

48-hr cytotoxicity, tested concentrations not 

specified 

TEC in 1 to 10 mg/L range 

157592) 

Neither nano-TiO2 nor 

conventional TiO2 at the tested 

condition caused changes in any 

of the three endpoints measured. 

Fish (zebrafish, Danio 

rerio), embryo and 

larvae 

(Zhu et al., 2008, 

Nano-TiO2: uncoated anatase, purity >99.5%, 

primary particle in spindle shape, published size 

≤ 20 nm, surface area not reported (Nanjing 

High Technology NANO CO., LTD, Nanjing, 

Jiangshu province, China); in suspension (in Milli 

Q water): mean measured size 230 nm, 

measured size range 100 to 550 nm, secondary 

particles formed by primary particles have 

irregular shapes 

96-hr exposure to 0, 1, 10, 50, 100, or 

500 mg/L nano-TiO2 or conventional TiO2 to 

fish eggs (started within 1.5 hr postfertilization); light cycle 14 hr light/10 hr 

dark; following endpoints were measured: 

(1) survival of embryo and larvae; (2) 

hatching rate at 84 hr post-fertilization; and 

(3) malformation (e.g., pericardial edema 

and tissue ulceration, body arcuation, etc.) 

in embryo and larvae 

193742) 

Conventional TiO2: anatase, purity >99.0%, 

published size: 10,000 nm (Third Chemical 

Regent Factory of Tianjin, Tianjin, China); in 

suspension (in Milli Q water): mean measured 

size 1,100 nm, measured size range 330 to 

2,250 nm, neither primary nor secondary 

particles have a uniform shape 

Fish (zebrafish, Danio 

rerio) 

(Griffitt et al., 2008, 

LC50 >10 mg/L for both adults 

and fry 

48-hr mortality on adult zebra fish and 

zebra fish fry (<24 hr post-hatch) at a 

gradient of concentrations up to 10 mg/L 

20.5-nm primary particle, mainly 220.8 or 

687.5 nm in moderately hard water, 20% 

rutile:80% anatase, BET surface area 45 m2

/g, 

157565) sonicated (Degussa P25) (photocatalytic) 

 5-14 
Protocol 

(No UV illumination, unless 

specified) 

Test Species 

(Reference) Material Study Outcome 

Fish (rainbow trout, LC50 >100 mg/L 

Oncorhynchus mykiss) 

(Warheit et al., 2007, 

667208

091075) 

140 nm in water, 79% rutile:21% anatase, 

coated (90-wt % TiO2, 7% alumina, and 1% 

amorphous silica) (DuPont uf-C TiO2) (photopassivative or photo-stable) (Warheit, 2008, 

OECD 203 (1992, ) (96 hr acute 

toxicity) 

0.1, 1.0, 10, and 100 mg/L (uf-C and fine 

TiO2) 157470). 

Chronic Exposure to Aquatic Organisms 

Invertebrate (water flea, 

Daphnia magna) 

(Adams et al., 2006, 

66-nm powder, ~35% rutile:65% anatase, 

average 330 nm in water, (Sigma product 

634662) (photocatalytic) (Lyon, personal 

communication, 2008, 

8-day exposure to suspension at 1, 10 or 

20 ppm (concentration over time was not 

reported) 

40% mortality at 20 mg/L 

157782) 157524) 

Invertebrate (water flea, 

Daphnia magna) 

(Wiench et al., 2007, 

667210

090635) 

50 nm × 10 nm, rutile, surface coating aluminum 

hydroxide, hydrated silica, dimethicone/ 

methicone copolymer, BET surface area 100 

m2

/g (T-LiteTM SF-S) (photostable UV filter) 

OECD 211 (2008, ) (21-day 

reproduction), test concentrations: 0.01, 

0.03, 0.1, 0.3, 1, 3, 10, 30, 100 mg/L 

NOEC 3 mg/L 

LOEC 10 mg/L 

OECD 202 (2004, 667207) (Chronic 

toxicity, 21-day immobility) 

LC0 = 30 mg/L 

Fish (rainbow trout, 

Oncorhynchus mykiss) 

(Federici et al., 2007, 

Respiratory distress, organ 

pathologies, and oxidative stress 

at as low as 0.1 mg/L; nano-TiO2

could be a surface acting toxicant

21 nm, 75% rutile:25% anatase, sonicated 

(Degussa P25) (photocatalytic) 

0-, 7-, or 14-day exposure to 0, 0.1, 0.5 or 

1.0 mg/L (mean measured TiO2

concentrations were 0.089, 0.431, and 

0.853 mg/L over the 12-hr period, equating 

to 89, 85, and 86% of the expected 

concentrations, respectively) 

091222) 

Acute Exposure to Terrestrial Organisms 

Photosynthetic enzyme 

complexes isolated from 

spinach leaves 

(Blaise et al., 2008, 

<100-nm powder (Sigma product 634662, IC20 >100 mg/L 

Canada or France), characteristics in water not 

reported 

15 min, tested concentrations not specified, 

measure the decrease in chlorophyll 

fluorescence emitted from the enzyme 

complexes as an indicator of inhibition of 

photosynthetic efficiency (Luminotox assay) 

(Bellemare et al., 2006, 

157592) 

157779) 

Mix in a 1:1 ratio with certified reference 

material sediment, 15 min, tested 

concentrations not specified, measure light 

output (Luminotox assay) (indirect 

toxicity/interaction) 

Nano-TiO2 did not affect the 

toxicity of certified reference 

material sediment 

Plant (spinach, Spinacia Nano-TiO2: 

oleracea) 

(Linglan et al., 2008, 

Nano-TiO2: 5 nm, anatase, not coated Soak the seeds in 0.25% nano-TiO2 or 

conventional TiO2 for 48 hr, and spray 

0.25% nano-TiO2 or conventional TiO2 onto 

the leaves from 2-leaf stage to 8-leaf stage 

at 0.25% 

Enhanced growth (size, single 

plant fresh weight, single plant 

dry weight) 157534) 

Conventional TiO2 Increased chlorophyll content 

Increased net photosynthetic rate

Increased mRNA, protein 

concentration, and activity of 

Rubisco activase 

Conventional TiO2: 

No significant changes 

Plant (spinach, Spinacia 

oleracea) 

(Zheng et al., 2005, 

Size not specified, rutile (Shanghai Chemical 

Co. of China product) 

Soak aged seeds for 48 hr at 0, 0.25, 0.5, 

1.0, 1.5, 2.0, 2.5, 4.0, 6.0, or 8.0 mg/L 

Increased germination rate, 

intensity of photosynthesis, 

chlorophyll synthesis, and 

Rubisco activase activity in a 

dose response manner (at up to 

~4.0 mg/L; peak effect at 

~2 mg/L; higher concentrations 

have opposite effects) 

157784) 

No statistically significant 

reduction in growth, transpiration 

or water use efficiency rates, 

therefore no findings of toxicity. 

Adsorption of Hombikat UV100 

particles more apparent than 

Degussa particles. 

Plant (willow trees) 

(Seeger et al., 2009, 

Nano-TiO2: Degussa P25 (20/80% 

rutile/anatase, average diameter 25 nm); 

Hombikat UV100 (100% anatase, average 

diameter <10 nm) 

190-hr exposure to 0, 1, 10, and 100 mg/L 

(TiO2) and 0, 1, 20, and 50 mg/L (TiO2) for 

Degussa, and 100 mg/L (TiO2) for Hombikat 

UV100. 

644124) 

Solutions shaken. Adsorption measured by 

light microscopy; growth, transpiration, and 

water use efficiency measured using 

standard willow tree acute toxicity test 

 5-15 
Protocol 

(No UV illumination, unless 

specified) 

Test Species 

(Reference) Material Study Outcome 

Plant (maize, Zea mays 

L.) Asli and Neumann 

(2009, 

Nano-TiO2: Degussa P25 

(mean diameter 30 nm) 

5-hr root exposure to 0.3 and 1.0 g/L of 

either nanomaterial in hydroponic solution 

over 5-day period; 6-wk root exposure to 

1.0g/L of either nanomaterial in clay soil 

Accumulation at the cell wall 

surfaces of primary roots and 

subsequent inhibition of cell wall 

pore size, water transport 

capacity, leaf growth and 

transpiration. No statistically 

significant effect observed on 

shoot growth rate when exposed 

to nanomaterials in soil. 

193771) Bentonite clay (1-60nm) 

Invertebrate (isopod, 

[woodlouse] Porcellio 

scaber) (Jemec et al., 

2008, 

3-day dietary exposure to nonsonicated 

nano-TiO2 at 0.1, 0.5, 1, 10, 100, 1,000, 

2,000, or 3,000 μg/g food or to sonicated 

nano-TiO2 at 1,000, 2,000, or 3,000 μg/g 

food (leaves soaked in nonsonicated or 

sonicated nano-TiO2 dispersion and then 

dried) 

15 nm in diameter, 15-75 nm in length, 

elongated spheroid shape, anatase, surface 

area 190-290 m2

/g, 99.7% pure (Sigma product). 

780- to 970-nm aggregates in nonsonicated 

dispersion, 350- to 500-nm aggregates in 

sonicated dispersion, sizes on dry leaves not 

reported 

Decreased activities of catalase 

and glutathione-S-transferase 

(GST) in digestive glands at 0.5, 

2,000, and 3,000 μg/g 

nonsonicated nano-TiO2, but not 

in middle doses of nonsonicated 

nano-TiO2 or any doses of 

sonicated nano-TiO2 

157552) 

No changes in feeding rate, 

defecation rate, food assimilation 

efficiency, weight, or mortality 

were noted up to 3,000 μg/g 

Expose synchronized worms in the L1 

stage to nano-TiO2 or conventional TiO2 in 

ultrapure water with pH adjusted to 7.0 with 

HNO3 and NaOH 

Invertebrate (nematode, 

Caenorhabditis elegans) 

(Wang et al., 2009, 

Nano-TiO2, anatase, primary particle diameter 

50 nm, measured BET surface area 325 m2

/g for 

primary particle, purity >99%, hydrodynamic 

diameter (of aggregates in pure water) range 

338 to 917 nm (medium 550 nm), zeta potential 

at pH 7.0 = -18.9 mV (Hongchen Material Sci & 

Tech, Co., China) 

Lethality to the vermiform 

nematode: 24-hr LC50 was 

significantly lower for nano-TiO2

(79.9 mg/L) than for conventional 

TiO2 (135.8 mg/L) 

193696) 

Exposure for 24 hr (for lethality to the 

vermiform nematode) or 5 days (for growth 

– length of the worm, and reproduction 

tests – number of eggs inside the worm 

body, and number of offspring per worm) at 

24.0, 47.9, 95.9, 167.8, and 239.6 mg/L 

Length of the worm, number of 

eggs inside the worm body, and 

number of offspring per worm 

were all significantly decreased 

at 47.9 mg/L or higher 

concentrations of nano-TiO2 and 

at 95.9 mg/L or higher 

concentrations of conventional 

TiO2

Conventional TiO2, anatase, measured primary 

particle diameter 285 nm (by TEM), measured 

BET surface area 7.3 m2/g, purity >99%, 

hydrodynamic diameter range 158 to 687 nm 

(medium 494 nm), zeta potential at pH 7.0 = - 

33.8 mV (ACROS, Acros Organics) 

N/A – Not applicable 

a

Authors reported cloudy appearance or difficulty to dissolve nano-TiO2 in preparing stock suspension. The testing concentrations (final concentrations in 

medium) were calculated by the volume of 10 mg/L stock suspension added into the medium. The actual concentrations of nano-TiO2 in medium were not 

reported. 

b

Authors reported cloudy appearance in 100 mg/L TiO2 suspension. After centrifugation, nano-TiO2 concentrations were no more than 10% of initial 

concentrations. For example, 200 μg/L nano-TiO2 was added into pond water, and nano-TiO2 was only 1 μg/L after centrifugation.

MTC – Microbial Toxic Concentration, calculated by 

comparing the area under and above the growth curve 

(Gabrielson et al., 2003, 

BET – Surface area measured by Brunauer, Emmett, and Teller analysis 

DIN – Deutsches Institut für Normung (German Institute for Standardization) 157862

EC50 – Effective concentration 50; the concentration at which 50% of subjects showed response 

EU – European Union 

IC20, IC25 – inhibitory concentration at which organisms showed 20%, 25% inhibition in measured 

endpoints 

ISO – International Organization for Standardization 

GST – Glutathione-S-transferase 

LC50 – Lethal concentration 50; the concentration at which 50% of subjects died 

LOEC – Lowest observed effect concentration 

MARA – Microbial array for risk assessment (assay) 

; Gabrielson et al., 

2003, 157863) 

NOEC – No observed effect concentration 

OECD – Organization for Economic Co-operation and 

Development 

P25 – Aeroxide® P25 

PAM – Pulse amplitude modulation 

TEC – Threshold effect concentration. The TEC for 

cytotoxicity is calculated using the NOEC and LOEC of 

cell viability reduction. TEC = (NOEC × LOEC)1/2 

TEM – Transmission electron microscopy 

UV – Ultraviolet (light/radiation), wavelengths in the 

range of 10 to 400 nm 

5.2.1.1. Effects on Bacteria and Fungi (Terrestrial and Aquatic) 

Data for the effects of photostable nano-TiO2 on bacteria and fungi are lacking. On the other 

hand, photocatalytic nano-TiO2 is known for its antibacterial and antifungal properties and has been 

tested for various applications, including drinking water treatment (Coleman et al., 2005, 089849); 

surface coatings and paints (Kuhn et al., 2003, 090597; Tsuang et al., 2008, 157483); and food 

 5-16 
packaging (Chawengkijwanich and Hayata, 2007, 157708). Examples of recent studies of 

photocatalytic nano-TiO2 in bacteria and fungi are provided in Table 5-3. 

Because most bacteria and fungi are nonpathogenic and are major decomposers in most 

terrestrial and some aquatic ecosystems, chemicals with antibacterial and antifungal properties are 

not necessarily beneficial when released into the environment. The health of decomposers is 

important for nutrient cycling in the environment, such as carbon and nitrogen cycling in soil (Neal, 

2008, 196069). Additionally, some bacteria and fungi form a symbiotic relationship with plants. A 

well-known example is the nitrogen-fixing bacteria (genus Rhizobium) that live on the roots of 

legumes. Legumes provide nutrients and a relatively anaerobic environment for the rhizobia, and 

obtain ammonia formed from atmospheric nitrogen by the rhizobia (Long, 1989, 644893). Thus, 

indiscriminant exposure to chemicals with antibacterial properties could harm plants by interfering 

with symbiotic bacteria. 

Sensitivity to photocatalytic nano-TiO2 toxicity varies among species of bacteria. Adams et al. 

(2006, 157782) reported that in the presence of sunlight, gram-negative Escherichia coli were more 

sensitive to nano-TiO2-induced growth inhibition than gram-positive Bacillus subtilis. With 

2,000 ppm of nano-TiO2 in the growth medium, E. coli growth was decreased by 46% while B. 

subtilis growth was inhibited by 99%. At 500 ppm, E. coli growth was decreased by only 15% and B. 

subtilis growth was not inhibited (Adams et al., 2006, 157782). The different dose-response 

relationships of gram-positive and gram-negative bacteria to nano-TiO2 suggest the potential for 

nano-TiO2 to alter microbial population balance (diversity), both in wastewater treatment plants and 

during various phases of use and disposal of nano-TiO2. One generally accepted explanation for 

nano-TiO2-induced toxicity in bacteria and fungi is the generation of ROS, which can cause cell wall 

or cell membrane damage (Kuhn et al., 2003, 090597; Neal, 2008, 196069), such as lipid 

peroxidation (Maness et al., 1999, 193538). Although, as discussed above, UV illumination increases 

photocatalytic nano-TiO2 toxicity, photocatalytic nano-TiO2 is also toxic in the dark (Adams et al., 

2006, 157782; Coleman et al., 2005, 089849). Because TiO2 generates ROS (mainly highly reactive 

hydroxyl radicals, ·OH) in the presence of UV light and oxygen (Reeves et al., 2008, 157506), 

mechanisms other than oxidative stress might also contribute to nano-TiO2 toxicity in the dark and 

possibly also under UV light. For example, several types of nano-TiO2 (anatase and a mixture of 

anatase/rutile) have been shown to adsorb protein and calcium (Ca2+) in the medium, and cause 

in vitro cytotoxicity in mammalian cell lines (Horie et al., 2009, 193433). 

5.2.1.2. Effects on Aquatic Organisms 

Data on the effects of nano-TiO2 in aquatic organisms are available for freshwater algae, 

freshwater invertebrates (water fleas and fairy shrimp), and freshwater fish (rainbow trout) 

(Table 5-3). Only two aquatic organism studies in the literature involve photostable nano-TiO2

(Warheit et al., 2007, 091075; Wiench et al., 2007, 090635). For other aspects of U.S. EPA Tier I 

aquatic toxicity testing (e.g., estuarine and marine organism acute toxicity, whole sediment acute 

toxicity, and bio-availability/bio-magnification toxicity) (U.S. EPA, 2008, 157481), studies have not 

yet been reported. 

Algae 

Algae are primary producers of chemical energy in ecosystems. In addition to being the food 

base in aquatic systems, algae provide much of the earth’s oxygen. Effects on algae are measured at 

the population level, for example, in terms of population growth. In algal tests, 72-hour exposures 

are considered acute exposure in European Union (EU) regulations, and 96-hour exposures are 

considered chronic by U.S. EPA (2008, 157481). A limited number of studies on the effects of either 

photocatalytic or photostable TiO2 in algae have been completed. 

 5-17 
For photostable nano-TiO2, EC50 values determined for 72-hour growth inhibition in green 

alga (Pseudokirchneriella subcapitata) were 21 mg/L (based on decreases in healthy cell numbers) 

and 87 mg/L (based on inhibition of growth rate) (Warheit et al., 2007, 091075). In contrast, 

exposure to concentrations of 0.01 to 1 mg/L of photostable nano-TiO2 increased growth rate by 

1-3% (green alga cell numbers increased 6-19%) (Warheit et al., 2007, 091075). U-shaped doseresponse relationships are not unique to nanomaterials, and it cannot be ruled out that increased 

growth at the low dose was a compensatory response to low levels of toxicity (Calabrese and 

Baldwin, 1998, 047938; Davis and Svendsgaard, 1990, 048278). Fine (approximately 380 nm) TiO2

showed almost no inhibition in growth rate (or cell number) at up to 1 mg/L, and EC50 values of 

16mg/L (based on decreases in healthy cell numbers) and 61 mg/L (based on inhibition of growth 

rate) (Warheit et al., 2007, 091075). Hartmann et al. (2010, 196322) also studied green algal growth 

inhibition, testing three different sizes of nano-TiO2 and observing toxicity in all three cases. The 

authors’ primary discussion, however, was on the difficulty of reproducing results due to the 

complex interactions of the systems; the determination of a dose-response relationship was 

complicated by the effects of concentration-dependent aggregation of the nanoparticles, subsequent 

sedimentation, and possible attachment to vessel surfaces. Hartmann et al (2010, 196322) concluded 

that their research underlines the potential for interactions with existing environmental constituents 

to affect the toxicity of nanoparticles. 

For photocatalytic nano-TiO2, the EC50 values determined for 72-hour growth inhibition in 

green algae ranged from approximately 30 mg/L to > 50 mg/L (Blaise et al., 2008, 157592; HundRinke and Simon, 2006, 090607). Hund-Rinke and Simon (2006, 090607) also tested the potential 

for TiO2 to reduce growth by physically shading algae, and reported that as much as 50 mg/L of 

photocatalytic nano-TiO2 physically above the algae did not decrease algal growth, that is, it did not 

cause a shading effect. When nano-TiO2 and algae are in the same liquid medium, photocatalytic P25 

nano-TiO2 was reported to adsorb onto the surfaces of green algae (Pseudokirchneriella 

subcapatitata) and to increase cellular weight by more than 130% (Huang et al., 2005, 157801). The 

concentration of P25 was not reported. If the attached nano-TiO2 directly blocks sunlight that 

otherwise could reach the algal cell surface or if this extra weight causes algae to stay in deeper 

water, the consequent reduction in sunlight could inhibit the algal growth. Because photostable 

nano-TiO2 would also block UV penetration, similar effects could occur with photostable nano-TiO2. 

Without experimental evidence, predicting the impact of nano-TiO2 on photosynthesis is difficult 

because nano-TiO2 exposure reportedly increases photosynthesis in terrestrial plants, namely 

spinach, as discussed later in this section. Nano-TiO2 could affect aquatic and terrestrial plants 

differently due to exposure routes, doses, and other factors. 

Although no marine organisms have been tested for nano-TiO2 toxicity, the physical 

attachment of nano-TiO2 particles on cells could pose a risk to aquatic organisms that reproduce by 

external fertilization. A wide variety of marine organisms fall into this category. Attached nano-TiO2

could decrease sperm cell mobility and consequently reproductive success. For comparison, carbon 

black nanoparticles have been reported to decrease sperm frequency of seaweed (marine 

macroalgae) and to affect seaweed embryo development (Nielsen et al., 2008, 644828). As discussed 

earlier (Section 5.1.1), the ionic strength due to salinity in seawater could influence the behavior and 

effects of nano-TiO2, such as more aggregation as compared to pure water. 

Nano-TiO2 was reported to increase algal cell weight 2.3-fold by adsorbing to the algal cell 

surface, but the tested nano-TiO2 concentrations in water were not reported (Huang et al., 2005, 

157801). If an increase in weight forces surface algae into deeper water, photosynthesis could be 

decreased1

 due to less sunlight available in deeper water than at the surface. The reduced light 

available to algae was also suggested to be the cause of more growth inhibition seen in algal cells 

treated with nano-TiO2, compared to conventional TiO2 (Aruoja et al., 2009, 193254). In Aruoja’s 

 

1

 On the other hand, nano-TiO2 taken up by spinach increased growth and photosynthesis by increasing the activities of enzymes important 

for photosynthesis (Linglan et al., 2008, 157534; Zheng et al., 2005, 157784). 

 5-18 
study (2009, 193254), nano-TiO2 formed large agglomerates with almost all algal cells entrapped. 

Conventional TiO2, on the other hand, formed small agglomerates with some algal cells entrapped 

and some algal cells free (Aruoja et al., 2009, 193254). Since these studies were both conducted in 

algal culture medium, it cannot be ruled out that some of the observed growth inhibition may be due 

to absorption of zinc and phosphate by nano-TiO2, decreasing the availability of these nutrients to 

the algae (Kuwabara et al., 1986, 625577). 

Aquatic Invertebrates 

The endpoints used most often in ecological studies with invertebrates are mortality and 

immobility; other endpoints include morphological changes, heart rate changes, and reproductive 

effects. Fairy shrimp, Thamnocephalus platyurus, are small freshwater crustaceans and filter feeders 

that live in temporary water bodies that dry out or periodically experience decreased water levels 

(Brausch et al., 2006, 193296; Löhr et al., 2007, 193518). In the dry season, T. platyurus survives by 

laying resting-stage eggs (known as cysts), which hatch into nauplii (first stage of crustacean larvae) 

within hours after being hydrated (Brausch and Smith, 2009, 193297). The lethality and 

immobilization in T. platyurus larvae and adults as well as the hatch rate of T. platyurus cysts are 

often used as endpoints for freshwater contaminant tests. Hydras (Hydra attenuata) are small simple 

animals with a tube-shape body (usually 1-20 mm long) and tentacles on one end of the body. 

Intoxication of hydras can be seen in tentacle morphology, which can be normal, clubbed (a sign of 

minor intoxication), shortened (severe intoxication), or completely retracted (lethal intoxication, 

because this inevitably leads to death) (Environment Canada, 2007, 157697). 

Acute and chronic toxicity of nano-TiO2 intended for sunscreen use was studied in Daphnia 

magna and reported in a poster at a scientific meeting by Wiench et al. (2007, 090635). In the acute 

exposure study, EC50 values (from 48-hour mortality tests) were above 100 mg/L for all tested forms 

of TiO2, which consisted of three photostable forms (uncoated T-LiteTM SF, coated T-LiteTM SF-S, 

and coated T-LiteTM MAX), a photocatalytic nano-TiO2, and a pigment-grade TiO2 (Wiench et al., 

2007, 090635). In the chronic exposure study, photostable coated T-Lite SF-S was given to D. magna 

at up to 100 mg/L for 21 days, and the authors reported that the LC50 was 30 mg/L. In this study, 

death was determined by the lack of swimming ability. 

For reproductive effects after 21 days, the no observed effect concentration (NOEC) value for 

T-Lite SF-S was 3 mg/L, and the lowest observed effect concentration (LOEC) value was 10 mg/L 

(Wiench et al., 2007, 090635). In a different study that used photostable nano-TiO2 intended to 

protect plastics against UV-induced degradation, 48-hour exposure to 100 mg/L of the nano-TiO2

induced 10% immobility in D. magna (Warheit et al., 2007, 091075). 

The effects of photocatalytic nano-TiO2 toxicity have been studied by several research teams 

in four types of water fleas (D. magna, D. pulex, Ceriodaphnia dubia, and Chydorus sphaericus), 

one type of fairy shrimp (T. platyurus), and one type of freshwater hydra (H. attenuata). For water 

fleas, the 48-hour mortality or immobility EC50 was generally greater than 100 mg/L (Lovern and 

Klaper, 2006, 088040 [unfiltered]) (Velzeboer et al., 2008, 157476; Wiench et al., 2007, 090635), 

with two exceptions. One study reported an LC50 greater than 10 mg/L, which in this case was the 

highest concentration tested (Griffitt et al., 2008, 157565). Another study reported a 48-hour LC50 of 

5.5 mg/L, using filtered nano-TiO2 samples, which have an average particle size of 30 nm after going 

through a 0.22-mm Nylaflo filter (Lovern and Klaper, 2006, 088040). In contrast, unfiltered 

nano-TiO2 samples had all sizes of nano-TiO2 clumps, ranging from 100 to 500 nm in diameter, and 

the mortalities never exceeded 11% at up to 500 mg/L (Lovern and Klaper, 2006, 088040). Chronic 

exposure for 8 days caused 40% mortality at 20 mg/L in daphnids (Adams et al., 2006, 157782). For 

fairy shrimp, the 24-hour mortality or immobility LC50 was higher than 100 mg/L (Blaise et al., 

2008, 157592; Heinlaan et al., 2008, 193414). In the only study of hydra, the EC50 of 96-hour 

morphological changes was <100 mg/L (Blaise et al., 2008, 157592). The relative sensitivity among 

 5-19 
these aquatic invertebrates to nano-TiO2 cannot be determined, due to the variability of tested 

nano-TiO2 formulations and experimental designs. 

When D. magna were exposed to photocatalytic P25 nano-TiO2 in water, nano-TiO2 was 

observed on the exoskeleton and antennae and in the digestive tract (Baun et al., 2008, 157598). 

Baun et al. (2008, 157598) noted that the aggregation of nanoparticles on the exoskeleton, at 

sufficient dose, might impede a daphnid’s mobility. Although not investigated in this study, the 

aggregation of nanoparticles on the antennae, a chemosensory organ important for feeding and 

reproductive behaviors, could adversely affect a daphnid’s growth and reproduction (Oberdörster et 

al., 2006, 088054). Because nano-TiO2 primary particles are smaller than the size range of particles 

daphnids feed on (400 to 40,000 nm), the presence of nano-TiO2 in the digestive tract suggests that 

daphnids feed on nano-TiO2 aggregates (Baun et al., 2008, 157598). Whether nano-TiO2 is taken up 

by other tissues, excreted, or transformed in daphnids is unclear (Baun et al., 2008, 157598). Even if 

nano-TiO2 is not absorbed into tissues, the presence of nano-TiO2 in the digestive tract of daphnids 

could still contribute to bioaccumulation in the food web (Section 4.6.1.2.). 

The behavior and heart rate of D. magna were evaluated in daphnids exposed to photocatalytic 

nano-TiO2 at 2.0 mg/L for 1 hour (Lovern et al., 2007, 091069). In this study, nano-TiO2 had an 

average particle diameter of 30 nm, and tetrahydrofuran, the organic solvent used to prevent 

aggregation, was not detected in the final nano-TiO2 suspension. The concentration of 2.0 mg/L was 

selected because it was the lowest observed effect level (LOEL) of D. magna mortality after 48-hour 

exposure (Lovern and Klaper, 2006, 088040). Behavior (e.g., hopping frequency, appendage 

movement as an indicator of feeding frequency, and postabdominal claw curling) and heart rates 

were not affected by the 1-hour nano-TiO2 exposure (Lovern et al., 2007, 091069). 

Fish 

Fish are used in toxicity tests to represent secondary energy consumers in aquatic systems. 

Commonly used fish species in ecotoxicity tests include freshwater rainbow trout (Oncorhynchus 

mykiss), bluegill sunfish (Lepomis macrochirus), fathead minnows (Pimephales promelas), and 

estuarine species sheepshead minnows (Cyprinodon variegatus). Data from zebra fish (Danio rerio), 

a model organism widely used in biological and toxicological studies, can also be useful. Fish study 

endpoints can include concentrations of chemicals, such as in fish bioaccumulation tests 

(Section 4.6.1.1, “Bioaccumulation”); mortality; behavioral markers (e.g., fatigue, abnormal 

buoyancy control, and swimming); and pathology. 

The toxicological studies of photostable nano-TiO2 in fish are very limited. The 96-hour acute 

toxicity of photostable nano-TiO2 (DuPont uf-C) in rainbow trout produced an LC50 value of greater 

than 100 mg/L (Warheit et al., 2007, 091075). However, DuPont uf-C is designed to protect plastics 

from UV-induced degradation, and is not known to be used in sunscreen; no fish studies of 

nano-TiO2 intended for sunscreen use were found. 

In contrast, photocatalytic nano-TiO2, which may be used in drinking water treatment, has 

been tested in fish for acute effects (Griffitt et al., 2008, 157565; Zhu et al., 2006, 157721) and 

chronic effects (Federici et al., 2007, 091222). Bioaccumulation (Zhang et al., 2006, 157722) and 

interaction with other heavy metals were discussed previously (Table 4.2). In the acute exposure 

study, the LC50 for a 48-hour exposure to an anatase/rutile mixture of uncoated nano-TiO2 was 

>10 mg/L for zebrafish (in both female adults and <24-hour post-hatch fry) (Griffitt et al., 2008, 

157565). For zebrafish eggs (blastula stage), acute exposures for 96 hours at up to 500 mg/L of 

either nano-TiO2 or conventional TiO2 (both uncoated anatase) did not cause developmental toxicity, 

as measured by survival rate of the zebrafish embryos and larvae, hatching rate of embryos, and 

malformation in embryos and larvae (Zhu et al., 2008, 193742). In the Zhu et al. (2008, 193742) 

study, nano-Al2O3 and conventional Al2O3 at up to 1,000 mg/L also did not cause developmental 

toxicity to zebrafish eggs, but both nano-ZnO and conventional ZnO caused decreases in survival 

rates and hatching rate as well as increases in tissue ulceration at 1 mg/L or higher concentrations. 

 5-20 
Sub-lethal toxicity was observed in juvenile rainbow trout after 14 days of exposure to 

photocatalytic P25 nano-TiO2 (Federici et al., 2007, 091222). Respiratory toxicity and pathological 

changes in the gill and intestine were seen after a 14-day exposure at concentrations as low as 

0.1 mg/L. Furthermore, there were signs of oxidative stress (increased concentrations of 

thiobarbituric acid substances, an indicator of lipid peroxidation and oxidative stress, in multiple 

tissues), and activation of anti-oxidant defenses (increased total glutathione levels in the gill). 

Na+

K+-ATPase activity was also increased in the gill and intestine. Disturbances were observed in 

the metabolism of copper and zinc, but not of Na+

, K+, Ca2+ or Mn. No major hematological 

disturbances were observed. Worth noting is that these effects occurred without appreciable Ti 

accumulation in the internal organs, suggesting no nano-TiO2 accumulation, as discussed earlier in 

Section 4.6.1.1. The authors suggested that surface-bound TiO2 (through surface adsorption) might 

play a role in toxicity, similar to the case of aluminum, a surface-acting toxicant that can cause 

systemic toxicity without significant internal accumulation. Federici et al. (2007, 091222) concluded 

that although nano-TiO2 was not a major hemolytic toxicant or disruptor of ion regulation in this 

study, respiratory distress, organ pathologies, and oxidative stress were adverse effects. 

Summary of Effects on Aquatic Organisms 

Sub-lethal effects of nano-TiO2 include decreases in daphnid reproduction by photostable 

nano-TiO2 (Wiench et al., 2007, 090635), as well as respiratory distress, pathological changes in gills 

and intestine, and behavioral changes in fish (rainbow trout) by photocatalytic nano-TiO2 (Federici et 

al., 2007, 091222). Several studies reported visible turbidity in nano-TiO2 stock suspensions, and the 

actual nano-TiO2 concentration in the liquid phase might be different from the concentration 

calculated from added nano-TiO2 (Velzeboer et al., 2008, 157476; Zhang et al., 2006, 157722; Zhang 

et al., 2008, 157462). Given that natural organic matter in the environment can affect the extent of 

aggregation and deposition of nanoparticles or modify nanoparticle surface charges (Navarro et al., 

2008, 157517)(Kim et al., 2009, 635778), the bioavailability and behavior of nano-TiO2 in the 

environment are likely to be different from bioavailability and behavior in pure water or simple 

media, although the direction of the difference is difficult to predict. 

5.2.1.3. Effects on Terrestrial Organisms 

Plants 

Information on nano-TiO2 interactions with plants is currently available for photocatalytic 

uncoated nano-TiO2 in spinach and willow trees (Table 5-3). Photocatalytic uncoated nano-TiO2 has 

been shown to enhance the growth of spinach in several studies (Lei et al., 2008, 157540; Linglan et 

al., 2008, 157534; Mingyu et al., 2007, 157667; Mingyu et al., 2007, 157666; Yang et al., 2006, 

157723; Zheng et al., 2005, 157784). When a nano-TiO2 suspension was used to soak the seeds and 

was sprayed on the leaves, the germination rate and growth of the plant were enhanced (Zheng et al., 

2005, 157784). These effects were at least partially due to nano-TiO2-induced increases in the 

activity of several enzymes important for photosynthesis (Linglan et al., 2008), adsorption of nitrate, 

transformation of inorganic into organic nitrogen (Yang et al., 2006, 157723), and anti-oxidative 

stress response (Lei et al., 2008, 157540). Conventional TiO2 suspensions showed either 

insignificant effects (in comparison with untreated controls) or much smaller effects than nano-TiO2

did (Linglan et al., 2008, 157534; Zheng et al., 2005, 157784). 

Seeger et al. (2009, 644124) exposed willow tree roots to two types of TiO2 nanoparticles 

(Degussa P25 and Hombikat UV100) suspended in deionized water at various concentrations. There 

were no statistically significant changes in transpiration rates, growth, or water use efficiency after 

190 hours of exposure to nano-TiO2 in solution. Investigators found that roots exposed to the 

solution with smaller nano-TiO2 particles (<10 nm average diameter) had nanoparticles compactly 

 5-21 
attached all over the roots’ surface, while roots in the solution with larger particles (average 25 nm 

diameter) showed minimally attached particles. However, the researchers did not determine whether 

the nanoparticles entered the trees through the xylem. The investigators concluded that these two 

types of nano-TiO2 are not toxic to willow trees, under the experimental conditions used (Seeger et 

al., 2009, 644124). 

In contrast, Asli and Neumann (2009, 193771) found that colloidal suspensions of TiO2

nanoparticles interfered with water transport capacity, leaf growth, and transpiration in maize (Zea 

mays L.) seedlings. The authors exposed maize roots to colloidal suspensions of inorganic bentonite 

clay (particle size 1-60 nm) and Degussa P-25 TiO2 nanoparticles (mean diameter of 30 nm) in 

hydroponic solutions and in soil. The authors found statistically significant reductions in hydraulic 

conductivity (i.e. water flow through roots) when adding either material at low concentrations 

(1 g/L) to hydroponic solutions surrounding maize roots over a 5-hour period. Also, transpiration 

was rapidly inhibited (over a 3-hour period), when both materials were added to the solution at the 

same concentration. Colloidal nanoparticles of both materials suspended in water flowing to roots 

appeared to attach to root cell walls, thereby reducing cell wall pore diameters and root hydraulic 

conductivities. However, when the authors grew maize for 6 weeks in clay soil irrigated with either 

bentonite or TiO2, they found no statistically significant effect on shoot growth; they hypothesized 

that this apparent lack of effect may be due to the fact that the total number of roots increased during 

this time, thereby increasing the plants' water supply capacity, which could counterbalance possible 

pore clogging actions by the nanoparticles. 

Terrestrial Invertebrates 

The only known studies on the effects of nano-TiO2 on terrestrial invertebrates include a study 

on an isopod, Porcellio scaber (Jemec et al., 2008, 157552), and a study on nematodes, 

Caenorhabditis elegans (Wang et al., 2009, 193696). Living in soil, isopods and nematodes 

contribute to nutrient cycling and decomposition, and have been used as indicators of soil pollutants. 

Jemec et al. (2008, 157552) investigated the effects of photocatalytic anatase nano-TiO2 on the 

terrestrial isopod Porcellio scaber, known as woodlouse. Woodlice, approximately 16 mm long, live 

in the upper layer of soil and surface leaf litter. They break down organic matter and contribute to 

soil health, and are commonly used in ecological studies. In the Jemec et al. (2008, 157552) study, 

woodlice ate dry leaves that had been soaked in nano-TiO2 dispersions (sonicated or nonsonicated). 

The sonication process decreased the mean agglomerate size from 780-970 nm in a nonsonicated 

dispersion to 350-500 nm. The activities of catalase and glutathione-S-transferase (GST), two antioxidative stress enzymes in the digestive gland (hepatopancreas) were measured. The activities of 

both enzymes were decreased at 0.5, 2,000, and 3,000 μg/g of nonsonicated nano-TiO2, but not at 

middle concentrations (1, 10, 100, and 1,000 μg/g) of nonsonicated nano-TiO2 or at any 

concentration (1,000, 2,000, and 3,000 μg/g) of sonicated nano-TiO2 (Jemec et al., 2008, 157552). 

No changes in feeding rate, defecation rate, food assimilation efficiency, weight, or mortality were 

noted at concentrations up to 3,000 μg/g of either sonicated or nonsonicated nano-TiO2 in the food. 

This study illustrates the importance of nano-TiO2 dispersion preparation method on nano-TiO2

toxicity. 

Wang et al. (2009, 193696) investigated the lethality, growth inhibition, and effects on 

reproduction of nano-TiO2 and conventional TiO2 in the nematode, C. elegans, a small free-living 

(i.e., not parasitic) roundworm that inhabits soil in temperate climates around the world and feeds on 

bacteria and fungi. In the laboratory, C. elegans is often cultured on agar plates or in liquid medium 

in a Petri dish and is often fed E. coli. In the Wang et al. (2009, 193696) study, C. elegans strain 

Bristol N2 (wild-type) in L1 stage (larvae before the first molting) was exposed to anatase nano-TiO2

and anatase conventional TiO2 in water. In addition to lethality and growth inhibition, decreased 

reproduction was observed at lower mass concentrations of nano-TiO2 than conventional TiO2. The 

tested reproduction parameters were eggs inside body and the number of offspring per worm, which 

 5-22 
includes offspring at all stages beyond the egg over the entire brood period. The mechanism of 

reproductive effects was not investigated. Due to the lack of toxicity of supernatant of nano-TiO2

(obtained by centrifuging the nano-TiO2 suspension), dissolution of the particle does not contribute 

to observed nano-TiO2 effects on C. elegans (Wang et al., 2009, 193696). 

5.2.1.4. Indirect and Interactive Ecological Effects 

In addition to the direct toxicity of nano-TiO2, indirect and potentially synergistic effects of 

nano-TiO2 could also be important. Nano-TiO2 could adsorb pollutants (Nagaveni et al., 2004, 

090578; Pena et al., 2006, 090573), carry the pollutants into areas in an organism that the pollutants 

alone would not naturally appear (Moore, 2006, 089839), and increase the uptake of other pollutants 

(a “Trojan horse” effect). Consequently, nano-TiO2 could enhance pollutant toxicity, and even cause 

toxicities different from those caused by exposure to the pollutant alone due to differences in 

distribution. Also, as discussed in Section 4.6.1.3, co-exposure to nano-TiO2 in water increased the 

uptake of arsenic (Sun et al., 2007, 193662) and cadmium (Zhang et al., 2007, 090114) in carp, but 

toxicity was not measured in these two studies. 

Nano-TiO2 was found to have no effect on the toxicity of sediment and its elutriate in a study 

using certified reference material sediment (Blaise et al., 2008, 157592). The effects of 11 

nanomaterials on sediment toxicity (as measured in 2 direct contact assays, the Microtox solid phase 

assay1 2 and the Luminotox solid phase assay ) and sediment elutriate toxicity (as measured with the 

MARA assay3

) were studied using a mixture of each nanomaterial and the certified reference 

material sediment at a 1:1 ratio. Photocatalytic nano-TiO2 was one of only three tested nanomaterials 

that did not increase the sediment or elutriate toxicity in any of the three assays (Blaise et al., 2008, 

157592). 

5.2.1.5. Summary 

Limited ecological toxicity information on nano-TiO2 is currently available. Most 

ecotoxicological studies have tested photocatalytic nano-TiO2 that would be suitable for water 

treatment, but only a few studies have used photostable nano-TiO2 intended for sunscreen. Coated 

photostable nano-TiO2 in sunscreen could lose its coating through processes such as aging, 

weathering, chemical alterations (e.g., change in pH), and metabolism or biotransformation in living 

organisms (e.g., digestion by daphnids). If so, the photocatalytic nano-TiO2 core could be exposed 

and thus even photostable nano-TiO2 could have photocatalytic properties. 

Effects of chronic exposure to nano-TiO2 have been investigated only in water fleas and fish. 

Although acute exposure effects have been studied in microorganisms and various aquatic macroorganisms, these studies focused on lethality or immobility and provided little insight on modes of 

action. For terrestrial organisms, only acute exposure to anatase nano-TiO2 was investigated and only 

in invertebrates (P. scaber and C. elegans) and spinach. Photocatalytic nano-TiO2 decreased 

reproduction in C. elegans without affecting body length. Although increased growth in spinach 

following acute exposure to anatase nano-TiO2 could be useful for agricultural purposes, the effects 

of such growth promotion in an ecological system remain unclear. Photocatalytic nano-TiO2

enhanced the uptake of arsenic and cadmium in fish, indicating the possibility of interactive effects 

between nano-TiO2 and co-occurring toxic substances. 

 

1

 Microtox assay measures the reduction in light output from bioluminescent bacteria, Vibrio fischeri. For solid-phase assays, the 

concentration that causes 25% inhibition (IC25) is calculated after 20 minutes of exposure. 

2

 Luminotox assay measures the inhibition of photosynthetic efficiency of photosynthetic enzyme complexes isolated from spinach leaves. 

For the Luminotox solid-phase assay, IC20 is calculated after 15 minutes of exposure. 

3

 MARA assay (microbial array for risk assessment assay) measures growth inhibition in baking yeast and ten species of bacteria. A 

microbial toxic concentration is calculated after 18 hours of exposure. 

 5-23 
5.3. Health Effects 

This section summarizes and evaluates the evidence of nano-TiO2-induced health effects from 

epidemiological studies, laboratory animal studies, and a few selected ex vivo and in vitro studies. 

For a review of nano-TiO2 in vitro effects, see Fond and Meyer (2006, 196337). Most health effects 

studies used pure nano-TiO2, and therefore their characteristics and effects may differ from 

nano-TiO2 as used in commercial products or products containing nano-TiO2. For instance, 

nano-TiO2 in sunscreen may include mostly agglomerates, instead of perfectly dispersed primary 

particles. As discussed in Section 5.1, many other factors also influence the effects. When available, 

data on factors with potential influence on health effects are provided in Tables 5-4 through 5-10. 

The health effects evidence is organized by human and laboratory animal studies and route of 

exposure, with noncarcinogenic effects discussed in Section 5.3.1 and carcinogenic effects discussed 

in Section 5.3.2. 

5.3.1. Noncarcinogenic Effects 

This section summarizes in vivo studies of nano-TiO2 noncarcinogenic effects through dermal, 

oral, respiratory, and other routes of exposure. The presentation is organized by exposure routes, 

because exposure routes play a profound role in toxicokinetics, toxicodynamics, and health effects. 

More studies have been completed on respiratory exposure (inhalation and instillation) than on other 

exposure routes. Studies investigating solely skin penetration (not health effects) are discussed in 

Section 4.6.3. Commercial sunscreens were tested in dermal exposure studies only. Most studies 

tested photocatalytic nano-TiO2, which could be suitable as an agent in drinking water treatment. 

Commercial sunscreens were tested in dermal exposure studies only. Known photostable nano-TiO2

and rutile nano-TiO2, which is expected to be photostable, were used in some studies (Chen et al., 

2006, 090139; Mohr et al., 2006, 097493; Nemmar et al., 2008, 157514; Oberdörster et al., 1992, 

045110; Pott and Roller, 2005, 157790; Wang et al., 2007, 090290; Wang et al., 2007, 157616; 

Warheit et al., 2007, 091075; Warheit et al., 2007, 090594). 

5.3.1.1. Studies in Humans 

No epidemiological studies or case reports are available for nano-TiO2 noncarcinogenic 

effects. A few case reports described noncarcinogenic effects in the respiratory system of workers 

exposed to TiO2 particles of unspecified size. For example, exposure to conventional TiO2 has been 

associated with pneumoconiosis (Yamadori et al., 1986, 193728), pulmonary fibrosis and 

bronchopneumonia (Moran et al., 1991, 157956), and pulmonary alveolar proteinosis (Keller et al., 

1995, 157938). TiO2 or Ti accumulation in the lung, sometimes years after workplace exposures, and 

Ti-loaded macrophages have also been reported in workers (Keller et al., 1995, 157938; Määttä and 

Arstila, 1975, 157979; Yamadori et al., 1986, 193728), as have Ti particles in the lymph nodes 

(Määttä and Arstila, 1975, 157979; Moran et al., 1991, 157956) and in the liver and spleen (Moran et 

al., 1991, 157956). None of these case reports, however, provided quantitative TiO2 exposure data or 

measured potentially confounding variables such as exposures to crystalline silica and tobacco 

smoke. 

One epidemiological study (Chen and Fayerweather, 1988, 193312) found no consistent 

relationship between TiO2 (size not specified) exposure and chronic respiratory disease or fibrosis, 

but no conclusions can be drawn because of serious limitations, including restricting subjects to 

workers eligible for pensions; lack of information on the duration of TiO2 exposure, asbestos or other 

chemical exposures; and the lack of detailed information on sampling. 

 5-24 
5.3.1.2. Animal Studies 

For the most part (except as noted below), laboratory animal toxicity studies have investigated 

the effects of acute or subchronic exposure to nano-TiO2. This section presents in vivo studies of 

noncancer effects nano-TiO2 (Tables 5-4 to 5-7) by route of exposure: dermal, oral, respiratory, and 

others. Most animal studies of nano-TiO2 focus on photocatalytic nano-TiO2, including P25. 

Although sunscreen nano-TiO2 formulations are intended to be photostable, the coatings that impart 

photostability to anatase or part-anatase nano-TiO2 in some sunscreen formulations are known to 

degrade over time (Barker and Branch, 2008, 180141; Dunford et al., 1997, 157929). 

Toxicity from Dermal Exposure 

Toxicity findings from studies of dermal exposure to nano-TiO2 or sunscreen that contains 

TiO2 are presented in Table 5-4. For healthy unflexed skin, adverse health effects are not expected 

from dermal exposure to photostable nano-TiO2 in sunscreen (NanoDerm, 2007, 157660; Scientific, 

2007, 196826). Photocatalytic nano-TiO2, however, sometimes is used in sunscreens (Barker and 

Branch, 2008, 180141; Dunford et al., 1997, 157929). Photocatalytic nano-TiO2 can generate ROS 

when exposed to UV light and can cause oxidative stress and cytotoxicity in cells (cultured human 

fibroblasts) and in cell-free in vitro experiments (Dunford et al., 1997, 157929; Lu et al., 2008, 

157526). To date, the effects of long-term or repeated use of sunscreen containing nano-TiO2 have 

not been investigated in vivo, and no case reports of skin damage from such use are currently 

available. As discussed earlier (Section 4.6.3), most available studies indicate penetration of the 

outer skin layer and the stratum corneum, but not penetration of living skin cells. 

After a single topical application of photostable nano-TiO2, laboratory rabbits showed no skin 

irritation 4 hours after application or sensitization 3 days after application (Warheit et al., 2007, 

091075). Furthermore, although some sunscreens containing TiO2 (size not specified) increased 

mouse skin absorption of herbicides and pesticides (2,4-D, paraquat, parathion or malathion), TiO2

alone actually decreased the mouse skin absorption of the tested herbicide, 2,4-D (Brand et al., 2003, 

157866). The investigators reported that a solvent in the sunscreen caused increased skin absorption 

of herbicides, and this secondary effect can be avoided by substituting phenyl trimethicone as the 

solvent (Brand et al., 2003, 157866). 

Some researchers, such as Nohynek et al. (2007, 090619), have noted a discontinuity between 

in vitro and in vivo testing results, particularly for skin toxicity. Some in vitro cultures or 

preparations (other than those using intact skin samples) lack the stratum corneum layer, which 

according to currently available data can block penetration, such that in vitro tests might overstate 

toxicity of chemicals like TiO2. Of the four investigations reviewed, only three report in vivo studies 

of health effects after dermal exposure to TiO2 (pages 16, 17, and 41-43 of NanoDerm, 2007, 

157660; Warheit et al., 2007, 091075; Wu et al., 2009, 193721), and only one of those three used 

nano-TiO2 intended for sunscreen (pages 16, 17, 41, and 43 of NanoDerm, 2007, 157660). Warheit 

et al. (2007, 091075) used ultrafine particles, roughly 100 nm in size. Three studies used a single 

application, and the longest exposure was only 3 days. The NanoDerm report (2007, 157660) 

concluded that “TiO2 exposure did not modify the viability, proliferation, apoptosis, and 

differentiation [or] adhesive properties of skin cells.” As discussed previously (Section 4.6.3), skin 

penetration studies have shown that some nano-TiO2 can stay in hair follicles for up to 10 days. 

The only report with repeated dermal exposure included 30 days of daily dermal exposure on 

porcine skin and 60 days of daily dermal exposure on hairless mouse skin (Wu et al., 2009, 193721). 

As discussed in Section 4.6.3, the 30-day exposure of 4-nm nano-TiO2, but not larger nano-TiO2, 

resulted in nano-TiO2 particles in the basal cells of epidermis, but not in dermis, of pigs. In addition, 

morphological changes at the subcellular level were seen in the basal cells in the 4 nm TiO2 group. 

The authors also tested 60-day dermal exposures to 10-, 21-, 25-, 60-, and 90-nm nano-TiO2 with 

various anatase and rutile ratios in hairless mice for nano-TiO2 penetration in skin, as well as 

distribution, signs of oxidative stress, and pathological changes in various organs (Table 5-4). Most 

 5-25 
changes were seen in the 10-, 21-, and 25-nm nano-TiO2 groups, and none was seen in control or 

90-nm nano-TiO2 group. Increased Ti concentrations were seen in skin, subcutaneous muscle, heart, 

liver, and spleen. Signs of oxidative stress were seen in skin and liver. Pathological changes were 

seen in the skin, liver, heart (only 10-nm group), spleen, and lung. While hairless mice are 

commonly used as a model for skin studies, hairless mice study results should be evaluated with care 

for human relevance. For instance, the thicknesses of stratum corneum and epidermis of hairless 

mice are approximately half and two thirds of that in humans, respectively; skin permeability was 

higher in hairless mice than in humans (Haigh and Smith, 1994, 625322). Furthermore, hairless mice 

(BALB/c nu/nu) are deficient in T cells (Ku and Lee, 2006, 625354), and their immune function 

deficiency may render them more susceptible to nano-TiO2-induced changes than other animals or 

humans. 

With relatively few in vivo dermal exposure studies investigating nano-TiO2 skin absorption 

and penetration (Table 4-4) and health effects (Table 5-4), several data gaps on the health effects of 

dermal exposure to nano-TiO2 are evident. First, information on the dermal penetration and effects of 

nano-TiO2 in flexed skin and structurally compromised skin is lacking. Flexed healthy skin (Rouse et 

al., 2007, 157644; Zhang and Monteiro-Riviere, 2008, 193735) and compromised skin (Zhang and 

Monteiro-Riviere, 2008, 193735), including UV-exposed skin (Mortensen et al., 2008, 155612), have 

been shown to allow nanoparticles (other than nano-TiO2, which was not tested) to penetrate deeper 

than healthy nonflexed skin. Sunscreen containing nano-TiO2 is expected to be used on flexed 

healthy skin and misused on sunburned skin or skin with micro-lesions, such as microscopic cuts due 

to shaving. Cytotoxicity was seen in cultured skin cells treated with nano-TiO2 (Lee et al., 2009, 

157457), and the authors postulated that, in skin with compromised epidermis structure (e.g., 

sunburned skin or “soaked” skin), contact could occur between nano-TiO2 from sunscreen and living 

cells in the skin and lead to adverse effects. Second, effects from long-term, repeated dermal 

exposures to nano-TiO2 in sunscreen, similar to real-life exposure, have not been studied. Finally, the 

toxicity of the various intermediate forms of nano-TiO2 in the production process (possible sources 

of occupational exposure, by dermal and other routes) has not been studied. 

 5-26 
Table 5-4. Summary of health effects of nano-TiO2 particles in mammalian animal models: 

dermal route 

Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Brand et al. 

(2003, 

Some (not all) tested sunscreens increased 

transdermal penetration of herbicide/pesticide. 

Commercially available 

sunscreens, some of 

which contained TiO2

(size not specified) 

Mouse For testing indirect dermal effect 

157866 Female 

hairless 

CRL:SKH1 

In vitro 

exposure 

Single and 

repeated 

exposures 

a) Commercially available sunscreens, 

applied at 2 mg/cm2

 to skin excised from 

mice and placed in a diffusion chamber. 

30 min after the sunscreen application, 

herbicide 2,4-D was applied on skin. 

) 

Solvent, not TiO2 or ZnO, is responsible for 

sunscreen-increased skin absorption of 

herbicide/pesticide. 

a) Sunscreen effect on transdermal penetration of 

herbicide 2,4-D: 4 out of 7 tested sunscreens that 

contain TiO2 (and 1 out of 2 sunscreens that contain 

no TiO2) increased transdermal penetration of 

herbicide 2,4-D. 

b) Combination of TiO2 with phenyl 

trimethicone, ZnO, and octyl 

methoxycinnamate (OM) 

c) TiSilc untinted sunscreen, which 

contains TiO2 was applied. 2,4-D was 

also applied. Both were applied on skin, 

and then again 4.5 hr after the first 

application. 

b) Formulation effects:TiO2 alone, TiO2 plus ZnO, 

and TiO2 in trimethicone (simulation of commercial 

formula) decreased 2,4-D transdermal penetration. 

c) Repeated application of both sunscreen and 

herbicide: The peak penetration of 2,4-D herbicide 

was higher at the second application of TiSilc 

sunscreen and 2,4-D, compared to the first 

application of TiSilc and 2,4-D. However, the 2,4-D 

penetrations of first and second applications of 

TiSilc and 2,4-D were the same when skin was 

washed after both (but not just one) applications of 

TiSilc and 2,4-D. 

d) TiSilc untinted sunscreen and 

pesticides: Paraquat, Malathion, and 

Parathion 

d) Sunscreen effect on transdermal penetration of 

other pesticides: Absorption of pesticides 

(Paraquat, Malathion, and Parathion) was also 

increased in skin pretreated with sunscreen TiSilc. 

No effects on cell proliferation (as measured by 

bromo-deoxy-uridine, BrdU, labeling); apoptosis (as 

measured by a double-staining method of Ki67 and 

TUNEL, terminal deoxynucleotidyl transferase 

biotin-dUTP nick end labeling); adhesive properties 

(as measured by the expression of P-cadherin, an 

adhesion molecule specific for basal epidermal 

keratinocytes); or differentiation (as measured by 

the expressions of keratin-1, keratin-10, and 

filaggrin) of epidermal keratinocytes. 

(NanoDerm 

(2007, 

Human 

foreskin grafts 

on SCID mice 

A commercially available 

sunscreen, hydrophobic 

emulsion containing 

nano-TiO2 (Anthelios XL 

SPF 60, La Roche 

Posay, France) 

For testing dermal effects 

157660

In vivo 

exposure 

Single 

exposure 

Sunscreen containing nano-TiO2 applied 

to skin at 2 mg/cm2 in occlusion for 1, 24, 

or 48 hr 

) 

Sacrificed after exposure time; punch 

biopsy from the human skin graft area 

Tested sunscreen containing nano-TiO2 did not 

affect viability, proliferation, apoptosis, 

differentiation, or adhesive properties of skin cells. 

Warheit et al. 

(2007, 

No dermal irritation effects, no clinical signs of 

toxicity, and no BW loss. 

Rabbit For testing acute dermal irritation 

091075 New Zealand 

White 

In vivo 

exposure 

Single 

exposure 

Nano-TiO2 (identified as 

uf-C , a pre-commercial 

version of DuPont Light 

Stabilizer 210), 79% 

anatase/21% rutile, not 

coated, approximately 90 

wt% TiO2, 7% alumina, 

and 1% amorphous 

silica, average particle 

size 140.0 ± 44 nm in 

water, average BET 

surface area 38.5 m2

/g 

Doses – 0 or 0.5 g ) 

Not considered a skin irritant. Single exposure for 4 hr (nano-TiO2 in 

0.25 mL deionized water on 6 cm2

 area of 

skin), covered by gauze 

Observation at 1, 24, 48, and 72 hr after 

exposure 

Increases in cell proliferation in the draining 

auricular lymph node of the ears treated with 50% 

and 100% nano-TiO2 compared to the vehicle 

control group. 

For testing dermal sensitization (local 

lymph node assay) 

Warheit et al. 

(2007, 

Mouse 

091075 Female, 

CBA/JHsd 

) 

0, 5, 25, 50, or 100% nano-TiO2 on both 

ears for 3 days In vivo 

exposure No dermal sensitization by nano-TiO2: Stimulation 

index (mean disintegrations per minute of each 

experimental group/mean disintegrations per 

minute of the vehicle control group) did not exceed 

3.0 in any nano-TiO2 treated groups. Consequently 

the EC3 value (the estimated concentration 

required to induce a threshold positive response, 

i.e., where stimulation index equals 3) for nano-TiO2

was not calculated. 

Positive control group: 25% 

hexylcinnamaldehyde in 4:1 acetone:olive 

oil for 3 days Repeated 

exposure 

(Vehicle of positive control) group: 4:1 

acetone:olive oil for 3 days 

Sacrifice on test day 5 

Diluting vehicle: N,N-Dimethyl formamide 

Positive control group had a dermal sensitization 

response. 

 5-27 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Pig Pig: After 30 days of treatment Wu (2009, 

Male 

100% anatase, 

uncoated, nano-TiO2

(Zhejiang Wanjin 

Material Technology Co., 

Ltd.): 

Porcine skin, in vivo, shaved pig ear 

starting at age of 4 wk, approximately 

24 mg of test formulation containing 5% 

nano-TiO2 (4 or 60 nm) and Tween 80 

was topically applied in the marked test 

area on the right ear skin for 30 

consecutive days. Punch biopsies 

collected at 24 hr after the last treatment 

for TEM. 

193721

In vivo 

exposure 

Repeated 

exposure 4 nm, hydrophobic 

surface, measured 

particle size 5 ± 1 nm, 

surface area 200 m2

/g 

10 nm, hydrophobic 

surface, measured 

particle size 10 ± 1 nm, 

surface area 160 m2

/g 

75% anatase/25% rutile, 

uncoated nano-TiO2 (P25 

from Degussa, 

Germany): 

21 nm, hydrophilic 

surface, surface area 50 

m2

/g 

100% rutile, uncoated, 

nano-TiO2 (Zhejiang 

Hongsheng Material 

Technology Co., Ltd.): 

25 nm, hydrophilic 

surface, measured 

particle size 25 ± 5 nm, 

surface area 80 m2

/g 

60 nm, hydrophobic 

surface, measured 

particle size 60 ± 10 nm, 

surface area 40 m2

/g 

90 nm, hydrophobic 

surface, measured 

particle size 90 ± 10 nm, 

surface area 40 m2

/g 

Nano-TiO2 was detected in all layers of epidermis 

(stratum corneum, stratum granulosum, prickle cell 

layer, and basal cell layers), but not in the dermis of 

porcine skin. Only 4 nm nano-TiO2 penetrated into 

the deeper layer of the epidermis (basal cell layer). 

Subcellular changes (extended intercellular space, 

impairment of desmosome, and vacuoles around 

nucleus in basal cells) were seen. No gross lesions 

(such as erythema or edema). 

) 

BALB/c hairless mice skin, in vivo, dorsal 

region starting at age of 7-8 wk. Test 

formulation containing 5% nano-TiO2

(10 nm, 21, 25, 60, or 90 nm), carbopol 

940, and triethanolamine was applied on 

the dorsal skin for 60 consecutive days at 

8 mg emulation (or 400 μg nano-TiO2) per 

cm2

 skin. 3 hr after application, the 

dressing was removed and residual 

nanomaterials were removed from the 

skin with lukewarm water and the skin 

was dried. 

Mouse Hairless mice: After 60 days of treatment 

Male and 

female 

BALB/c 

(hairless) 

Mice treated with 10, 21, and 25 nm nano-TiO2 had 

decreased BW and increased relative liver weight 

to BW. Mice in the 10- and 21-nm groups also had 

increased relative spleen weight to BW. 

In vivo 

exposure Decreased SOD activities (indicator of antioxidant 

defense) in the skin and liver (10 and 21 nm). 

Increased lipid peroxidation (as measured by 

malondialdehyde) in the skin and liver (10, 21, 

25 nm) (in skin only - 60 nm). Decreased collagen 

content of skin (as measured by hydroxyproline) 

(10, 21, 25, and 60 nm). 

Repeated 

exposure 

Increased Ti in the skin, subcutaneous muscle, 

liver, heart, and spleen, but not in the blood or 

subcutaneous saccus lymphaticus in the 10-, 21-, 

25-, and 60-nm groups. Almost negligible changes 

in the brain and kidney, with the exception of 

increased Ti in the brain after 21 nm nano-TiO2

exposure. Increased Ti in the lung may be 

significant in the 21- and 60-nm groups. 

Pathological changes in skin (excessive 

keratinization, thinner dermis) (particularly in the 

10- and 21-nm groups, also in the 25- and 60-nm 

groups), liver (focal necrosis – 21-, 25-, and 60-nm 

groups; liquefaction necrosis – 10-nm group), heart 

(small trace of white blood cells – 10-nm group), 

spleen (minor increase in local macrophages – 10-, 

21-, 25-, and 60-nm groups), and lung (slight 

alveolar thickening -- in 10, 21-, 25-, and 60-nm 

groups). No pathological changes in the brain. 

90-nm group showed no changes. 

BET – Brunauer, Emmett, Teller method of calculating surface area OM – Octyl methoxycinnamate 

BrdU – Bromo-deoxy-uridine SOD – superoxide dismutase 

EC3 – Estimated concentration required to induce a threshold positive response, where 

stimulation index equals 3 

TUNEL –Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling 

Toxicity from Oral Exposure 

Currently only three toxicological studies of nano-TiO2 through oral exposure are available 

(Table 5-5). Two of them observed the toxicity for up to 2 weeks after a single oral gavage of 

nano-TiO2 (Wang et al., 2007, 090290; Warheit et al., 2007, 091075), and the other investigated 

genomic instability after nano-TiO2 exposure through drinking water for 5 or 10 days (Trouiller et 

al., 2008, 157484). 

The Warheit et al. study (2007, 091075) was intended to provide basic hazard screening 

information on well-characterized types of nano-TiO2 through a “base set” of tests spanning 

mammalian toxicity, genotoxicity, and aquatic (ecological) toxicity endpoints. The acute oral toxicity 

aspect of this project involved female rats receiving a single oral gavage of up to 5,000 mg/kg 

photostable nano-TiO2 (uf-C) (3 rats per dose). The authors reported “no biologically important BW 

loss” and no gross lesions at necropsy 14 days after the gavage. Given that this was a basic screening 

study, no information on organ weights, histological examinations, or blood tests (hematological or 

biochemical) was obtained, and thus it was not meant to rule out systemic toxicity or functional 

changes. However, the study does provide evidence that up to 5,000-mg/kg nano-TiO2 was not lethal 

as tested. 

 5-28 
In the Wang et al. study (2007, 090290), male and female mice received a single oral gavage 

of 5,000 mg/kg TiO2 as 25-nm rutile spindles, 80-nm rutile spindles, or 155-nm anatase octahedrons 

(Table 5-5 for more details). The large dose was selected because of the expected low toxicity and 

was administrated according to OECD testing procedures. No obvious acute toxicity was evident 

over a 2-week period. However, liver and kidney toxicity were indicated by biochemical parameters 

in the serum and by pathological examination. Although no abnormal pathology was observed in the 

heart, lung, testicle/ovary, and spleen tissues, myocardial damage was suggested by increases in 

serum lactate dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (α-HBDH), although 

such increases might also reflect damage to other organs. Morphological changes in the brain were 

seen in the hippocampus in both the 80-nm and 155-nm groups. The main organs with elevated TiO2

concentrations (measured only in female mice) were the liver, spleen, kidneys, lungs, and brain. 

Although the liver is expected to receive most of the TiO2 absorbed from the gastrointestinal tract 

through the portal vein, elevated TiO2 levels in the liver were observed only in the 80-nm group. The 

reason for this size-specific elevation in hepatic TiO2 concentration remains unknown. 

The preliminary results of the Trouiller et al. (2008, 157484) study showed increased DNA 

and chromosomal damage in various tissues of adult mice given 60-600 μg/mL photocatalytic 

nano-TiO2 (P25) in drinking water for 5 days. In a separate experiment, the offspring of mice that 

were given nano-TiO2 in drinking water for ten days in the second half of the pregnancy showed 

increases in DNA deletions in the eye-spot assay (Trouiller et al., 2008, 157484), which detects 

reversion of the mouse pink-eyed unstable (pun) mutation through DNA deletions of duplicated pinkeyed dilution (p) gene in the offspring of C57BL/6Jpun/pun mice (Reliene and Schiestl, 2003, 

157857). This study showed not only genotoxicity and clastogenicity, but also multi-generation 

effects of photocatalytic nano-TiO2 through oral exposure. Although the concentrations investigated 

in this study are very high, the suggested modes of action and effects of exposure during pregnancy 

are noteworthy, particularly for photocatalytic nano-TiO2. This work is also relevant to discussions 

of the carcinogenicity of nano-TiO2 (Section 5.3.2). The application of genotoxicity data to the 

question of potential carcinogenicity is based on the premise that genetic alterations are found in all 

cancers. Mutagenicity/genotoxicity is the ability of chemicals to alter the genetic material in a 

manner that permits changes to be transmitted during cell division. Although most tests for 

mutagenicity detect changes in DNA or chromosomes, some specific modifications of the epigenome 

including proteins associated with DNA or RNA, can also cause transmissible changes. Genetic 

alterations can occur via a variety of mechanisms including gene mutations, deletions, translocations, 

or amplification; evidence of mutagenesis provides mechanistic support for the inference of potential 

for carcinogenicity in humans. 

 5-29 
Table 5-5. Summary of health effects of nano-TiO2 particles in mammalian animal models: oral 

route 

Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

No mortality, no biologically important BW losses, and no 

gross lesions present in the rats at necropsy. 

For testing acute effects Warheit et al. 

(2007, 

Rat 

Female, strain/stock 

not specified 

Nano-TiO2 (identified as uf-C, a 

pre-commercial version of 

DuPont Light Stabilizer 210), 

79% anatase/21% rutile, not 

coated, approximately 90 wt% 

TiO2, 7% alumina, and 1% 

amorphous silica, average 

particle size 140.0 ± 44 nm in 

water, average BET surface 

area 38.5 m2

/g 

091075 Doses – 175, 550, 1,750, 

or 5,000 mg/kg (3 rats per 

dose) 

) 

Grey colored feces were observed in rats dosed at 

1,750 mg/kg (1 of 3 rats) and 5,000 mg/kg (All 3 rats). 

Single oral gavage Oral LD50 >5,000 mg/kg for female rats. 

Observation for 14 days 

postexposure 

Single oral gavage (acute Hepatic Toxicity: 

effects) 

Mouse 

Male and female 

CD-1 (ICR) 

Nano-TiO2 (Hangzhou Dayang 

Nanotechnology Co. Ltd.), 

rutile, uncoated, 25 nm 

(measured average size 

21.1 ± 5.1 nm), surface area 

43.0 m2

/g, column/spindle 

shape, purity >99% (Chen, 

personal communication, 2008, 

Wang et al. 

(2007, 090290

157588) 

Nano-TiO2 (Hangzhou Dayang 

Nanotechnology Co. Ltd.), 

rutile, uncoated, 80 nm 

(measured average size 

71.4 ± 23.5 nm), surface area 

22.7 m2

/g, column/spindle 

shape, purity >99% (Chen, 

personal communication, 2008, 

157588) 

Fine TiO2 (Zhonglian Chemical 

Medicine Co.), 155 nm 

(measured average size 

155.0 ± 33.0 nm), surface area 

10.4 m2

/g, anatase, uncoated, 

octahedrons, purity >99% 

(Chen, personal 

communication, 2008, 157588) 

Dose – 5,000 mg/kg 

10 female and 10 male 

mice per TiO2 size group 

Necropsy at 2 wk after the 

gavage 

Increases in coefficients (wet organ weight/BW) of liver 

(females in 25-nm and 80-nm groups), serum ALT (females 

in 25-nm group), serum ALT/AST (females in 25-nm group 

and males in 155-nm groups), and serum LDH (females in 

25-nm and 80-nm groups).a

 Decreases in AST in males in the 

155-nm group (Chen, personal communication, 2008, 

) 

 

157588). 

Pathological changes: hydropic degeneration around the 

central vein, spotty necrosis of hepatocytes (males and 

females in 80-nm and 155-nm groups). 

Nephrotoxicity: 

Increases in serum BUN (females in 25-nm group; no tin 

males) and serum LDH (females in 25-nm and 80-nm 

groups; male data not available) (Chen, personal 

communication, 2008, 157588).a

Pathological changes: swelling in renal glomerules and 

proteinic liquid in renal tubule (males and females in 80-nm 

group). 

Possible Brain Toxicity: 

Pathological changes: increases in vacuoles in the neuron of 

the hippocampus (males and females in 80-nm and 155-nm 

groups). The vacuoles could be from reversible fatty 

degradation (Chen, personal communication, 2008, 157588). 

Possible Myocardial Damage: 

Increase in serum LDHa (females in 25-nm and 80-nm 

groups; male data not available), α-HBDH (females in 25-nm 

and 80-nm groups; male data not available) (Chen, personal 

communication, 2008, 157588). Based on the data in this 

study alone, it cannot be ruled out that LDH and α-HBDH 

were from kidney or liver. 

Pathological Results: 

No pathological changes in heart. 

No pathological changes in heart, lung, testicle/ovary or 

spleen in male and female mice exposed to either 80 nm or 

155 nm TiO2. No pathological changes in any organs of mice 

exposed to 25 nm TiO2. 

Distribution: 

TiO2 distribution in female mice: increased Ti concentrations 

in liver (80-nm group), spleen (25-, 80-, and 155-nm groups), 

kidney (25- and 80-nm groups), lung (80-nm group) and brain 

(25-, 80-, and 155-nm groups). For the 80-nm group, highest 

Ti concentration was in liver (3,970 ng/g), followed by spleen, 

kidney, and lung (~375-625 ng/g). For 25-nm group, highest 

Ti concentration was in spleen (~500 ng/g). 

 5-30 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Increased genomic instability (adult mice): Trouiller et al. 

(2008, 

Mouse 

Wild-type and 

C57BL/6Jpun/pun

Nano-TiO2 (P25), 

photocatalytic, 80% 

anatase/20% rutile, not coated 

For testing genotoxicity in 

two generations 157484

Wild-type adult mice: 60, 

120, 300 and 600 μg/mL in 

drinking water for 5 days 

(Based on the assumption 

of 5 mL water intake per 

day per mouse with a BW 

of 30 g, the total doses 

would be 50, 100, 250 and 

500 mg/kg BW) 

C57BL/6Jpun/pun mice for 

eye-spot assay: 10-day 

exposure, pregnant mice 

were given nano-TiO2 in 

drinking water from 

8.5-18.5 days post 

conception. Offspring were 

sacrificed at 20 days old. 

DNA damage was increased in cells in peripheral blood at 

600 μg/mL. DNA damage was measured by alkaline Comet 

assay, which detects DNA single strand breaks, double 

strand breaks, alkaline liable sites, and other lesions. 

) 

DNA double strand breaks (measured by γH2AX immunostaining) were increased in bone marrow at all tested doses. 

Chromosomal damage (measured by micronucleus assay) 

was increased in peripheral blood at 600 μg/mL. 

Oxidative DNA damage (measured by HPLC) was increased 

in liver at 600 μg/mL. 

Increased genomic instability (offspring): 

Increases in DNA deletions at the pink-eyed unstable (pun) 

locus which result from homologous recombination or double 

strand breaks between the DNA fragments that contain 

duplicated pink-eyed dilution (p) gene (Reliene and Schiestl, 

2003, 157857) as measured by the eye-spot assay at 

500 mg/kg. 

Increased inflammation: 

Increases in (mRNA levels of) pro-inflammation markers, 

TNF-α, IFN-γ, and IL-8 (KC) (but not anti-inflammatory 

markers, TGF-β, IL-10 or IL-4) in peripheral blood at 

500 mg/kg as measured by real time RT-PCR. 

a

LDH may be from heart, liver, kidney, skeletal muscle, brain, blood cells, and lungs. A test for LDH isotypes can help to narrow down the source. The primary 

sources for various LDH isotypes in humans are: LDH-1 from heart muscle and red blood cells; LDH-2 from white blood cells; LDH-3 from lung; LDH-4 from 

kidney, placenta, and pancreas; and LDH-5 from liver and skeletal muscle (MedlinePlus, 2009, 193814). 

IL-8 (KC) – IL-8 stands for interleukin-8 and KC for chemokine (CXC motif) 

ligand 1 (CXCL1) α-HBDH – Alpha-hydroxybutyrate dehydrogenase 

γH2AX – Phosphorylated form of histone H2AX (phosphorylation of H2AX at 

serine 139) IL-10 – Interleukin-10 

LDH – Lactate dehydrogenase, a general marker of cell injury (Ma-Hock et al., 

2009) 

ALT – Alanine aminotransferase 

AST – Aspartate aminotransferase 

LD50 – Lethal dose 50; the dosage that is lethal to 50% of the tested 

population BET – Brunauer, Emmett, Teller method of calculating surface area 

BUN – Blood urea nitrogen RT-PCR – Reverse transcription polymerase chain reaction HPLC – High performance liquid chromatography TGF-β – Transforming growth factor-beta IFN-γ – Interferon-gamma TNF-α – Tumor necrosis factor-alpha IL-4 – Interleukin-4 

Toxicity from Respiratory Exposure 

This section discusses the health effects of nano-TiO2 exposure through the respiratory tract 

(Table 5-6). Two methods of exposure commonly employed for studies of respiratory toxicity are 

inhalation and instillation. Instillation can be performed in various ways, but essentially involves the 

direct administration of a substance to the lungs rather than allowing the subject to inhale the 

material. Intratracheal instillation “can be a useful and cost-effective procedure for addressing

specific questions regarding the respiratory toxicity of chemicals, as long as certain caveats are 

clearly understood and certain guidelines are carefully followed” (Driscoll et al., 2000, 011376). 

Among the advantages of instillation are that it permits researchers to control the doses administered 

into the lung and allows fast administration of test material to the lower respiratory tract. Instillation 

studies can be useful for identifying most types of effects (other than upper respiratory tract effects, 

such as nasal effects) and for comparing the relative potency of compounds, and for this reason are 

of interest for screening different materials for toxicity. Additionally, instillation studies require 

smaller amounts of test material, and chances of incidental ingestion exposure (as in whole-body 

chamber inhalation) are lower than in inhalation studies (Driscoll et al., 2000, 011376; Osier et al., 

1997, 086056). On the other hand, instillation exposure involves invasive delivery, bypassing of the 

upper respiratory tract, confounding effects from the instilled vehicle, and the use of higher doses or 

dose rates than those tested in inhalation experiments. Confounding effects are also a concern from 

 5-31 
anesthesia (needed for instillation, but not inhalation), which could affect the retention and clearance 

of the test material (Driscoll et al., 2000, 011376). Furthermore, studies have shown that exposure to 

the same particle through intratracheal instillation and inhalation can yield different responses. For 

example, compared to inhalation, instillation caused more particles to be deposited in the basal 

regions of the lung and caused particles to be distributed less homogenously (Osier et al., 1997, 

086056). Also, results from instillation cannot be extrapolated quantitatively for estimating 

inhalation results (Driscoll et al., 2000, 011376). 

Interpreting and comparing results from studies with different respiratory exposure methods 

(such as inhalation, instillation, and aspiration) requires caution. Differences among exposure 

methods could influence uptake doses and particle distributions in the body. Also, the test material 

preparation required for different exposure methods (such as aerosol and suspension medium 

preparation) could affect nanomaterial aggregation. Conclusions drawn from studies using different 

methods should disclose confounding factors to avoid misleading readers. As an illustration, 

consider a study that exposed mice to single-walled carbon nanotubes (SWCNT) through inhalation 

and pharyngeal aspiration (Shvedova et al., 2008, 157491). Even though the doses were designed to 

generate the same deposited dose in the lung, the aerosol generation and agglomerate sizes of the test 

material differed. The authors carefully stated their conclusion at the end of discussion: “Because of 

exposure to smaller SWCNT structures by inhalation of a dry aerosol versus aspiration of a particle 

suspension containing micrometer-size agglomerates, inhalation exposure was more potent than 

aspiration of an equivalent mass of SWCNT.” 

The tendency of nano-TiO2 to agglomerate raises an issue for interpreting experimental 

toxicology studies when the respiratory tract is the portal of entry. Upon inhalation, insoluble 

particles will deposit in the lung according to the aerodynamic diameter of the particulate unit (i.e., 

the agglomerate) and the physiological/morphometric characteristics of the subject. Once deposited 

as a result of inhalation or intratracheal instillation, additional factors (e.g., physicochemistry of the 

particles, biochemistry of the fluid lining of the lung, and other pharmacokinetic factors of the 

subject) may impact particle size and composition and determine the ultimate dose to the target 

cell/molecule. The influence of the lung milieu on agglomeration is discussed in more detail below. 

It should be noted that the concentrations in available respiratory toxicity studies of nano-TiO2

are presumably much higher than likely ambient or occupational exposure levels. High 

concentrations of fine-mode particles are known to cause the phenomenon of “particle overload.” In 

its simplest terms, at sufficiently high concentrations, the body’s ability to clear inhaled particles is 

severely compromised to the point that effects occur that would not occur at high-end “real-world” 

exposures (ISLI Risk Science Institute Workshop Participants, 2000, 002892). Thus, under particle 

overload conditions, exposure-response relationships and even the type of responses produced can be 

unreliable. However, the nanoparticle-specific exposures evoking particle overload have not been 

fully described. 

Effects in Respiratory Tract 

As discussed below and summarized in Table 5-6, pulmonary effects studied through 

inhalation or instillation of nano-TiO2 include pulmonary inflammation, recruitment of neutrophils 

and macrophages, nano-TiO2 aggregate-loaded macrophages, disruption of alveolar spaces, alveoli 

enlargement, proliferation of alveolar type II pneumocytes, and increases in alveolar epithelial 

thickness. Selected instillation studies are highlighted here primarily for effects not investigated in 

inhalation studies (i.e., effects outside the respiratory tract and interactions with other factors). 

Some of the factors that affect nano-TiO2 respiratory tract toxicity were investigated by 

Oberdörster et al. (2000, 036303). Toxicity of nano-TiO2 could be decreased by cross-tolerance to 

oxidative stress, because nano-TiO2 given through an intratracheal instillation caused less 

inflammation in rats previously exposed (and adapted) to Teflon fumes than in rats that were not 

adapted. Furthermore, nano-TiO2 induced more severe pulmonary inflammation in compromised 

 5-32 
rats, which had been given an endotoxin to mimic gram-negative bacterial infections, than in healthy 

rats. 

Inhalation and Instillation in the Same Study 

Grassian et al. (2007, 093170) exposed mice to nano-TiO2 through either inhalation or 

intranasal instillation. After instillation exposures to similar surface area doses (based on primary 

particle surface areas) of 5-nm anatase nano-TiO2 and 21-nm anatase/rutile nano-TiO2, mice showed 

a more severe inflammation response to 21-nm nano-TiO2 than to 5-nm TiO2. This example shows 

that surface area alone is not a sufficient dose metric in all studies (Grassian et al., 2007, 093170; 

Warheit et al., 2007, 091075), especially when the crystal form and other factors are not the same. In 

the Grassian et al. (2007, 093170) study, the aggregates of 21-nm and 5-nm nano-TiO2 differed in 

both size and density, either of which could affect the surface area that would interact with the 

tissues. Although the same nano-TiO2 was used in both inhalation and intranasal instillation, direct 

comparisons of exposure routes effects were not feasible for two reasons. First, the exposure doses 

were not the same, whether the doses were expressed as particle concentrations in air or solution, 

estimated particle mass per mouse, or estimated particle surface area per mouse. Second, different 

vehicles (water for inhalation and saline for instillation) were used and the sizes of agglomerates 

were larger in inhalation aerosols than in instillation.

In a study by Osier et al. (1997, 086056), acute intratracheal inhalation of high levels 

(125 mg/m3

) of fine and nano-TiO2 caused less severe pulmonary response than intratracheal 

instillation. Intratracheal inhalation involved delivering aerosols to the trachea of anesthetized rats. 

Inhalation Studies 

The effects in the respiratory tract after inhalation of nano-TiO2 were consistent among 

studies. With increases in exposure duration, pulmonary lesions in rodents evolve from reversible 

pulmonary inflammation (in rats, mice, and hamsters) to impaired particle clearance or overload (in 

rats and mice, but not hamsters) and cellular proliferation (in rats and mice, but not hamsters). In 

rats, but not in mice or hamsters, chronic exposure leads to pulmonary alveolar fibrosis, metaplasia, 

and eventually lung tumors. 

In acute and subacute studies in mice and rats, the severity of pulmonary inflammation 

increased with increases in exposure time, and symptoms (pulmonary inflammation and increases in 

cell proliferation in bronchi and bronchioles) were reversible when exposure ended (Grassian et al., 

2007, 090606; Ma-Hock et al., 2009, 193534). 

In subchronic studies of nano-TiO2 exposure for 12 or 13 weeks, pulmonary inflammation, 

pathological changes in the lung (including fibrosis), and impairment of alveolar macrophagemediated test particle clearance were reported (Baggs et al., 1997, 048642; Bermudez et al., 2002, 

055578; Bermudez et al., 2004, 056707; Hext et al., 2002, 157878; Hext et al., 2005, 090567; 

Oberdörster et al., 1994, 046203). Similar to pulmonary lesions after acute and subacute exposure, 

pulmonary lesions after subchronic inhalation exposure were also decreased with recovery time, but 

some lesions, such as fibrotic reactions in the lung, were not completely reversed even after 1 year of 

recovery. 

Species differences to nano-TiO2 effects were observed among rats, mice, and hamsters 

(Baggs et al., 1997, 048642; Bermudez et al., 2002, 055578; Bermudez et al., 2004, 056707; Hext et 

al., 2002, 157878; Hext et al., 2005, 090567; Oberdörster et al., 1994, 046203). Pulmonary responses 

after 13 weeks of exposure were generally most severe in rats, followed by mice, and least severe in 

hamsters. Rats and mice, but not hamsters, experienced overload at 10 mg/m2

 nano-TiO2. 

Furthermore, only rats had fibroproliferative lesions and alveolar epithelial bronchiolization (a type 

of metaplasia). 

In chronic studies of nano-TiO2 inhalation in rats (Creutzenberg et al., 1990, 157963; 

Gallagher et al., 1994, 045102; Heinrich et al., 1995, 076637) and mice (Heinrich et al., 1995, 

 5-33 
076637), lung tumors occurred in rats, but not in mice (for more on carcinogenicity effects in these 

studies, see Section 5.3.2) . In the study of Creutzenberg et al. (1990, 157963), decreased pulmonary 

clearance (overload) was clearly demonstrated by using two sizes of tracer particles after nano-TiO2

exposure. During the 24-month exposure to nano-TiO2 (see Table 5-6 for concentrations), rats 

inhaled (nose-only) two types of radioactive tracers at 3, 12, and 18 months after the beginning of 

the experiment. The half-times for pulmonary clearance of the smaller tracer particles (0.35-μm 

59Fe2O3) were more than three times longer in rats exposed to nano-TiO2 at all three tested time 

points, indicating overload. For the larger tracer particles (3.5-μm 85Sr polystyrene), overload was 

seen at 3 and 12 months, and the clearance was back to control level at 18 months, which may be 

due to increased lung weight, altered lung structure, and altered breathing pattern, all of which could 

consequently change the deposition of 85Sr polystyrene particles (Creutzenberg et al., 1990, 157963). 

Systemic Effects and Specific Effects in Heart, Liver, Kidney, and Microvasculature 

The effects of respiratory exposure to nano-TiO2 are not limited to the respiratory system. In 

rats exposed to 5-mg nano-TiO2/kg BW of rutile nano-TiO2 rods through a single intratracheal 

instillation, observed effects included increases in the numbers of monocytes and granulocytes in the 

blood (signs of systemic inflammation); decreases in the number of platelets in the blood (platelet 

aggregation); and cardiac edema (Nemmar et al., 2008, 157514). In mice exposed to rutile and 

anatase nano-TiO2 through intranasal instillation, pathological changes were observed in the kidney, 

and temporary liver injury was suggested by changes in serum biomarkers (Wang et al., 2008, 

157473). 

Endothelium-dependent arteriolar dilation was impaired (decreased) by both fine TiO2 and 

nano-TiO2 inhaled by rats, more so by nano-TiO2 than fine TiO2 at similar lung load mass doses 

(Nurkiewicz et al., 2008, 156816). This microvascular dysfunction was attributed to fine TiO2- and 

nano-TiO2-induced increases in ROS in the microvascular wall, increases in nitrotyrosine expression 

in spinotrapezius microcirculation, and decreases in microvascular NO production (Nurkiewicz et 

al., 2009, 191961). In both fine TiO2-and nano-TiO2-treated groups, vascular smooth muscle 

sensitivity to NO was not altered, but the microvascular NO bioavailability was compromised 

(Nurkiewicz et al., 2009, 191961). 

Effects in Brain 

Since 1970, scientists have known that inhaled ultrafine air pollutants and engineered 

nanoparticles translocate into the brain (Oberdörster et al., 2004, 055639). Inflammatory responses, 

altered neurotransmitter levels, and pathological changes have been observed in rodent brains after 

inhalation of manganese oxide (Elder et al., 2006, 089253); instillation of nano carbon black 

(Tin Tin Win et al., 2008, 157486); and inhalation of ultrafine elemental 13C particles (Oberdörster et 

al., 2004, 055639). A few recent studies showed that anatase and rutile nano-TiO2 translocate into the 

brain following intranasal instillations (Wang et al., 2007, 157616; Wang et al., 2008, 157474). 

The only available studies of nano-TiO2 effects on the central nervous system are from a 

research group that has administered high doses of nano-TiO2 to mice using intranasal instillation 

(Wang et al., 2007, 157616; Wang et al., 2008, 157474; Wang et al., 2008, 157473). These 

researchers have reported increased oxidative stress and inflammatory response, altered 

concentrations and metabolism of neurotransmitters, and pathological changes in the mouse brain. 

When mice were given 25-nm rutile, 80-nm rutile, or 155-nm anatase nano-TiO2 though intranasal 

instillation (50 mg nano-TiO2/kg BW every 2 days for 2, 10, 20, or 30 days), changes in 

neurotransmitter levels in the brain were observed only in mice exposed to 80-nm and 155-nm 

nano-TiO2, whereas brain TiO2 concentrations were similar for all three sizes of nano-TiO2 (Wang et 

al., 2007, 157616). After intranasal instillation of 80-nm rutile or 155-nm anatase nano-TiO2 (500 μg 

per mouse every other day for up to 30 days), the highest Ti concentrations in the brain were in the 

hippocampus and olfactory bulb, the two regions where most pathological changes were also seen 

 5-34 
(Wang et al., 2008, 157474; Wang et al., 2008, 157473). The hippocampus and astrocytes seem to be 

the targets of nano-TiO2 toxicity in the brain (Wang et al., 2008, 157474; Wang et al., 2008, 157473). 

At the ultra-structural level, mitochondria appear to be a target of nano-TiO2 in nerve cells after both 

in vivo and in vitro exposures (Long et al., 2006, 089584; Wang et al., 2008, 157473). For the whole 

brain, inflammatory responses and oxidative stress, including lipid peroxidation and protein 

oxidation, were detected as elevated levels of oxidative markers and cytokines in mice exposed to 

80-nm rutile and 155-nm anatase nano-TiO2 (Wang et al., 2008, 157474; Wang et al., 2008, 157473). 

Levels of several neurotransmitters, including norepinephrine, 5-hydroxytryptamine, 

homovanillic acid, 5-hydroxyindole acetic acid, dopamine, and glutamic acid, were altered after 

intranasal instillation of nano-TiO2 (Wang et al., 2007, 157616; Wang et al., 2008, 157474; Wang et 

al., 2008, 157473). Nitric oxide, which serves as a neurotransmitter and an important player in 

inflammatory responses, was also increased in the brain of mice exposed to 80-nm and 155-nm 

nano-TiO2 (Wang et al., 2008, 157474). Additionally, the activity of cholinesterase, which inactivates 

the neurotransmitter acetylcholine, increased (Wang et al., 2008, 157474). These changes showed 

that the concentrations and metabolism of neurotransmitters in the brain were affected by nano-TiO2

given through intranasal instillations. 

Table 5-6. Summary of health effects of nano-TiO2 particles in mammalian animal models: 

respiratory route

Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Inhalation and Instillation in the same report 

Single inhalation exposure for 

4 hr 

Increases in the numbers of total cell (high 5 nm, low 

and high 21 nm) and macrophage (high 5 nm and 

21 nm) in BAL fluid immediately after exposure (not 

20 hr after exposure). 

Grassian et al. 

(2007, 

Mouse 

Male C57BL/6 

Nano-TiO2

(Nanostructured and 

Amorphous Materials), 

anatase, 5 nm, measured 

BET surface area 

219 ± 3 m2

/g, surface 

functionalization: O, O-H, 

H2O. Aerosol size: 

119 ± 1.56 nm (inhalation 

high dose), 

122.9 ± 1.55 nm 

(inhalation low dose) 

093170) 

Particle concentration in 

chamber: 

No changes in histology of the lung, total protein, LDH 

activity, or neutrophil number in BAL fluid. 5 nm TiO2: 

Low: 0.77 mg/m3

 (necropsy 

immediately after exposure) Nano-TiO2 distribution (only 4 high groups examined): 

agglomerates were seen in macrophages, alveolar 

epithelial cells, and alveolar interstitium. Little difference 

between 5 and 21 nm exposures or necropsy time. 

High: 7.22 mg/m3

 (necropsy 

immediately after exposure); 

7.35 mg/m3

 (necropsy 20 hr 

after the end of exposure) Calculated/estimated particle mass per mouse (μg) and 

particle surface area (cm2

Nano-TiO ): 2 (Degussa), 

anatase/rutile, 21 nm, 

BET surface area 

41 ± 1.1 m2

/g, surface 

functionalization: O, O-H, 

H2O. Aerosol size: 

138.8 ± 1.44 m2/g 

(inhalation high dose), 

152.9 ± 1.38 m2

/g 

(inhalation low dose) 

21 nm TiO2: 5 nm TiO2 Low: 1.3 μg/mouse and 3.2 cm2

 (immediately Low: 0.62 mg/m after exposure) 3

 (necropsy 

immediately after exposure) 5 nm TiO2 High: 12.5 μg/mouse and 30.3 cm2

(immediately after exposure) 12.7 μg/mouse and 30.7 

cm2

 (20 hr after exposure) 

High: 7.16 mg/m3

 (necropsy 

immediately after exposure); 

7.03 mg/m3

 (necropsy 20 hr 

after the end of exposure) 21 nm TiO2 Low: 1.1 μg/mouse and 2.2 cm2

(immediately after exposure) 

21 nm TiO2 High: 12.4 μg/mouse and 24.8 cm2 

(immediately after exposure) 12.2 μg/mouse and 24.4 

cm2 (20 hr after exposure) 

 5-35 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

21 nm TiO2 induced more inflammation than 5 nm TiO2: 

Increases in neutrophil number (21 nm low, medium and 

high; 5 nm medium and high); total cell number and IL-6 

(21 nm medium and high); LDH activity and IL-1β

(21 nm high) in BAL fluid. 

Single intra-nasal instillation 

Particle concentration in 

instillation solutions: 

5 nm TiO2: 

Low: 0.1 mg/mL No pathological changes in lung; no changes in TNF-α in 

Medium: 0.4 mg/mL BAL fluid. 

21 nm anatase/rutile TiO2 and 5 nm anatase TiO2 do not 

share the same dose-response curve for neutrophil 

concentration in BAL fluid as a function to either particle 

mass or surface area. 

High: 0.6 mg/mL 

21 nm TiO2: 

Low: 0.5 mg/mL 

Medium: 2.0 mg/mL Calculated/estimated particle mass per mouse (μg) and 

particle surface area (cm2

High: 3.0 mg/mL ): 

5 nm TiO2 Low: 5 μg/mouse and 12.1 cm2

Necropsy 24 hr after instillation 

5 nm TiO2 Medium: 20 μg/mouse and 48.4 cm2

5 nm TiO2 High: 30 μg/mouse and 72.6 cm2

21 nm TiO2 Low: 25 μg/mouse and 12.5 cm2

21 nm TiO2 Medium: 100 μg/mouse and 50 cm2

21 nm TiO2 High: 150 μg/mouse and 75 cm2

Osier et al. 

(1997, 

Compared to fine TiO2, nano-TiO2 caused more 

pulmonary responses and slightly higher (not significant) 

lung TiO2 burden. 

Acute intratracheal instillation 

and intratracheal inhalation 

Rats 

Female F344 

Fine TiO2 (Fisher 

Scientific), mean primary 

particle size 250 nm, 

anatase 

086056) 

Intratracheal inhalation 

exposure for 2 hr at 125 mg/m3 Compared to intratracheal instillation, intratracheal 

inhalation to TiO2 generally caused less severe and less 

persistent pulmonary responses and slightly (not 

significant) higher TiO2 lung burden. 

Nano-TiO2 (Degussa), 

mean primary particle size 

21 nm, anatase 

Intratracheal instillation 

exposure to the equivalent 

amount of TiO2 as in the lung at 

day 0 of intratracheal inhalation 

(500 μg fine TiO2 or 750 μg 

nano-TiO2 in 0.2 mL saline) 

Increases in polymorphonuclear leukocytes in BAL cell 

pellet on day 1 after intratracheal inhalation of fine TiO2; 

on days 1, 3, and 7 after intratracheal instillation of 

nano-TiO2; and days 0 and 1 after intratracheal 

inhalation of nano-TiO2. Necropsy 0, 1, 3 or 7 days 

postexposure (3 rats per group) 

Decreases in macrophage inflammatory protein-2 levels 

in BAL supernatant on days 0, 1, and 3 after 

intratracheal inhalation of nano-TiO2; and day 1 after 

intratracheal instillation of nano-TiO2. Increases in 

macrophage inflammatory protein-2 levels in BAL cell 

pellets on days 1, 3, and 7 after intratracheal instillation 

of nano-TiO2; and on days 0 and 1 after intratracheal 

inhalation of nano-TiO2. 

Increases in TNF-α protein was detected by 

immunocytochemistry (but not by ELISA) on days 0 and 

1 after intratracheal inhalation of water (control); days 1 

and/or 3 after intratracheal instillation of fine or 

nano-TiO2 and intratracheal inhalation of fine TiO2; and 

at all time points after intratracheal inhalation of 

nano-TiO2. 

Inflammatory cell influx (polymorphonuclear leukocytes 

in BAL) was correlated with macrophage inflammatory 

protein-2 levels in BAL cell pellet (but not in BAL 

supernatant), but not correlated with TNF-α protein 

levels in BAL cell pellet or supernatant or in lung 

sections stained immunocytochemically. 

Inhalation 

Lung burden: SiO2: 0.32 mg immediately after exposure. 

Nano TiO2/fine TiO2: 5.33/6.62 mg, 4.15/1.2 mg, 

3.14/1.66 mg immediately, 6 mo, 12 mo after exposure, 

respectively. 

Baggs et al. 

(1997, 

Rats Subchronic inhalation 

Male F344 

Nano-TiO2, ~20 nm, 

anatase (Degussa) 048642

Fine TiO2, ~250 nm, 

anatase (Fisher Scientific) 

Crystalline SiO2, ~800 nm 

Nano-TiO2: 23.5 mg/m3

; fine 

TiO2: 22.3 mg/m3

; SiO2

1.3 mg/m3

) 

6 mo after exposure, in the lung: SiO2 caused moderate 

focal interstitial fibrosis and moderately severe focal 

alveolitis; nano TiO2 caused slightly less fibrosis and fine 

TiO2 caused least fibrosis. Increases in stainable 

collagen in all three treated groups, compared to 

untreated groups. 

6 hr/day, 5 days/wk for 3 mo 

6- or 12-mo recovery before 

sacrifice 

12 mo after exposure, in the lung: SiO2

–

treated rats 

showed decreased fibrosis; nano TiO2 and fine TiO2

treated rats showed largely normal amount of interstitial 

fibrosis but increases in alveolar macrophage number. 

Increases in stainable collagen only in SiO2. 

 5-36 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Fine TiO2: 

Bermudez et al. 

(2002, 

Rat Subchronic inhalation 

Female CDF 

(F344)/CrlBR 

Fine TiO2 (DuPont), rutile; 

aerosol 1.36 - 1.44 μm 

MMAD 

Lung burden of fine TiO2: 

Fine TiO2: 0, 10, 50 or 

250 mg/m3 Immediately after exposure: lung burden of fine TiO2: mice > rats > hamsters at 50 and 250 mg/m3: 

rats > mice > hamsters at 10 mg/m3

. The lung burden 

decreased with time after exposure. 

055578) 

Nano-TiO2 (P25), 

photocatalytic, average 

primary particle size 

21 nm, 1.37 μm MMAD; 

aerosols: 1.29-1.44 μm 

MMAD 

Nano-TiO2: 

Bermudez et al. 

(2004, 

nano-TiO2: 0, 0.5, 2, or 

10 mg/m3 Mouse 

Female 

B6C3F1/CrlBR 

056707

Hamster 

Female Syrian 

golden (Lak:LVG 

[SYR] BR) 

6 hr/day, 5 days/wk for 13 wk 

0 (immediately after exposure), 

4, 13, 26, or 52 (up to 46 and 

49 for hamsters exposed to fine 

TiO2 and nano-TiO2, 

respectively) wk of recovery 

before sacrifice 

The retention in lung-associated lymph nodes: 

rats > mice > hamsters at all concentrations. The burden 

in the lymph nodes increased with time after exposure 

(rats of all dose groups, mice of low and mid-dose 

groups, and hamsters of high-dose group). 

) 

Comparison of 

fine and 

nano-TiO2 data 

reported in 

Bermudez et al. 

(2002, Pulmonary clearance kinetics of fine TiO2: mice and rats 

in high-dose groups retained 75% initial burden after 

52 wk of recovery, while hamsters retained only 10% 

initial burden after 26 wk of recovery. Overload in rats 

and mice at 50 or 250 mg/m3

. 

055578) 

and 

Bermudez et al. 

(2004, 056707): 

Hext et al. (2002, Lung burden of nano-TiO2: 157878; 2005, 

Lung burden of nano-TiO2: rats ≥ mice > hamster. 090567

Immediately after exposure, at 10 mg/m3

, rats and mice 

had same lung burdens for nano-TiO2. At 2 or 0.5 mg/m3,

rats had more lung burden. Mice and rats, but not 

hamsters, have pulmonary particle overload at 

10 mg/m3

. 

) 

Pulmonary clearance kinetics of nano-TiO2: At 10 mg/m3

, 

rats and mice had linear fashion decreases of lung 

burden to ~50% after 52-wk recovery, while hamsters 

had a biphasic fashion decrease to 3% after 48-wk 

recovery. At 2 and 0.5 mg/m3

, rats, mice and hamsters 

had biphasic decreases in lung burn, and rats only had 

detectable nano-TiO2 after the whole recovery period. 

Burden in the lymph nodes associated with lung: During 

the whole recovery time, burden increased with time in 

rats of 10 and 5 mg/m3

 groups, and in mice of 10 mg/m3

group. No nano-TiO2 was detected in hamster lymph 

nodes at any time point or treatment group. 

General health of rats, mice and hamsters: 

Rats and mice at all treated groups had decreases in 

weight gain after exposure, and recovery occurred 3-

4 wk postexposure. Mice exposed to 250 mg/m3

 fine 

TiO2 had a consistent lower weight during the recovery 

period, but rats exposed to 250 mg/m3

 fine TiO2 had a 

consistent heavier weight. Hamster exposed to fine TiO2

had decreases in weight gain after exposure and 

recovery 6 wk postexposure. Hamsters exposed to 

nano-TiO2 had weight loss after exposure and a slow 

recovery over the remainder of the study. Hamsters had 

higher morbidity and mortality rates across treatment 

groups than rats and mice; this was probably due to agerelated renal diseases. 

 5-37 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Pulmonary inflammation after fine TiO2 exposure: Rats, 

mice and hamsters had pulmonary inflammation, and 

only hamsters had full recovery. 

Rats generally had more severe inflammation, and 

hamsters had the least. 

Fine TiO2 exposure: Increases in neutrophil %, 

lymphocyte %, and macrophage number in BAL fluid in 

rats and mice (in mid- and high-dose groups); increase 

in neutrophil % in rats at the lowest exposure. Hamsters 

had increased macrophage number, neutrophil %, and 

lymphocyte % at the highest concentration; they had an 

increased neutrophil % at the medium concentration. 

Within 26 wk of recovery, hamsters showed normal 

neutrophil % and macrophage number; within 46 wk of 

recovery, hamsters had normal lymphocyte %. Mice and 

rats showed partial recovery in neutrophil and 

macrophage response and no recovery in lymphocyte 

response after 52 wk of recovery. 

Fine TiO2 exposure: LDH levels in BAL fluid transiently 

increased in mice and rats 

Pulmonary inflammation after nano-TiO2 exposure: Rats 

and mice had pulmonary inflammation. 

Nano-TiO2 exposure: Rats and mice, but not hamsters, 

in the 10 mg/m3 groups had increased numbers of 

macrophage and neutrophil and concentrations of LDH 

and protein in BAL fluid. 

Pulmonary lesions were most severe in rats, and least in 

hamsters. 

Fine TiO2 exposure: Alveolar cell proliferation was seen 

in rats (0 week postexposure at mid- and high-dose 

groups, 4 and 13 wk postexposure at high-dose group) 

and mice (13 and 26 wk postexposure at high-dose 

group), but not in hamsters. 

Only rats had a progressive fibroproliferative lesion and 

alveolar epithelial metaplasia (bronchiolization). 

Fine TiO2 exposure: At 52 wk postexposure, mouse 

lungs had particle-laden macrophages in alveolar and 

relatively normal alveolar septal structures. Rat lungs 

had particle-laden macrophages inside alveolar cells, 

fibrosis and thickening in interstitial tissue, and little 

alveolar epithelial metaplasia (bronchiolization) of lining 

epithelium. Hamster lungs did not show retained particle 

burden or macrophage accumulation. 

Nano-TiO2 exposure: Alveolar epithelial proliferation, 

alveolar bronchiolization (alveolar epithelial proliferation 

of metaplastic epithelial cells around macrophages 

loaded with particles), alveolar septal fibrosis and 

interstitial particle accumulation in rats, but not mice nor 

hamsters, of the 10 mg/m3

 group. With increasing time 

postexposure, the lesions became more severe. 

Species and particle differences: 

Overload was seen in rats and mice (but not hamsters) 

exposed to 50 and 250 mg/m3

 fine TiO2 or 10 mg/m3

nano-TiO2. 

Lung TiO2 burdens and tissue responses in mice, rat and 

hamsters exposed for 13 wk to 10 mg/m3

 nano-TiO2 or to 

50 mg/m3

 fine TiO2 were similar for all three species. 

 5-38 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Nano-TiO2 (P25), 

photocatalytic, 80% 

anatase/20% rutile, 

primarily particle size 

15-40 nm, 0.8 μm MMAD 

Creutzenberg et 

al. (1990, 

Rat Rats: 

Female Wistar 

Chronic inhalation 

Increases in lung weight, and retention of inhaled 

nano-TiO2 in lungs and lung-associated lymph nodes 

(mean lung retention was 39.3 mg/lung at the end of 

exposure). The retention slowly decreased postexposure 

(from 40 mg/lung after 18 mo of nano-TiO2 exposure to 

3.3 mg/lung at 4 mo postexposure). 

Rats: 24 mo exposure: 

7.2 mg/m3

 for the first 4 mo, 

followed by 14.8 mg/m3

 for 

4 mo, 9.4 mg/m3

 for 16 mo, and 

clean air for 6 mo 

(concentration sometimes are 

reported as 7.5, 15, 10 mg/m3

) 

157963) 

Mouse 

Female NMRl 

Increased half-time of pulmonary clearance of tracer 

18 or 19 hr/day, 5 days/wk in particles 

whole body chamber For inhaled 0.35 μm labeled tracer particles 

Mice: 13.5-mo exposure: Same 

treatment as in rats for the first 

8 mo, followed by 9.4 mg/m3

 for 

5.5 mo, and clean air for 9.5 mo

After 3-, 12-, 18-mo nano-TiO2 exposure and 18-mo 

exposure plus 3-mo recovery, clearance half times were 

208, 403, 357, and 368 days, respectively. 

The controls had 61-96 days for all time points. 

For inhaled 3.5 μm labeled tracer particles 

After 3-, 12-, 18-mo nano-TiO2 exposure and 18-mo 

exposure plus 3-mo recovery, clearance half times were 

1,222, 229, 58 and 48 days, respectively. 

The controls had 58-70 days for all time points. 

The decreases in clearance half time after 12- and 

18-mo exposure, compared to controls, was possibly 

dye to increases in lung weight, altered lung structure 

and breathing pattern, which lead to more in the tracheobronchial region of the long and apparently higher 

clearance rates. 

Gallagher et al. 

(1994, 

Rats did not have increases in DNA adducts in the lung: 

045102 No increases in DNA adduct 2 (nuclease P1-sensitive 

adduct) in the lung. 

) 

Decreases in DNA adduct 1 (age-related, putative 

I-compound) in peripheral lung DNA compared to filtered 

air-exposed rats, probably due to adduct dilution through 

cell proliferation induced by particle exposure. 

Heinrich et al. 

(1995, 

Rats: 

076637 Increased mortality (60% vs. 42% in control) and lung 

wet weight, decreased mean lifetime and BW. 

) 

Increased incidence of lung tumors [18-mo exposure: 5 

out of 20 rats exposed to TiO2 (0 out of 18 in control) had 

lung tumors. 24-mo exposure: 4/9 rats in TiO2 (0/10 in 

control)]. 

Mice: 

No increase lung tumors. 

Increased mortality (33% vs. 10% in control) and lung 

wet weight, decreased BW. 

Carcinogenic in rats, but not in mice. 

 5-39 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Nano-TiO2

(Nanostructured and 

Amorphous Materials), 

anatase, measured 

average primary particle 

size 3.5 ± 1.0 nm, BET 

surface area 219 ± 3 m2

/g, 

surface functionalization: 

O, O-H, H2O 

(manufacturer reported 

primary particle 5 nm, 

surface area 210 m2

/g) 

Mouse Acute inhalation 

Male C57BL/6 

No adverse effect/Minimal pulmonary inflammation. Grassian et al. 

(2007, 090606

Aerosol size geometric 

mean 120-128 ± 1.6-

1.7 nm for acute (two 

concentrations) and 

subacute (one 

concentration) exposures 

Doses – 0, 0.77, or 7.22 mg/m3

Single exposure of 4 hr in 

whole-body chamber 

No recovery time 

No treatment effects on most parameters measured to 

gauge inflammatory response (neutrophil number in BAL 

fluid, total protein, and LDH activity were not changed), 

and no effects on lung histopathology. 

) 

Increased total cell count and macrophage count in BAL 

fluid at highest dose. 

Moderate but significant pulmonary inflammatory 

response that lasted for at least 2 wk but resolved by 

wk 3 after exposure. 

Subacute inhalation 

Doses – 0 or 8.88 mg/m3

 

4 hr/day for 10 days in wholebody chamber No changes in most parameters measured to gauge 

inflammatory response [total protein, LDH activity, and 

cytokine (IFN-γ, IL-6, or IL-1β) concentrations in BAL 

fluid were not changed, and no effects on lung 

histopathology. 

0, 1, 2, or 3 wk of recovery 

before sacrifice 

Increased macrophage count in BAL fluid in treated 

group at wk 0, 1, and 2 postexposure but not at wk 3 

postexposure. 

Macrophages in BAL fluid were loaded with TiO2

particles and less so at wk 3 postexposure. 

Absolute lung weight was increased at 50 mg/m3

immediately after exposure, but not after 16-day 

recovery. 

Ma-Hock et al. 

(2009, 

Rat Short-term inhalation 

Male Wistar 

Nano-TiO2 (Baker & 

Collinson, Inc.), uncoated, 

14% rutile/86% anatase, 

hydrophobic surface, 

average primary particle 

25.1 ± 8.2 nm (range 13 to 

71 nm) measured under 

TEM. BET surface area 

51.1 ± 0.2 m2

/g. Zeta 

potential was 16.5 ± 2.2 

mV in 1 mM KCl. 

193534

Aerosols: 0.7-1.1 μm 

MMAD (geometrical 

standard deviations 

2.3-3.4). Small and large 

agglomerates in the 

atmospheres, ranging 

from below 100 nm to 

several hundred nm. 

Estimated number 

concentrations of particles 

<100 nm represents only 

0.1-0.4% of the total 

particle mass for all three 

atmospheres. 

0, 2, 10, and 50 mg/m3

 (actual 

concentrations 0, 2.4, 12.1, and 

50.0 mg/m3

), 6 hr/ for 5 days, 

head-nose exposures to dust 

aerosols 

) 

Lung burden: 118.4, 544.9 and 1,635 μg/lung 

immediately after inhalation of 2, 10 and 50 mg/m3

nano-TiO2, respectively. 16 days of recovery later, the 

lung burdens were 93.4, 400.4 and 1,340 μg/lung, 

respectively. Calculated clearance half-times were 47, 

36 and 56 days for 2-, 10- and 50-mg/m3

 groups, 

respectively. 

No recovery (immediately after 

the last exposure [0 days]), 3- 

or 16-day recovery after the last 

exposure. In other words, 

necropsy on study days 5, 8, 

and 21, respectively. 

In the mediastinal lymph nodes, TiO2 was only detected 

in the 50-mg/m3

 group, and the nano-TiO2

concentrations were higher at 16 days after the last 

exposure (mean 11.01 μg in collected lymph nodes) than 

immediately after exposure (mean 2.34 μg). No TiO2 was 

detected in the liver, kidney, spleen or basal brain with 

olfactory bulb (detection limit 0.5 μg per organ). 

BAL fluid: increases in total cell count at 50mg/m3

 and 

polymorphonuclear neutrophils at 10 mg/m3

 and 

50 mg/m3, but no changes in eosinophil, lymphocyte, or 

macrophage counts, total protein content, enzyme 

activities, and levels of 9 (out of tested 60) cell 

mediators. Among the 9 mediators, effects were only 

observed at 10 mg/m3

 or higher immediately after 

exposure. After 3 days of recovery, effects were still 

observed, but for clusterin and haptoglobin, they were 

observed at 2 mg/m3

. Cell mediator levels were the 

same as controls after 16 days of recovery in 2 and 

10 mg/m3

 groups, but not in 50 mg/m3 group. 

Clinical pathology in blood: minor effects on serum cell 

mediator. No increase in serum troponin I, a biomarker 

for myocardial damage in rodents. 

Increased cell replication in large/medium bronchi and 

terminal bronchioles at all three groups immediately after 

exposure and after 3 days of recovery (not after 

16 days). Macrophage diffusion also decreases over 

time. No change in lung cell apoptosis. 

Changes were most prominent immediately after the last 

exposure or 3 days afterward, and some endpoints 

returned to control levels by 16 days of recovery. 

 5-40 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Nano-TiO2 caused more severe and prolonged (~1 yr) 

pulmonary inflammatory response (i.e., increase in 

alveolar macrophages, polymorphonuclear neutrophils, 

and lavagable protein) than fine TiO2. 

Rat Subchronic inhalation 

Male F344 

Nano-TiO2, 20 nm, 

anatase (Degussa); in 

aerosols: agglomerates 

0.71 ± 1.9 μm MMAD 

Oberdörster et 

al. (1994, Nano-TiO2: 23.5 ± 2.9 mg/m3

; 

fine TiO2: 22.3 ± 4.2 mg/m3 046203) 

6 hr/day, 5 days/wk, for 12 wk Fine TiO2, 250 nm, 

anatase (Fisher 

Scientific); in aerosols: 

agglomerates 

0.78 ± 1.7 μm MMAD 

When inflammatory response was expressed as number 

of polymorphonuclear neutrophils and dose was 

expressed as surface area for retained particles (i.e., 

lavagable particles), nano-TiO2 and fine TiO2 shared the 

same dose response curve. 

Recovery for 4, 8, 12, 29 or 

64 wk before sacrifice 

More severe and prolonged impairment of alveolar 

macrophage-mediated particle clearance in rats exposed 

to nano-TiO2 than rats exposed to fine TiO2. Seven mo 

after TiO2 exposure, fine TiO2 exposed (but not 

nano-TiO2 exposed) rats showed normal clearance 

rates. 

Pathological changes in the lung: nano-TiO2 caused 

greater epithelial effects (Type II cell proliferation, 

occlusion of pores of Kohn) and more interstitial fibrotic 

foci than fine TiO2. 

Dosimetry: 

Nano-TiO2 and fine TiO2 had a similar mass deposition in 

the lower respiratory tract and same retention in the 

alveolar space up to 1 yr after exposure. 

Nano-TiO2 showed longer total pulmonary retention 

(retention halftime: ~500 days for nano-TiO2, ~170 days 

for fine TiO2), more translocation to the pulmonary 

interstitium and regional lymph nodes, a greater fraction 

being retained, and a larger fraction of alveolar burden in 

the interstitium (suggesting nano-TiO2 depends mainly 

on mucocillary clearance, while fine-TiO2 depends on 

clearance to the gastrointestinal tract) than fine TiO2. 

 5-41 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Ti and S distribution in tissues: Immediately after 5-day 

inhalation/after 14-day recovery 

Short-term inhalation: 6 hr/day 

for 5 consecutive days, headnose exposure 

Rat 

Male Wistar, 

Strain Crl: 

WI(Han) 

Nano-TiO2, 20-30 nm 

(measured by TEM), 70% 

anatase, 30% rutile, BET 

surface area 48.6 m2

/g, 

uncoated, isoelectric point 

(IEP) was pH 7 in 10 mM 

KCl, MMAD 1.0 μm in 

aerosol 

van Ravenzwaay 

et al. (2009, 

193689

Fine TiO2, median size 

200 nm in ethanol 

(measured by DLS), rutile, 

BET surface area 6 m2

/g, 

IEP <pH 3 in 10 mM KCl 

(Kronos International), 

MMAD 1.1 μm in aerosol 

Quartz dust DQ12, 

median size 315 nm in 

ethanol, BET surface area 

5.9 m2

/g, IEP <pH 3 in 

10 mM KCl (Doerentrup 

Quarz GmbH, Germany), 

MMAD 1.2 μm in aerosol 

Aerosol concentration (mg/m3

): 

Nano-TiO2: target 100 

(measured concentration 

88.0 ± 6.4) 

Fine TiO2: 250 (measured 

274.0 ± 30.5) 

Quartz dust DQ12: 100 

(measured 96.0 ± 5.4). 

Count concentration of particles 

<100 nm (particles/cm3

): 

Nano-TiO2: 205,920 

Fine TiO2: 54.600 

Quartz dust DQ12: 21.292 

Calculated mass fraction 

measured <100 nm: 

Nano-TiO2: 0.5% 

Fine TiO2: 0.05% 

Quartz dust DQ12: 0.03% 

For distribution of the tested 

substance in the body, the 

following tissues were tested 

immediately after the last 

exposure and after 14-day 

recovery: lung, mediastinal 

lymph nodes, liver, kidney, 

spleen and basal brain with 

olfactory bulb (3 rats/group/time 

point) 

BAL at 3 or 14 days after the 

last exposure (5 rats/group/time 

point) 

Histological examination 

(6 rats/group/time point) and 

TEM of lung and mediastinal 

lymph nodes (3 rats/group/time 

point): immediately after the 

exposure and after 14-day 

recovery 

Nano-TiO2: 2,025/1,547 μg TiO2 in lung, 2.2/8.5 μg TiO2

in mediastinal lymph nodes. 

) 

Fine TiO2: 9,182/7,257 μg TiO2 in lung, 8.2/108 μg TiO2

in mediastinal lymph nodes. 

Quartz DQ 12: 2,190/1,975 μg quartz in lung, 19/56 μg 

quartz in mediastinal lymph nodes. 

No TiO2 or quartz were detected in any groups in liver, 

kidney, spleen, or basal brain with olfactory bulb 

(detection limits: 0.3 μg Ti = 0.5 μg TiO2 per tissue, 5 μg 

Si = 11 μg SiO2 per tissue). 

Deposition of inhaled fine and nano-TiO2 in lung: 

Fine and nano-TiO2 were mainly in the lumen of the 

alveoli and bronchi (extracellular) and some were in the 

cytoplasm of alveolar macrophages. 

Nano-TiO2 was mostly agglomerates in lung, and 

agglomerates were roughly the same size as those in 

the atmosphere. No sign of disagglomeration of the 

inhaled agglomerates. 

Biological effects of fine TiO2, nano-TiO2 and quartz: 

All treated groups: 

BAL had increased total cell count (most increases in 

polymophonuclear neutrophils, slight increases in 

lymphocytes and monocytes); increased total protein; 

increased activity lactate dehydrogenase, alkaline 

phosphatase, γ-glutamyltransferase and N-acetyl-βglucosaminidase. The changes in BAL parameters in the 

quartz group were not reversible, but changes in fine 

and nano-TiO2 groups were partly reversible by 14 days 

of recovery. 

Lung: diffuse histiocytosis 

Nano-TiO2 group: Reversible increases in absolute lung 

weight; mild neutrophilic inflammation in lung; 

inflammation declined by 14 days of recovery; 

lymphoreticulocellular hyperplasia in the mediastinal 

lymph nodes. 

Fine TiO2 group: Reversible increases in absolute lung 

weight; particle-loaded macrophages in the mediastinal 

lymph nodes. 

Quartz: Increase absolute lung weight, which maintained 

throughout recovery; multifocal infiltration of 

granulocytes in lung; after recovery time, pulmonary 

histological changes increased severity, and mediastinal 

lymph nodes had increased macrophage number and 

granulomatous inflammation. 

 5-42 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Short-term inhalation Nurkiewicz et al. 

(2008, 

Rats 

Female 

SpragueDawley, 

Hla:(SD)CVF 

Fine TiO2, primary particle 

<5 μm, 99% rutile 

(reported vendor), BET 

surface area 2.34 m2

/g 

(reported in Sager et al., 

2008, 

Histology of the lung: 

156816

157499) (SigmaAldrich, product #224227); 

MMAD of the aerosols 

402 nm with a GSD of 2.4, 

CMD of the aerosols 

710 nm 

Nano-TiO2 (P25), primary 

particle 21 nm, 80% 

anatase, 20% rutile 

(reported by vendor), BET 

surface area 48.08 m2

/g 

(reported in Sager et al., 

2008, 157499); MMAD of 

the aerosols 138 nm with 

a GSD of 2.2, CMD of the 

aerosols 100 nm 

Whole body chamber exposure 

Exposures selected which do 

not alter BAL markers of 

pulmonary inflammation or lung 

damage 

Exposure to fine TiO2: aerosol 

concentration × exposure time 

(actual deposition in lung) 

15 mg/m3

 × 480 min (90 μg) 

16mg/m3

 × 300 min (67 μg) 

12 mg/m3

 × 240 min (36 μg) 

6 mg/m3

 × 240 min (20 μg) 

3 mg/m3

 × 240 min (8 μg) 

Exposure of nano-TiO2: aerosol 

concentration × exposure time 

(calculated/actual deposition in 

lung) 

10 mg/m3

 × 720 min that took 

place over 3 days (38 μg) 

12 mg/m3

 × 240 min (19 μg) 

6 mg/m3

 × 240 min (10 μg) 

3 mg/m3 × 480 min (10 μg) 

12 mg/m3

 × 120 min (10 μg) 

3 mg/m3

 × 240 min (6 μg) 

1.5 mg/m3

 × 240 min (4 μg) 

Sham exposure (control): 

0 mg/m3

 × 240 min 

24 hr postexposure, sample 

collection, including 

exteriorizing spinotrapezius 

muscle with rats under 

anesthesia while leaving its 

nerves supply and all feed 

vessels intact for the test of 

arteriolar dilation 

No significant inflammation. ) 

Particle accumulation in alveolar macrophage. Anuclear 

alveolar macrophages were seen in both nano-TiO2 and 

fine TiO2 exposed rats, but not in sham-exposed rats. 

Anuclear alveolar macrophages are presumed to be an 

apoptotic change. 

Endothelium-dependent arteriolar dilation as measured 

after intraluminal infusion of the Caionophore A23187 in 

exteriorized spinotrapezius muscle: 

Both fine TiO2 and nano-TiO2 exposures impaired 

arteriolar dilation in a dose-dependent manner, and 

nano-TiO2 exposure produced greater impairment than 

fine TiO2 at similar pulmonary load doses. No-effect dose 

of fine TiO2 was 8 μg (as in lung deposition), and for 

nano-TiO2 was 4 μg. 

On a mass base, nano-TiO2 was approximately one 

order of magnitude more potent than fine TiO2; on total 

particle surface area base calculated by BET surface 

area, fine TiO2 would be more potent than nano-TiO2

(the authors suspected overestimation of the total 

nano-TiO2 surface area delivered, since no 

agglomeration was considered). 

Additional nano-TiO2 exposure conditions (12 mg/m3

 × 

2 hr; 4 mg/m3

 × 6 hr; 8 mg/m3 × 3 hr) yielded the same 

level of impairment of systemic arteriolar dilation, 

suggesting the response is dependent on the exposure 

concentration (of product) × time. 

Nurkiewicz et al. 

(2009, 

Same impairment of arteriolar dilation at 67 μg fine TiO2

and 10 μg nano-TiO2more than 50% decrease compared 

to sham treated controls after Ca2+ ionophore A23187 

injection at 20 and 40 psi ejection pressures. 

Same exposure conditions as 

above (Nurkiewicz et al., 2008, 191961

156816) for endogenous 

microvascular NO production 

tests, but only three groups in 

all other tests: aerosol 

concentration × exposure time 

(actual deposition in lung) 

) 

No change in arteriolar dilation in response to sodium 

nitroprusside (NO donor) in either 67 μg fine TiO2 or 10 

μg nano-TiO2 exposed rats, indicating no change in 

vascular smooth muscle sensitivity to NO. 

Sham exposure (control): 

0 mg/m3

 × 240 min Increased ROS amount in the microvascular wall in both 

67 μg fine TiO2 and 10 μg nano-TiO2 groups at the same 

Fine TiO2: 16mg/m level as measured by ethidium bromide fluorescence. 3

 × 300 min 

(67 μg) Increased nitrotyrosine expression in 10 μg nano-TiO2

treated rats (not measured in fine TiO2 group) in lung 

(3 folds) and spinotrapezius microcirculation (4 folds), as 

compared to sham exposure, suggesting nitrosative 

injury in lung and systemic microcirculation. 

Nano-TiO2: 6 mg/m3

 × 240 min 

(10 μg) 

24 hr postexposure, sample 

collection, including and 

exteriorizing spinotrapezius 

muscle as described in 

Nurkiewicz et al. (2008, 

Decreased Ca2+ ionophore A23187-stimulated 

endogenous microvascular NO production in fine TiO2

and nano-TiO2 treated groups in a dose-dependent 

manner: Similar to sham control, the NO production was 

sensitive to nitric oxide synthase inhibition caused by 

NG-monomethyl-L-arginine. 

156816) and excising 

spinotrapezius muscles from 

separate groups of rats for 

measurement of NO, 

microvascular oxidative stress, 

and nitrotyrosine staining 

Radical scavenging (by superoxide dismutase mimetic 

2,2,6,6-tetramethylpiperidine-N-oxyl and catalase); 

inhibition of NADPH oxidase (by apocynin); and 

inhibition of myeloperoxidase (by 4-aminobenzoic 

hydrazide) all restored stimulated NO production and 

partially restored arteriolar dilation (stimulated by Ca2+

ionophore A23187) in 67 μg fine TiO2 and 10 μg 

nano-TiO2 groups. 

 5-43 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Instillations 

Chen et al. 

(2006, 

Single intratracheal instillation Gross morphology and histology of the lung: 

Emphysema-like lung injuries were seen at 0.1 and 

0.5 mg/mouse (more severe at 0.5 mg) at 3 days, 1 wk, 

and 2 wk after the instillation. 

Mouse 

Male ICR 

Nano-TiO2 (Degussa), 

rutile, highly dispersed 

and hydrophilic fumed 

nano-TiO2, diameter 

19-21 nm (average 

primary particle size 

21 nm), surface area of 

50 ± 15 m2

/g, purity 

≥ 99.5% 

090139

To avoid aggregation, the 

nano-TiO2 suspension 

was ultrasonicated before 

it was used to treat 

animals or cells; each 

sample was vortexed just 

before an aliquot was 

drawn for instillation. 

However, authors did not 

report the sizes of 

aggregates before or after 

sonication. 

0, 0.1, or 0.5 mg/mouse ) 

3 days (for hyper-acute 

response), 1 wk (acute) or 2 wk 

(chronic) of recovery before 

sacrifice 

Pulmonary changes included disruption of alveolar 

space, alveolar enlargement, proliferation of alveolar 

type II pneumocyte, increases in alveolar epithelial 

thickness, and accumulation of particle-laden 

macrophages. 

1 wk after instillation, 0.1 mg/mouse increased alveolar 

macrophage infiltration, type II pneumocyte proliferation, 

and apoptosis in macrophage and type II pneumocyte. 

Gene expression in lung 1 wk after instillation of 0, 0.1, 

and 0.5 mg/mouse: 

cDNA microarray showed up-regulation in pathways 

involved in cell cycle regulation, apoptosis, chemokines, 

and complementary cascades. 

RT-PCR showed up-regulation in plgf, chemokines 

(cxcl1, cxcl5, and ccl3), tumor necrosis factor-related 

apoptosis-inducing ligand (TRAIL) and prostaglandin E 

receptor 4. 

Western blot and ELISA showed increases in placenta 

growth factor (PIGF) protein (a prechemokine that 

regulates the expression of several chemokines, leading 

to inflammatory cascade) in cells and in serum. 

Increased primary benign tumors and malignant cancers 

in lung in all tested doses. 

Mohr et al. 

(2006, 

Rat 

Female Wistar 

(HsdCpb:WU) 

Nano-TiO2 (P25), 

photocatalytic, hydrophilic, 

80% anatase/20% rutile, 

primarily particle size 

25 nm, BET specific 

surface area 52 m2

/g 

Repeated weekly intratracheal 

instillation 097493) 

Pott and Roller 

(2005, 

Instilled doses: 

 5 instillations × 3 mg 

 5 instillations × 6 mg 

 10 instillations × 6 mg 

157790)

a

Nano-TiO2 (Degussa T805 

/P805),a

 crystal form not 

specified, coated with an 

organic silicon compound; 

21 nm; 32.5 m2

/g 

Repeated weekly intratracheal 

instillation 

High initial acute mortality, lowered dose to 0.5 mg. 

No conclusion on carcinogenicity. 

Instilled doses: 

 15 instillations × 0.5 mg 

 30 instillations × 0.5 mg 

Fine TiO2, hydrophilic, 

anatase, primary particle 

200 nm, BET specific 

surface area 9.9 m2

/g 

Repeated weekly intratracheal 

instillation 

Increased primary benign tumors and malignant cancers 

in lung in all tested doses. 

Instilled doses: 

 10 instillations × 6 mg 

 20 instillations × 6 mg 

Single intratracheal instillation 

(acute effects) 

Nemmar et al. 

(2008, 

Rat 

Male Wistar 

Nano-TiO2, rutile, primary 

particle diameter 4-6 nm, 

rod shape (synthesized in 

the lab by a soft chemistry 

technique); BET surface 

for instilled nano-TiO2 rods 

was 14.64 cm2

 for dose of 

1 mg/kg, 82.30 cm2

 for 

5 mg/kg. Aggregates 

appeared to be in a radial 

arrangement and usually 

less than 1 μm. 

Pulmonary inflammation: increases in macrophage and 

neutrophil numbers in BAL fluid at 5 mg/kg. most 

nano-TiO2 aggregates in BAL fluid were inside 

macrophages. 

157514) 

1 or 5 mg/kg nano-TiO2 or 

vehicle only (150 μL) 

Pulmonary and cardiac edema: increases in the wet 

weight-to-dry weight ratios of lung and of heart at 1 and 

5 mg/kg. 

Single intratracheal instillation 

nano-TiO2 was suspended in 

saline containing 0.01% Tween 

80 (a surfactant and emulsifier) Systemic inflammation: increases in monocyte and 

granulocyte (but not lymphocyte) numbers in blood at Blood collection and necropsy 5 mg/kg. at 24 hr after instillation Platelet aggregation: decreases platelet number in blood 

of rats exposed to 5 mg/kg nano-TiO2, suggesting 

platelet aggregation [in vitro supporting evidence: adding 

2 or 10 μg/mL (but not 0.4 μg/mL) nano-TiO2 directly into 

untreated rat whole blood caused platelet aggregation]. 

 5-44 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Rats 

Male F344 

Nano-TiO2, ~20 nm, 

anatase 

Anatase nano-TiO2 induced more inflammatory response 

and higher interstitial access in the lung than anatase 

fine TiO2 of the same mass dose. 

Oberdörster et 

al. (1992, 

Single intratracheal instillation 

(acute effects) 

045110 Fine TiO2, ~250 nm, 

anatase 

500 μg of either anatase 

nano-TiO2 or anatase fine TiO2 

) 

A single intratracheal instillation, 

followed by 24-hr recovery 

Single intratracheal instillation 

(acute effects) 

Nano-TiO2, ~20 nm, 

anatase (free anatase 

nano-TiO2 ) 

Free anatase nano-TiO2 and serum-exposed anatase 

nano-TiO2 caused pulmonary inflammatory reaction 

(same level) and interstitial distribution. Free anatase nano-TiO2, 

104 μg Phagocytized anatase nano-TiO2 alone did not 

contribute significantly to inflammatory reaction, because 

the reaction can be explained by the alveolar 

macrophages and polymorphonuclear neutrophils. 

Alveolar macrophage 

collected 24 hr after 

donor-rat received 200 μg 

anatase nano-TiO2 via 

intratracheal instillation 

(containing phagocytized 

anatase nano-TiO2) 

Phagocytized anatase 

nano-TiO2 104 μg + 9.5 × 106 

alveolar macrophages + 3.9 × 

106 polymorphonuclear 

neutrophils 

Phagocytized anatase nano-TiO2 showed less interstitial 

distribution than free anatase nano-TiO2. 

Alveolar macrophages 6.8 × 

106 Alveolar macrophage 

collected from untreated 

rat lung Polymorphonuclear neutrophils 

2.2 × 106 PMNs from peripheral 

blood of untreated rats Serum-exposed anatase 

nano-TiO2 100 μg Serum-exposed anatase 

nano-TiO2 (incubated in 

rat serum for 1 hr and 

then washed twice) 

A single intratracheal instillation, 

followed by 24-hr recovery 

When inflammatory response was expressed as number 

of PMN and dose was expressed as surface area for 

retained particles (i.e., lavagable particles), all particles 

shared the same dose-response curve, except anatase 

and rutile nano-TiO2 at high doses. 

A single intratracheal instillation 

of 500 μg each; anatase fine 

TiO2 was also tested at 1,000 

μg; anatase nano-TiO2 was also 

tested at 65, 107, 200, and 

1,000 μg 

Fine TiO2, ~250 nm, 

anatase 

Nano-TiO2, ~20 nm, 

anatase 

Fine TiO2, ~220 nm, rutile 

(from Dr. Siegal at 

Argonne National 

Laboratory, Argonne, IL) 

When inflammatory response was expressed as lavage 

protein and dose was expressed as retained particle 

surface area, all particles shared the same dose 

response curve. 

24-hr recovery 

Nano-TiO2, ~12 nm, rutile Higher fractions of nano-TiO2 (anatase and rutile 

nano-TiO2) were interstitialized (translocated into 

interstitium or epithelium cells) than other particles. 

Carbon black, ~30 nm 

(Cabot, 660R) 

 5-45 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Single Intratracheal instillation 

(acute effects) 

Rat Nano-TiO2, ~20 nm, 

surface area is estimated 

to be 10 times of surface 

area of ~250 nm TiO2 

Pulmonary inflammation (neutrophil % in lung lavage) 

was seen at 24 hr postexposure. At the same mass 

dose, nano-TiO2 induced more inflammation than fine 

TiO2. When doses are expressed as surface area, fine 

TiO2 and nano-TiO2 shared the same dose-response curve. 

Oberdörster 

(2000, 036303 [strain/stock not 

specified] 

) 

Nano-TiO2: 30, ~150, 500 μg 

Fine TiO2: ~150, 500, 2,000 μg Fine TiO2, ~250 nm 

Repeated inhalation of PTFE 

fume (5 × 105 particles/cm3

 = 

~50 μg/cm3

, 5 min/day for 

3 days) followed by a single 

intratracheal instillation of 

100 μg nano-TiO2

Nano-TiO2 Cross tolerance: nano-TiO2 induced less pulmonary 

inflammation (neutrophil % in BAL fluid) in rats that had 

adapted to PTFE fumes for previous 3 days than in rats 

that were not adapted (not exposed to PTFE fume). The 

author suggested this cross tolerance is from adaptation 

to oxidative stress. 

Polytetrafluoroethylene 

(PTFE) (Teflon) fume, 

count median diameter 

~18 nm 

Nano-TiO2, ~20 nm Inhalation of LPS followed by a 

single intratracheal instillation of 

nano-TiO2 and fine TiO2 (acute 

effects) 

LPS alone: mild pulmonary inflammation (~10% 

neutrophil in lung lavage at 24 hr postexposure). The 

treatment of LPS was to mimic an early stage of 

infection with gram negative bacteria (compromised 

host). 

Fine TiO2, ~250 nm 

Lipopolysaccharide (LPS), 

an endotoxin found in 

gram negative bacteria LPS: ~12 min exposure, ~70 

endotoxin units (estimated 

alveolar dose) 

50 μg nano-TiO2, but not fine TiO2, further increased 

inflammatory response in compromised hosts with mild 

pulmonary inflammation. Nano-TiO2 and fine TiO2: 50 μg 

Neutrophil % in rats exposed to (LPS and then 

nano-TiO2) > (LPS and then fine TiO2), LPS alone, 

nano-TiO2 alone > fine TiO2 alone, negative control. 

Within 30 min of inhalation of 

LPS or saline, intratracheal 

instillation of nano-or fine TiO2

It is unclear whether fine TiO2 at a dose that increases 

inflammatory response would further increase 

inflammatory response in compromised hosts. 

24 hr of recovery 

Rehn et al. 

(2003, 

Transient pulmonary inflammatory responses to both 

types of nano-TiO2 (mostly only at 1.2 mg dose, some at 

0.6 mg groups) (most responses returned to normal by 

day 90). 

Single intratracheal instillation 

(subchronic effects) 

Rat 

Wistar 

Nano-TiO2 (P25), 

photocatalytic, 80% 

anatase/20% rutile, 

untreated, hydrophilic 

surface, primarily particle 

size ~20 nm 

090613) 

Doses: 0, 0.15, 0.3, 0.6, or 

1.2 mg nano-TiO2 (positive 

control: 0.6 mg quartz DQ12) in 

0.2 mL saline supplemented 

with 0.25% lecithin 

P25 induced more pulmonary inflammatory responses 

than T805 in some tests, but T805 induced more 

proliferation changes in the lung (as percentage of Ki67-

positive cells) than P25 on days 3 and 21. 

Nano-TiO2 (Aeroxide® 

T805), photostable, 80% 

anatase/20% rutile, 

silanized, 

trimethoxyoctylsilanetreated hydrophobic 

surface, primarily particle 

size ~20 nm 

3, 21, or 90 days of recovery 

Neither P25 nor T805 increased oxidative DNA adduct 

(as 8-oxoguanine) in the lung on day 90. 

Quartz induced persistent inflammatory response and 

increased 8-oxoguanine on day 90. 

Crystalline silica and 

quartz particles (DQ-12) 

as positive reference 

Renwick et al. 

(2004, 

Single intratracheal instillation 

(acute effects) 

Nano-TiO2 at 500 μg (but not nano-TiO2 at 125 μg or fine 

TiO2 at either 125 or 500 μg) increased neutrophil 

number (inflammation), LDH activity (cytotoxicity), GGT 

activity (epithelial damage), total protein in 

bronchoalveolar lavage fluid (membrane permeability), 

and macrophage activity to migrate toward chemotaxin 

C5a (chemotaxis). 

Rat 

Male Wistar 

Nano-TiO2 (Degussa), 

mean diameter 29 nm, 

BET surface area 

49.78 m2

/g 

056067) 

0, 125, and 500 μg particles in 

saline 

Fine TiO2 (Tioxide Ltd), 

mean diameter 250 nm 

BET surface area 6.6 m2

/g 

24 hr of recovery before 

sacrifice 

Both nano- and fine TiO2 (at 500 μg, but not at 125 μg) 

decreased phagocytic function of macrophage. Carbon black, mean 

diameter 260.2 nm, BET 

surface are 7.9 m2

/g Carbon black caused same changes as fine TiO2, with 

the exception of increases in LDH activity at 500 μg. Ultrafine carbon black, 

mean diameter 14.3 nm, 

BET surface 253.9 m2

/g 

Ultrafine carbon black caused same changes as 

nano-TiO2, but increases in inflammation and LDH and 

GGT activities were significant at 125 μg (nano-TiO2

caused significant changes at 500 μg only). 

 5-46 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Like fine TiO2, nano-TiO2 rods and nano-TiO2 dots 

caused only transient pulmonary inflammation, and not 

significant lung toxicity. 

Warheit et al. 

(2006, 

Single intratracheal instillation 

(subchronic effects) 

Rat 

Male 

Crl:CD(SD)IGS 

BR 

Fine TiO2 (DuPont): 

primary particle ~300 nm, 

anatase, ~99 wt % 

TiO2/~1 wt % alumina, 

BET surface area ~6 m2

/g 

(R-100) 

088436) 

0, 1 or 5 mg/kg of each testing 

material in PBS with polytron 

dispersant 

All 5 mg/kg TiO2 (fine, nano rods, and nano dots), but not 

1 mg/kg TiO2, caused transient, short-lived inflammation 

(increases in neutrophil % in BAL fluid at 24 hr 

postexposure only; increases in LDH by 5 mg/kg 

nano-TiO2 rods at 24 hr postexposure only). 

BAL fluid analysis at 24 hr, 

1 wk, 1 mo, and 3 mo 

postexposure (5 rats per group 

per dose per time point) 

Nano-TiO2 rods 

(synthesized 

hydrothermally): primary 

particle length 92-233 nm 

× width 20-35 nm, 

anatase, BET surface 

area 26.5 m2

/g 

No changes in lung weight, tracheobronchial cell 

proliferation (measured in high dose groups only) or lung 

morphology (pathological changes). Morphological studies at the 

same time points (4 rats per 

group per high dose per time 

point; 4 rats per group per low 

dose for the first two time 

points) 

TiO2 in macrophages was seen in all three types of TiO2. 

Transient lung parenchymal cell proliferation in low and 

high fine TiO2 at 1 wk postexposure (different from 

previous studies in similar conditions). 

Nano-TiO2 dots 

(synthesized 

hydrothermally): primary 

particle diameter 

5.8-6.1 nm, sphere, 

anatase, BET surface 

area 169.4 m2

/g 

Quartz caused sustained pulmonary inflammation and 

early sign of pulmonary fibrosis. 

Sustained pulmonary inflammation (increases in 

neutrophil % in BAL fluid at 1 mg/kg at 24 hr after 

exposure, 5 mg/kg at all time points) (increases in LDH 

at 5 mg/kg at all time points) (increase in neutrophils and 

foamy alveolar macrophages). 

Quartz (Min-U-Sil quartz): 

median primary particle 

~1.5 μm (range 1 to 

3 μm), crystalline silica, 

BET surface area 4 m2

/g Prelude of fibrosis (thickening of lung tissue) (persistent 

lung parenchymal cell proliferation at 5 mg/kg at 1 mo 

and 3 mo postexposure). 

Absolute lung weight was increased at 5 mg/kg at 1 wk, 

1 mo, and 3 mo postexposure. Increased 

tracheobronchial cell proliferation at 5 mg/kg (not 

measured in low dose) at 24 hr postexposure only. 

No sustained adverse pulmonary effects for photostable 

nano-TiO2 (both types of coated rutile). 

Single intratracheal instillation 

(subchronic effects) 

Warheit et al. 

(2007, 

Rat 

Crl:CD®(SD)IG

S BR 

Nano-TiO2 (DuPont), 

photostable, rutile, coated 

with alumina, (~98% TiO2, 

~2% alumina), average 

particle size of 136 nm in 

water and average BET 

surface area of 18.2 m2

/g 

(uf-1) 

090594) 

0, 1, or 5 mg/kg Pulmonary inflammation and cytotoxic effects at highest 

exposure of photocatalytic nano-TiO2 increased 

bronchoalveolar lavage fluid LDH and BAL fluid 

microprotein concentrations. 

90-day recovery period 

Increased tracheobronchial and lung parenchymal cell 

proliferation rates at highest exposure of photocatalytic 

nano-TiO2. Nano-TiO2 (P25) (Evonik), 

photocatalytic, 80% 

anatase/20% rutile, not 

coated, average particle 

size 129.4 nm in water, 

average BET surface area 

53.0 m2

/g 

Lung inflammation/cytotoxicity/cell proliferation and 

histopathological responses: quartz > nano-TiO2 P25 

(anatase and rutile) > fine TiO2 (rutile) = nano-TiO2 uf-1 

(rutile) = nano-TiO2 uf-2 (rutile). 

Nano-TiO2 (DuPont), 

photostable, rutile, coated 

with silica and alumina 

surface coating (~88 wt % 

TiO2, ~7 wt % amorphous 

silica and ~5 wt % 

alumina), average particle 

size of ~149.4 nm in 

water, average BET 

surface area 35.7 m2

/g 

(uf-2) 

Fine TiO2 (DuPont), 

photostable, rutile, coated 

with alumina (~99% TiO2

and ~1% alumina), an 

average particle size 

382 nm in water, average 

BET surface area 5.8 m2

/g 

Quartz 

 5-47 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Wang et al. 

(2008, 

Mouse Repeated intranasal instillation 

Female 

CD1(ICR) 

Nano-TiO2 (Hangzhou 

Dayang Nanotechnology 

Co. Ltd.), rutile, 80 nm, 

measured average size 

71.4 ± 23.5 nm, purity 

>99% 

TiO2 distribution (measured after 15 instillations): first 

into olfactory bulb, and then to hippocampus. Ti 

concentrations: hippocampus, olfactory bulb 

> cerebellum, cerebral cortex > thalamus. 

157474

Fine TiO2 (Zhonglian 

Chemical Medicine Co.), 

anatase, 155 nm, 

measured average size 

155.0 ± 33.0 nm, purity 

>99% 

~500 μg TiO2 in pure water per 

mouse every other day for 2, 

10, 20, or 30 days (1, 5, 10 or 

15 instillations, respectively) 

) 

Wang et al. 

(2008, 157473

Necropsy 1 day after last 

instillation 

For translocation of TiO2 into 

brain: 6 mice per group for each 

time point. 

For effects in brain: 10 mice per 

group 

Serum biomarkers for liver function (ALT, AST, ALP), 

kidney function and cholesterol levels: No consistent 

change. Only changes were increased ALT (80-nm 

group after 1 and 5 instillations, 155-nm group after 5 

instillations), increased AST (80-nm group after 5 

instillations) and increase ALP (155-nm group after 1 

instillation). 

) 

Pathological changes in kidney: atrophy of renal 

glomerulus, infiltration and dwindling of interstitially 

inflammatory cells in the lumen of Bowman’s capsules. 

No changes in organ weight. No pathological changes in 

heart, liver, spleen, cerebral cortex or cerebellum. No 

change in proinflammatory cytokine TNF-α in serum. 

Brain: 

Oxidative stress: GSH-Px and GST activities and GSH 

levels were increased in the 80-nm group after 5 

instillations, but not in other groups or other time points. 

Malondialdehyde levels (indicator for lipid peroxidation) 

and soluble protein carbonyl content (indicator for 

protein oxidation; measured only after 15 instillations) 

were increased in both the 80- and 155-nm groups after 

15 instillations. SOD activity was decreased in 155 nm 

after 15 instillations. Catalase activity (measured only 

after 15 instillations) was increased in the 80- and 

155-nm groups. 

Pathological changes in olfactory bulb and C1A regions 

of hippocampus: Olfactory bulbs showed increased 

neuron numbers, irregular arrangement of neuron cells, 

and ultra-structural changes in both the 80- and 155-nm 

groups. CA1 region of the hippocampus showed 

enlarged and elongated pyramidal cell soma, dispersed 

arrangement and loss of neurons, fewer Nissl bodies, 

fewer mitochondria, and increased rough endoplasmic 

reticulum. 

Astrocytes may be damaged (only measured after 15 

instillations): Hippocampus had increased glial fibrillary 

acidic protein (GFAP) levels, particularly in CA4 region. 

Activity of cholinesterase (which inactivates 

acetylcholine, a neurotransmitter) was increased. Both 

changes were in the 80- and 155-nm groups. 

Neurotransmitters: Levels of glutamic acid (a 

neurotransmitter) and nitric oxide (NO, as 

neurotransmitter and from inflammatory response) were 

increased in both the 80- and 155-nm groups (measured 

only after 15 instillations). 

Cytokines: Increased THF-α and IL-1β, but not IL-6 

(155 nm after 15 instillations). 

 5-48 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Repeated intranasal instillation 

(subacute effects) 

Wang et al. 

(2007, 

Mouse 

CD-1(ICR) 

Nano-TiO2 (Hangzhou 

Dayang Nanotechnology 

Co. Ltd.), rutile, 25 nm, 

purity >99% 

No changes in water and food consumption or BW. 

157616

Nano-TiO2 (Hangzhou 

Dayang Nanotechnology 

Co. Ltd.), rutile, 80 nm, 

purity >99% 

Fine TiO2 (Zhonglian 

Chemical Medicine Co.), 

anatase, 155 nm, purity 

>99% 

10 μL of 50 mg/kg TiO2 or water 

every 2 days 

Blood and brain were collected 

from anesthetized mice after 2, 

10, 20, or 30 days 

Brain TiO2 content (measured in all brain samples): 

increased in treated mice and was highest in 25 nm 

treated group at 2 and 10 days; decreased slightly and 

was similar in all treated groups at 20 and 30 days. 

) 

Neurotransmitters (measured in 20- and 30-day brain 

samples): 

Changed in 80 nm and 155 nm TiO2-treated mice 

compared to control, but not in 25 nm TiO2-treated mice. 

All changes were after 20 days, with the exception of 

decreased dopamine in 80-nm group after 30 days. 

After 20 days: Norepinephrine was significantly 

increased in 80 and 155 nm TiO2-treated mice; 

5-hydroxytryptamine was significantly increased in 

155 nm TiO2-treated mice; homovanillic and 

5-hydroxyindole acetic acid were decreased in 80 and 

155 nm TiO2-treated mice; dopamine was decreased in 

80 nm TiO2-treated mice. 

a

According to Pott and Roller (2005, 157790): “Titanium dioxide T805 from Degussa was ordered from Sigma-Aldrich, but the supplier only offered an 

amount of at least 40 kg P805. Neither Sigma-Aldrich nor Degussa answered at all clearly when questioned insistently as to the difference between T805 

and P805. So, it is not proven that P805 is identical with T805 from Degussa.” The primary particle size and surface area in the table were from Pott and 

Roller (2005, 157790). Currently available T805 is photostable nano-TiO2 (80% anatase, 20% rutile) that has been treated with octylsilane to achieve a 

hydrophobic surface. Degussa T805 primary particle is still 21 nm, but specific surface area (BET) is 45 m2

/g (Llames, personal communication, 2008, 

157529). 

ALP – Alkaline phosphatise, a marker of type II epithelial cell toxicity (Ma-Hock IL-6 – Interleukin-6 

et al., 2009, 193534) or liver toxicity IFN-γ – interferon-gamma 

ALT – Alanine transaminase LDH – Lactate dehydrogenase, a general marker of cell injury (Ma-Hock et 

AST – Aspartate aminotransferase al., 2009, 193534) 

BAL – Bronchoalveolar lavage LPS – Lipopolysaccharide 

BET – Brunauer, Emmett, Teller method of calculating surface area MMAD – Mass median aerodynamic diameter 

CMD – Count median diameter MTP – Microsomal triglyceride 

DLS – Dynamic light scattering NADPH – Nicotinamide adenine dinucleotide phosphate 

ELISA – Enzyme-linked immunosorbent assay P25 – Aeroxide® P25 

F344 – Fischer 344 rat strain PBS – Phosphate buffered saline 

GFAP – Glial fibrillary acidic protein PIGF – Placenta growth factor 

GGT – gamma-γ–glutamyltransferase, a marker for damage to Clara and type 

II epithelial cells (Ma-Hock et al., 2009, 

PMN – Polymorphonuclear neutrophils 

193534) PTFE – Polytetrafluoroethylene 

GSD – Geometric standard deviation ROS – Reactive oxygen species 

GSH – Reduced glutathione RT-PCR – Real-time polymerase chain reaction 

GSH-Px – Glutathione peroxidase SOD – Superoxide dismutase 

GST – Glutathione-S-transferase TEM – Transmission electron microscopy 

IEP – Isoelectric point TNF-α – Tumor necrosis factor-alpha 

IL-1β – Interleukin-1 beta 

Toxicity by Other Exposure Routes 

Ocular exposure, i.v., and subcutaneous (s.c.) injection have also been investigated in 

nano-TiO2 toxicity studies (Table 5-7). Ocular exposure to sunscreen containing nano-TiO2 could 

occur accidentally when sunscreen spray and sunscreen lotion are applied. At least one brand of 

sunscreen lotion that contains nano-TiO2 is in a tear-free formula and marketed for children 

(Project on Emerging Nanotechnologies, 2007, 157648). A single ocular exposure to a photostable 

nano-TiO2 caused conjunctival redness for 1 or 2 days in rabbits (Warheit et al., 2007, 091075). 

One journal article and two professional meeting abstracts were identified on the effects of 

injected nano-TiO2 in rats and mice. In the Fabian et al. (2008, 157576) study, an intravenous 

injection of 5 mg/kg nano-TiO2 with unknown photoreactivity did not induce changes in blood tests 

diagnostic for inflammatory responses, kidney toxicity, or liver toxicity. Two meeting abstracts 

presented immunological effect studies in mice exposed to nano-TiO2 through subcutaneous and 

intravenous injections (Miller et al., 2007, 157668; Weaver, personal communication, 2008, 157467). 

Preliminary results showed that photocatalytic nano-TiO2 in suspension (Degussa W740X) appeared 

 5-49 
to have very limited inflammatory ability, and very high doses (560 mg/kg for intravenous injections 

and 5,600 mg/kg for subcutaneous injections) were needed to produce immunological effects 

(Weaver, personal communication, 2008, 157467). 

Prenatal exposure to nano-TiO2 has been reported to affect the offspring in mice in one journal 

article (Takeda et al., 2009, 193667) and one poster at a scientific meeting (Trouiller et al., 2008, 

157484). 

Table 5-7. Summary of health effects of nano-TiO2 particles in mammalian animal models: other 

(injection, ocular) route 

Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Injection 

No inflammation, kidney toxicity, or liver toxicity 

detected: no changes in concentrations of 

cytokines, enzymes and other indicators in the 

blood (total of 67 parameters) for inflammatory 

responses, kidney function, and liver function. 

A single intravenous Fabian et al. (2008, 

injection via tail vein 

Rat 

Male Wistar 

(strain 

Crl:WI(Han) 

Nano-TiO2, primary particle 

20-30 nm (measured by TEM), BET 

surface area 48.6 m2

/g, 70% 

anatase/ 30% rutile, uncoated, IEP 

was pH 7 in 10 mM KCl 

157576); van 

Ravenzwaay et al. 

(2009, Saline (control) or 5 mg/kg 

nano-TiO2 193689) 

Nano-TiO2 stock 0.5% in rat 

serum, then diluted in saline, 

injection of ~1 mL of test 

substance preparation/kg of 

rat BW 

TiO2 distribution: TiO2 concentrations 1 day after 

injection: liver > spleen >> lung > kidney. The 

time for the TiO2 concentration to return to normal 

levels were in the same sequence. Liver had the 

same TiO2 levels after 14 and 28 days. Spleen 

had slightly decreased TiO2 levels 14 and 28 days 

after injection. Lung and kidney had no elevated 

TiO2 14 days after injection. No TiO2 was detected 

in blood cells, plasma, brain or lymph nodes 

(mediastinal, mesenteric, popliteal) at any three 

time points tested (detection limit 0.3 μg Ti = 0.5 

μg TiO2 per tissue). 

Fine TiO2 (Kronos International), 

median size 200 nm in ethanol 

(measured by DLS), rutile, BET 

surface area 6 m2

/g, IEP <pH 3 in 

10 mM KCl 

Aggregates in serum are 

mostly <1,000 nm, with 

10 wt% <100 nm 

Necropsy at 1, 14, and 

28 days after the injection 

(12 rats total for 4 treatment 

groups) 

Ti concentrations were 

measured in lung, liver, 

kidney, spleen, brain, blood 

cells, plasma, and popliteal 

lymph nodes at 1, 14, and 

28 s after injection 

Mouse i.v. injections 

Balb/c 

Nano-TiO2 (Degussa W740X), 

dispersion of photocatalytic 

uncoated nano-TiO2 (80% anatase/ 

20% rutile) at 40 wt%, primary 

particle 4.7 nm, mean aggregate 

size ≤ 100 nm; (Evonik, 2007, 

Lung, liver, and spleen showed white Miller et al. (2007, 

discoloration and phagocytosis of nano-TiO2

aggregates by macrophages under light 

microscope. 

157668

157577; Llames, personal 

communication, 2008, 157528; 

Weaver, personal communication, 

2008, 157467) 

5.6 mg/mouse/day for 

2 days (total dose 

11.2 mg/mouse) 

) 

1 or 3 days of recovery 

before sacrifice 

In 6-wk-old male offspring from nano-TiO2-

exposed dams 

Mouse Takeda et al. (2009, 

Slc:ICP, 

pregnant 

female and 

male offspring 

Nano-TiO2, 25-70 nm, anatase, 

surface area 20-25 m2/g, purity 

99.9% (from Sigma-Aldrich) 

s.c. injections of 100 μL of 

1 mg/mL nano-TiO2 (i.e., 

0.1 mg nano-TiO2) into each 

time-pregnant Slc:ICP 

mouse once per day at 3, 7, 

10 and 14 days post-mating 

193667) 

Nano-TiO2 particles were seen in the testis and 

brain (olfactory bulb and the cerebral cortex – 

frontal and temporal lobes) 

Decreased daily sperm production, epididymal 

sperm motility, and the number of Sertoli cells. 

Abnormal testicular morphology (seminiferous 

tubules) 

Male offspring were 

weighed, and sacrificed at 

4 days or 6 wk of age for 

evaluation. 

Markers of apoptosis (activation of caspase-3 and 

crescent-shaped cells), occlusion of small 

vessels, and perivascular edema observed in the 

brain 

 5-50 
Animal Testing Material Treatment Conditions Summary of Major Effects Reference 

Subcutaneously injected mice: Weaver et al. 

(2007, 

Mouse 

Sex, 

strain/stock 

not specified 

Nano-TiO2 (Degussa W740X), 

dispersion of photocatalytic 

uncoated nano-TiO2 (80% anatase/ 

20% rutile) at 40 wt%, primary 

particle 4.7 nm, mean aggregate 

size ≤ 100 nm; (Evonik, 2007, 

Subcutaneous injections: 

total 0 or total 5,600 mg/kg 

over 2 days 

193713

157577; Llames, personal 

communication, 2008, 157528; 

Weaver, personal communication, 

2008, 157467) 

Intravenous injections: total 

0 or total 560 mg/kg over 

2 days 

1 or 5 days of recovery 

Day 1: No changes in any cell population in 

peripheral blood, except CD8+ T cells. 

) 

Day 5: Increases in granulocytes in circulation 

and spleen; decreases in circulating lymphocyte 

percentages; no changes in macrophage 

percentages or any cell population in draining 

lymph nodes. 

Lack of Con-A stimulated T-cell proliferation in 

lymph nodes. 

Intravenously injected mice: 

Macrophage in the marginal zone of the spleen 

white pulp contained nano-TiO2 aggregates, 

suggesting interaction between T-cells and 

nano-TiO2. 

No changes in Con-A stimulated T-cell 

proliferation. 

Ocular exposure 

Warheit et al. 

(2007, 

Acute ocular irritation Reversible conjunctival redness in the treated eye 

(normal by 24 or 48 hr after administration of 

nano-TiO2). 

Rabbit 

Male New 

Zealand 

White 

Nano-TiO2 (identified as uf-C , a 

pre-commercial version of DuPont 

Light Stabilizer 210), 79% 

anatase/21% rutile, approximately 

90 wt% TiO2, 7% alumina, and 1% 

amorphous silica, average particle 

size 140.0 ± 44 nm in water, 

average BET surface area 38.5 

m2

/g 

091075 Doses – 0 or 57 mg to one 

eye of each animal 

) 

No corneal injury evident, no clinical signs 

observed, and no BW loss occurred. Single exposure (the eye 

remained unwashed 

following treatment) 

Observation at 1, 24, 48, 

and 72 hr following 

administration of the 

nano-TiO2 

BET – Brunauer, Emmett, Teller method of calculating surface area DLS – Dynamic light scattering 

BW – Body weight IEP – Isoelectric point 

CD8 – Cluster of differentiation 8 P25 – Aeroxide® P25 

CD8 + T cell – Cytotoxic T cell with CD8 surface protein TEM – Transmission electron microscopy 

5.3.1.3. Summary of Noncarcinogenic Effects 

Some of the noncarcinogenic effects shared by conventional and nano-TiO2 were similar in the 

nature or type of the effects, but differed in dose-response. For example, pulmonary inflammation in 

laboratory animals and overload in rats were observed after respiratory tract exposures to either 

conventional TiO2 or nano-TiO2, and nano-TiO2 often caused more severe or more persistent 

responses than conventional TiO2 at the same mass concentrations/doses. Systemic effects were also 

observed: increased inflammatory cell numbers and decreased platelet numbers in the blood, renal 

pathology, potential hepatic toxicity, and changes in the brain morphology and neurotransmitters. 

Except for the effects in the brain, the aforementioned effects outside the lung have been reported 

only once and have not been confirmed by other laboratories. While topically applied photostable 

nano-TiO2 is not expected to cause adverse effects in healthy skin, data are lacking on the effects in 

healthy flexed human skin and damaged human skin. 

5.3.2. Carcinogenic Effects 

The carcinogenicity of TiO2 to humans has been reviewed by various international health 

organizations and workplace regulatory agencies. Currently, TiO2 (including nano-TiO2, but not 

considered separately) is classified as “possibly carcinogenic to humans” (Group 2B) by the 

International Agency for Research on Cancer (IARC) (Baan, 2007, 157717; IARC, 2010, 157762) 

and as “carcinogenic” (Class D2A) by the Workplace Hazardous Materials Information System 

(WHMIS), a program administered by the Canadian Centre for Occupational Health and Safety 

(CCOHS) (2006, 157774). 

 5-51 
In a 2005 NIOSH draft evaluation, TiO2 was not designated as a “potential occupational 

carcinogen,” due to insufficient evidence (NIOSH, 2005, 196072). For nano-TiO2, NIOSH expressed 

concern in the 2005 draft about the potential carcinogenicity of ultrafine TiO2 (primary particle 

<0.1 μm) if exposure levels were at the current mass-based occupational limits of 1.5 mg/m3

 for 

respirable dust or 15 mg/m3

 for total dust, and recommended controlling exposure to as low as 

feasible below the recommended exposure limit (NIOSH, 2005, 196072). Based on an assessment of 

the lung tumor response in the rat and supported by consideration of the other pulmonary effects of 

TiO2, NIOSH draft recommended exposure limits are 1.5 mg/m3 for fine TiO2 (primary particle 

<10 μm)1

 and 0.1 mg/m3 for ultrafine (nano) TiO2. As mentioned in Section 4.2.2, these numbers 

were derived from converting particle mass to surface area dose (6.68 m2

/g for fine TiO2 and 48 m2/g 

for ultrafine TiO2), and the risk estimates will vary for other particle sizes and surface areas (pages 

60-61 of NIOSH, 2005, 196072), as well as crystal form and other factors not considered by NIOSH 

(Section 5.1) 

This section reviews studies in humans and in animals on carcinogenicity of nano-TiO2 and 

briefly discusses the mode of action of conventional TiO2 and nano-TiO2 carcinogenicity. 

Conventional TiO2 has been shown to induce lung tumors through inhalation in rats at 250 mg/m3

(6 hours/day, 5 days/week for 24 months) (Lee et al., 1985, 193501; Lee et al., 1985, 067628), but 

not at 50 mg/m3

 or below (Lee et al., 1985, 193501; Lee et al., 1985, 067628; Muhle et al., 1991, 

063996). No increases in tumors were observed in mice receiving a single intratracheal instillation of 

0.5 mg of TiO2, in mice and rats fed with TiO2 in the diet at up to 5.0% daily for 103 weeks, or in 

hamsters given 3 mg of TiO2 via intratracheal instillation weekly for 15 weeks (Baan, 2007, 

157717). Similarly, epidemiological studies did not show increased lung cancer in people exposed to 

conventional TiO2 (Boffetta et al., 2001, 157891; Chen and Fayerweather, 1988, 193312; Fryzek et 

al., 2003, 157864; Ramanakumar et al., 2008, 157507; Siemiatycki, 1991, 157954). The 

carcinogenicity studies of conventional TiO2 are not discussed in detail in this document, and readers 

are referred to studies cited here and in the IARC monographs Working Group report (Baan, 2007, 

157717). 

5.3.2.1. Studies in Humans 

Several epidemiological studies of TiO2 carcinogenicity have been reported: two populationbased case-control studies (one for lung cancer (Boffetta et al., 2001, 157891) and the other for 

20 types of cancer (Siemiatycki, 1991, 157954); two retrospective cohort mortality studies (Boffetta 

et al., 2004, 157849; Fryzek et al., 2003, 157864); one mortality, morbidity, and case-control study 

(lung cancer and chronic respiratory diseases) (Chen and Fayerweather, 1988, 193312); and a casecontrol study (lung cancer) (Ramanakumar et al., 2008, 157507). Based on these studies, IARC 

(2010, 157762), the CCOHS (2006, 157774), and NIOSH (2005, 196072) concluded that the 

evidence is insufficient to conclude that TiO2 exposure increases the risk of lung cancer in human 

beings. Furthermore, none of these studies were designed for nano-TiO2 exposure, and none of them 

provided information on TiO2 particle sizes. Even if the TiO2 in these studies included some particles 

in the nanoscale range, the risks posed by nano-TiO2 (ultrafine primary particles) and the relationship 

between particle size and lung cancer risk in humans cannot be discerned from these studies. 

5.3.2.2. Animal Studies 

Carcinogenicity of nano-TiO2 was observed in three animal studies using photocatalytic 

nano-TiO2 in rodents (Borm et al., 2000, 041486; Heinrich et al., 1995, 076637; Pott and Roller, 

2005, 157790). Increased lung tumor incidences were observed in rats (Borm et al., 2000, 041486; 

 

1

 “Fine” particles in the NIOSH draft (2005, 196072) are defined as all particle sizes that are collected by respirable particle sampling, i.e., 

50% collection efficiency for particles of 4 μm, with some collection of particles up to 10 μm. 

 5-52 
Heinrich et al., 1995, 076637; Pott and Roller, 2005, 157790), but not in mice (Heinrich et al., 1995, 

076637), exposed to P25 through inhalation or intratracheal instillation. Photocatalytic nano-TiO2

given through intraperitoneal injections did not increase tumors in the abdominal cavity in rats (Pott 

et al., 1987, 029823). Intramuscular implantation of nano-TiO2 with unknown photo-reactivity also 

did not increase tumors at the sites of implantation in rats (Hansen et al., 2006, 090611). Data 

specifically on photostable nano-TiO2 carcinogenicity are inconclusive (Pott and Roller, 2005, 

157790). As mentioned in Chapter 4, because internal transport of the materials will influence the 

ultimate dose to the organism, it should be noted that multiple routes of exposure will be considered 

that may not be likely or possible primary exposure routes but that could have relevance when 

internal transport is considered (e.g., i.v., i.p., and i.m.). 

Intratracheal Instillation 

Female Wistar CRP/WU rats received fine and nano-TiO2 via intratracheal instillations, and 

the tumor incidence and pulmonary inflammation were measured 2.5 years after administration 

(Borm et al., 2000, 041486). Fine TiO2 (250 nm) was given 6 times at 10 mg each, and the 

photocatalytic nano-TiO2 (21 nm, 80% anatase, 20% rutile, uncoated, P25) was given 5 times at 

6 mg each (Borm, personal communication, 2008, 157591). At these total doses (60 mg for fine TiO2

and 30 mg for nano-TiO2), lung clearance might be expected to be severely compromised. The 

authors found evidence of alveolar and interstitial inflammation 2.5 years after instillation. The 

histologically confirmed tumor incidences were 27% for fine TiO2 and 66% for nano-TiO2, while the 

macroscopic tumor incidences were only 20.9% for fine TiO2 and 50% for nano-TiO2. In vehicletreated controls, the microscopic tumor incidences were between 5 and 6%. Although particles that 

induce high tumor incidences generally also cause high inflammatory cell counts, nano-TiO2 caused 

a high tumor incidence and low inflammatory cell counts. Borm et al. (2000, 041486) suggested that 

tumor formation was directly related to high interstitialization rather than overload and subsequent 

tissue response, similar to the premise that lung burden is correlated to surface area of the particles 

(Oberdörster et al., 1994, 046203). 

Pott and Roller (2005, 157790) reported increases in pulmonary tumors in rats exposed to 

hydrophilic fine TiO2 and hydrophilic nano-TiO2, but were unable to draw conclusions about the 

carcinogenicity of hydrophobic nano-TiO2. Female Wistar (HsdCpb:WU) rats received weekly 

intratracheal instillations of three types of TiO2: hydrophilic nano-TiO2 (P25), hydrophobic 

nano-TiO2 (Aeroxide® P805/Degussa P805; see Footnote c in Table 5-8), and hydrophilic fine TiO2

(232033 from Sigma). If the products used in the study are the same as those currently available, 

both the hydrophilic nano-TiO2 and fine TiO2 were photocatalytic and the hydrophobic nano-TiO2

was photostable. The tested TiO2 physicochemical properties, doses, and key results are listed in 

Table 5-8. The types of primary benign lung tumor were adenoma and epithelioma, and the primary 

malignant tumors were adenocarcinoma and squamous cell carcinoma. At the tested doses, 42-46 

rats out of 48 rats/group survived in the hydrophilic nano-TiO2 and hydrophilic fine TiO2 groups, and 

statistically significant increases in benign or malignant lung tumors, or both, were observed in these 

two groups. 

 5-53 
Table 5-8. Treatments and pulmonary tumor incidences in rats exposed to fine and nano-TiO2

through intratracheal instillation in Pott and Roller (2005) study 

Crystal form; 

primary 

particle size; 

specific 

surface area 

(BET) 

Dose 

(number of 

instillations 

× mg per 

instillation) 

Lungs with 

primary 

benign 

tumors (%) 

Lungs with 

primary 

malignant 

tumors (%) 

Lungs 

with 

tumors, 

total (%) 

Lungs with 

metastases 

of other 

tumors (%) 

Rats at 

start/at 

riska

Photostability Survival 

50% (wk) Treatment 

Nano-TiO2, 

hydrophilic 

(P25) 

Majority anatase; 

25 nmb

 (21 nm 

and 30 nm were 

also reported); 

52 m2

/g 

Photocatalytic 5 × 3.0 48/42 114 21.4 31.0 52.4 14.3 

5 × 6.0 48/46 114 17.4 50.0 67.4 15.2 

10 × 6.0 48/46 104 23.9 45.7 69.6 15.2 

Nano-TiO2, 

hydrophobic 

(Degussa 

P805)c

 (Sigma 

AL 900032)c

(Data of Degussa 

T805)c

 Crystal 

form not 

specified, coated 

with an organic 

silicon 

compound; 

21 nm; 32.5 m2

/g 

(Currently 

available 

Degussa 

T805 is a 

photostable 

UV filter) 

15 × 0.5 24/11 86 0.0 0.0 0.0 9.1 

30 × 0.5 48/15 114 6.7 0.0 6.7 6.7 

Fine TiO2, 

hydrophilic 

(Sigma AL 

232033) 

Anatase; 200 nm; 

9.9 m2

/g 

(Untreated 

anatase is 

photocatalytic) 

10 × 6.0 48/44 108 15.9 13.6 29.5 11.4 

20 × 6.0 48/44 113 38.6 25.0 63.6 2.3 

No treatment -- -- -- 48/46 113 0.0 0.0 0.0 13.0 

BET – Brunauer, Emmett, Teller method of calculating surface area 

P25 – Aeroxide® P25 

UV – Ultraviolet (light/radiation), wavelengths in the range of 10 to 400 nm 

a

Rats at risk were “sufficiently examined rats which survived at least 26 wk after first instillation” according to Pott and Roller (2005, 157790). 

b

Regarding particle characteristics, Pott and Roller (2005, 157790) noted “There are no clearly measured values or more than one piece of information.” The value listed in the 

table was assumed to be close to the correct value and was used for further calculations by Pott and Roller (2005, 157790). 

c

According to Pott and Roller (2005, 157790): “Titanium dioxide T805 from Degussa was ordered from Sigma-Aldrich, but the supplier only offered an amount of at least 40 kg P 

805. Neither Sigma-Aldrich nor Degussa answered at all clearly when questioned insistently as to the difference between T805 and P805. So, it is not proven that P805 is 

identical with T805 from Degussa.” The primary particle size and surface area in the table were from the Pott and Roller (2005, 157790) study. Currently available T805 is 

photostable nano-TiO2 (80% anatase, 20% rutile) that has been treated with octylsilane to achieve a hydrophobic surface. The primary particle size is still 21 nm, but the specific 

surface area (BET) is 45 m2

/g. 

Hydrophobic nano-TiO2 (Degussa P805) showed high acute mortality in the Pott and Roller 

(2005, 157790) study. Nano-TiO2 P805 was given at a much lower amount in each instillation than 

nano-TiO2 P25 and fine TiO2, because instilled P805 showed acute lethality. A single intratracheal 

instillation of P805 at 0.5, 1.0, and 1.5 mg caused death in 25%, 58%, and 92% female Wistar rats, 

respectively, within 24 hours. Pott and Roller (2005, 157790) originally ordered Degussa T805 for 

their study, and were unable to confirm that the received P805 was the same as T805. The 

physicochemical properties of T805, but not P805, were used for calculation and reported in the 

study (Pott and Roller, 2005, 157790). In contrast to the high acute toxicity of hydrophobic 

nano-TiO2 reported in the Pott and Roller (2005, 157790) study, very low toxicity of hydrophobic 

nano-TiO2 was reported in an earlier study by Rehn et al. (2003, 090613). Rehn et al. (2003, 090613) 

reported that a single intratracheal instillation of P805 at 0.15, 0.3, 0.6, or 1.2 mg caused no death in 

female Wistar rats. Furthermore, P805 induced only mild, reversible inflammatory responses in the 

lung, and was less biologically active than P25 (Rehn et al., 2003, 090613). The reasons for the 

discrepancy in the toxicity of hydrophobic nano-TiO2 (P805 versus T805 manufactured by Degussa) 

remain unclear. 

 5-54 
Inhalation 

Heinrich et al. (1995, 076637) reported increased lung tumor rates in rats (but not in mice) that 

inhaled photocatalytic nano-TiO2. Animals were exposed to P25 aerosols (18 hours/day, 

5 days/week) in whole-body exposure chambers. Generated by a dry dispersion technique, the 

nano-TiO2 aerosol had a MMAD of 0.80 μm, with a geometric standard deviation of 1.80. 

For female Naval Medical Research Institute (NMRI) mice, the nano-TiO2 exposure was 

stopped after 13.5 months and followed by clean air exposure for 9.5 months. The 13.5-month 

nano-TiO2 aerosol exposure was 4 months at 7.2 mg/m3

, 4 months at 14.8 mg/m3, and 5.5 months at 

9.4 mg/m3

. Although nano-TiO2 exposures decreased lifespan in mice (50% mortality at 17 months 

after birth, compared to 20 months in controls), the exposures did not increase lung tumor incidence 

at the end of the study (13.8% in nano-TiO2 exposed, compared to 30% in controls). Even though the 

reported spontaneous lung tumor rate seemed to be higher than historical data (20.7% in the natural 

lifespan of female NMRI mice (Lohrke et al., 1984, 157978); 12% broncho-alveolar lung adenoma 

and 10% bronchiolo-alveolar lung carcinoma in female Han:NMRI mice up to 104 weeks old 

(Rittinghausen et al., 1997, 157924), 13.8% would not be considered as an increase even compared 

to historical controls. 

For female Wistar rats, the nano-TiO2 exposure was stopped after 24 months, and followed by 

clean air exposure for 6 months. The 24-month nano-TiO2 aerosol exposure consisted of 4 months at 

7.2 mg/m3

, 4 months at 14.8 mg/m3, and 16 months at 9.4 mg/m3. At the end of the 30-month study, 

32 of 100 nano-TiO2-exposed rats had benign or malignant lung tumors (20 benign squamous cell 

tumors, 13 adenocarcinoma, 4 adenoma, and 2 squamous cell carcinoma), while only 1 of 217 

control rats had lung adenocarcinoma (Heinrich et al., 1995, 076637). The lung particle loading was 

23.2 mg/lung after 6 months, and 39.2 mg/lung after 24 months (Gallagher et al., 1994, 045102). The 

exposure to nano-TiO2 did not increase the levels of DNA adducts in the lung (Gallagher et al., 1994, 

045102). This study showed that inhaled photocatalytic nano-TiO2 is a lung carcinogen in female 

rats, but no dose-response relationship can be calculated due to the dosing design. In a parallel study, 

decreased pulmonary clearance (overload) was clearly demonstrated (Creutzenberg et al., 1990, 

157963). 

The aerosol concentrations used in the Heinrich et al. (1995, 076637) study, ranging from 

7.2 mg/m3

 to 14.8 mg/m3, are occupationally relevant. For example, the OSHA PEL (Occupational 

Safety and Health Administration permissible exposure limit) is 15 mg/m3

 and the ACGIH TLV 

(American Conference of Governmental Industrial Hygienists threshold limit value) is 10 mg/m3

. 

Intraperitoneal Injection 

Pott et al. (1987, 029823) intraperitoneally injected Wistar and Sprague-Dawley rats with 

photocatalytic nano-TiO2 (P25)1

 and examined abdominal cavities for tumors. The treatment doses 

ranged from a single intraperitoneal injection of 5 mg nano-TiO2 to 5 injections of 20 mg nano-TiO2

(for a total of 100-mg nano-TiO2) over 5 weeks (Table 5-9). Tumor incidences were based on rats 

with sarcoma, mesothelioma, or carcinoma in the abdominal cavity. Rats with uterine tumors were 

excluded from the rats-with-tumor count, because 5-10% of the controls had malignant tumors of the 

uterus and some with metastases. Tumor incidences in the abdominal cavity in nano-TiO2-treated 

rats ranged from 0% to 10% in the 5 experiments using nano-TiO2 (Table 5-9). Although controls 

were not available in all experiments, Pott et al. (1987, 029823) concluded there were no increases in 

tumor incidence (in the abdominal cavity) in nano-TiO2 treated rats. 

 

1

 Data from Pott et al. (1987, 029823) reported the P25 as anatase and did not specify particle size in the 1987 publication. Currently 

available P25 is 80% anatase and 20% rutile (primary particle size approximately 21 nm), and a representative of Degussa stated that the 

company has never changed the formula since Degussa P25 was introduced to the market (Clancy, personal communication, 2008, 

193844). 

 5-55 
Table 5-9. Incidence of tumor in the abdominal cavity of rats intraperitoneally injected with 

photocatalytic nano-TiO2. 

Animal, age at the 

beginning of the 

experiment 

Rats with sarcoma, mesothelioma, or 

carcinoma, other than uterine tumors, in the 

abdominal cavity (percentage) 

Nano-TiO2 treatment 

Rats sacrificed when in bad health or 2.5 yr after treatment 

Wistar rat, i.p. injection of 18 mg/rat, once per wk for 5 wk (total dose 

90 mg/rat) 

6 of 113 rats examined (5.3%) 

9-wk old 

Sprague-Dawley rats, i.p. injection of 5 mg/rat 2 of 52 rats examined (3.8%) 

8-wk old 

Wistar rats, i.p. injection of 5 mg/rat 0 of 47 rats examined (0%) 

4-wk old 

Wistar rats, i.p. injections of 2, 4, and then 4 mg/rat (total dose 10 mg/rat) 0 of 32 rats examined (0%) 

5-wk old 

Preliminary results at 28 mo after i.p. injection 

Wistar rats, i.p. injection of 20 mg/rat, once per wk for 5 wk (total dose 

100 mg/rat) 

5 of 53 rats (36 rats examined and 17 rats survived) 

(9.4%) 8-wk old 

i.p. – intraperitoneal 

Source: Data from Pott et al. (1987, 029823). 

Intramuscular Implantation 

No tumors were observed in rats receiving implantations of either conventional TiO2 or 

nano-TiO2 for up to 12 months (Hansen et al., 2006, 090611). Each of the 10 male Sprague-Dawley 

rats was surgically implanted with conventional TiO2 (a 9-mm × 2-mm disk containing 100% rutile) 

subcutaneously on the left side, and with nano-TiO2 (20-160 nm, mean size 70 nm, 90% anatase and 

10% rutile) intramuscularly on the right side of paravertebral muscle. The implanted doses were one 

disk of conventional TiO2 and 0.1 mL nano-TiO2. Four rats were sacrificed after 6 months, and the 

remaining six were sacrificed after 12 months. Inflammation (but not granuloma) was observed at 

the site of conventional TiO2 implantation, and granuloma (localized nodular inflammation; 

noncancerous inflammation) was observed at the site of nano-TiO2 implantation at both 6 and 

12 months. No tumors were observed at either time. 

5.3.2.3. Modes of Action for Carcinogenicity 

The mode of action of lung cancer induced by poorly soluble particles with no specific toxicity 

is believed to be particle deposition in respiratory epithelium, decreased lung clearance (to the 

degree of overload), persistent inflammation, cellular injury and persistent cell proliferation, fibrosis, 

and secondary genotoxicity (mutation) in the lung cells. TiO2 is traditionally considered chemically 

inert and falls into the category of poorly soluble particles with no specific toxicity. When doseresponse is expressed as surface area (dose) to tumor proportion (response), TiO2, nano-TiO2, and 

other poorly soluble particles with no specific toxicity appear to share the same dose-response curve1

(Dankovic et al., 2007, 157704). 

With the exception of mutation, all the events described in the previous paragraph (Baan et al., 

2006, 186864; Muhle and Mangelsdorf, 2003, 157859) have been reported in rats exposed to both 

 

1

 Because the nano-TiO2 data used in this dose-response curve were from studies using the same photocatalytic nano-TiO2 product, this 

dose-response curve might not be applicable to nano-TiO2 with a different crystalline type/ratio, purity, shape, surface treatment, or some 

other characteristic. Although such factors are known to affect nano-TiO2 toxicity, their role in carcinogenicity remains unknown. 

 5-56 
fine TiO2 and photocatalytic nano-TiO2 through inhalation or instillation (Borm et al., 2000, 041486; 

Heinrich et al., 1995, 076637; Hext et al., 2002, 157878; Pott and Roller, 2005, 157790). Figure 5-1 

illustrates that, at low or medium exposure levels, lungs with normal clearance show inflammation 

that diminishes over time after exposure ceases. When the exposure level is high enough to decrease 

clearance, rats show persistent pulmonary inflammatory responses (even after exposure ends), cell 

proliferation and fibrosis, and eventually tumors. In mice, when the exposure is high enough to cause 

decreases in clearance, pulmonary inflammatory responses gradually decrease after the exposure 

ceases and no persistent pathological changes or tumors are observed in the lung. In hamsters, no 

overload has been observed and therefore no prediction of the outcome of overload in hamsters is 

presented here. 

Increased mutation frequency in hypoxanthine-guanine phosphoribosyl transferase (hprt) was 

seen in type II alveolar cells isolated from rats exposed to 100 mg/kg fine TiO2 through intratracheal 

instillation (Driscoll et al., 1997, 053253). No studies that investigated mutations in lungs of rats 

exposed to nano-TiO2 are available. In vitro studies also support the mode of action stated above. 

Both macrophage- and neutrophil-enriched BAL cell populations from rats exposed to high 

concentrations of fine TiO2 showed increased mutations in cultured cells (rat alveolar type II 

epithelial cell line; RLE-TN) in vitro (Driscoll et al., 1997, 053253). Because catalase, an enzyme 

that catalyzes the decomposition of hydrogen peroxide to water and oxygen, decreased BAL-cellinduced mutation in RLE-TN cells, ROS released from inflammatory cells could contribute to 

secondary genotoxicity and eventually to the carcinogenicity of TiO2 (Driscoll et al., 1997, 053253). 

This sequence of events, however, does not appear to occur in mice. At an inhalation dose that 

causes overload, nano-TiO2 does not appear to increase lung tumors in mice. More specifically, 

overload occurs in mice at an inhalation concentration of 10 mg/m3

 nano-TiO2 (P25), based on the 

increase of clearance half-life of nano-TiO2 from 40 days at 2 mg/m3

 to 395 days at 10 mg/m3, after 

13 weeks (6 hours/day, 5 days/week) of exposure (Hext et al., 2002, 157878). After 13.5 months of 

inhalation exposure to the same type of nano-TiO2 (P25) at approximately 10 mg/m3

 (including 

4 months of exposure at 14.8 mg/m3

), mice showed no increased lung tumors over the 2-year study 

period (Heinrich et al., 1995, 076637). 

Although the evidence available to date for nano-TiO2 carcinogenesis is consistent with the 

mode of action of other poorly soluble particles and suggests that particle overload is a sufficient 

condition for nano-TiO2 to induce lung cancer, this does not definitively establish that particle 

overload is a necessary condition for nano-TiO2-induced lung cancer. For example, it has been 

suggested that nano-TiO2-induced lung tumors are directly related to high interstitialization1

 rather 

than overload (Borm et al., 2000, 041486). Given the paucity of nano-TiO2 cancer studies and the 

lack of consensus on exposure-dose metrics, the question arises whether there may be other effects 

or modes of action unique to nano-TiO2 or nanomaterials in general that are yet to be found. 

 

1

 High interstitialization can be a part of the process of particle overload, but high interstitialization may not lead to overload. 

 5-57 
Deposition in respiratory tract

TiO2 or nano-TiO2 in air

Normal clearance Decreased clearance (overload)

Mice

Cellular injury, persistent cell 

proliferation, fibrosis

Rats

Lung tumors

Mutation

Inflammation diminishing over 

time post-exposure

Persistent inflammation

Deposition in respiratory tract

TiO2 or nano-TiO2 in air

Normal clearance Decreased clearance (overload)

Mice

Cellular injury, persistent cell 

proliferation, fibrosis

Rats

Lung tumors

Mutation

Inflammation diminishing over 

time post-exposure

Persistent inflammation

Figure 5-1. The pulmonary effects of TiO2 or nano-TiO2 exposure through 

inhalation or instillation. 

Although the carcinogenicity of TiO2 and nano-TiO2 in rats at high doses has been shown in 

inhalation and instillation studies, the relevance of this rat-specific response to human health is under 

debate. Rats have been suspected to be more sensitive to poorly soluble particle-induced lung cancer 

because they are more prone to pulmonary inflammation (Muhle and Mangelsdorf, 2003, 157859). 

Furthermore, lung tumors induced by poorly soluble low-toxicity particles are limited to rats with 

severely compromised particle clearance in lung (overload) (Hext et al., 2005, 090567). In human 

exposures, people working in dusty environments, such as coal miners, could encounter high 

concentrations of particles and may have impaired lung clearance (Baan et al., 2006, 186864). Coal 

miners, however, are likely to be exposed to a mixture of particles (i.e., not limited to poorly soluble 

low-toxicity particles). Evidence of persistent or chronic inflammation in humans exposed to TiO2 is 

suggested only by case studies of workers exposed to TiO2 and other minerals (Keller et al., 1995, 

157938; Moran et al., 1991, 157956; Yamadori et al., 1986, 193728). 

5.3.2.4. Summary of Carcinogenic Effects 

The results of nano-TiO2 carcinogenicity studies in animals are summarized in Table 5-10. No 

data are available for nano-TiO2 carcinogenicity in humans or for photostable nano-TiO2 in animals. 

TiO2 (not specific to nano-TiO2) was classified as “possibly carcinogenic to humans” (Group 2B) by 

an IARC monographs Work Group in 2006 (Baan, 2007, 157717), and “carcinogenic” (Class D2A) 

by WHMIS (CCOHS, 2006, 157774). NIOSH (2005, 196072) proposed not designating TiO2 as a 

“potential occupational carcinogen” because of insufficient evidence, but expressed concern about 

 5-58 
 5-59 

the potential carcinogenicity of ultrafine TiO2 (nano-TiO2) at the current exposure limits. Based on 

calculated lung cancer risks, the draft NIOSH recommendation was for an exposure limit of 

0.1 mg/m3

 for ultrafine TiO2 and 1.5 mg/m3 for fine TiO2 (less than 2.5 μm), as time-weighted 

average concentrations. The relevance of rat-specific nano-TiO2 carcinogenicity to human health 

remains to be elucidated. 

Table 5-10. Results of nano-TiO2 carcinogenicity studies in animals 

Exposure route Species Result Lowest effective dose 

(highest ineffective dose) References 

Photocatalytic nano-TiO2 

Intratracheal 

instillation 

Wistar rats, female Increased lung tumors (benign 

and malignant) 

5 instillations at 6.0 mg/instillation Borm et al. (2000, 

041486) 

5 instillations at 3.0 mg/instillation Pott and Roller (2005, 

157790) 

Inhalation Wistar rats, female Increased lung tumors Approximately 12 mg/m3 for 24 moa Heinrich et al. (1995, 

076637) 

NMRI mice, female No increases in lung tumors (Approximately 10 mg/m3 for 13.5 mo)b Heinrich et al. (1995, 

076637) 

Intraperitoneal 

injection 

Wistar and 

Sprague-Dawley 

rats 

No increase in abdominal tumors (5 intraperitoneal injections at 18 mg/rat per 

injection) 

Pott et al. (1987, 

029823) 

Nano-TiO2 with unspecified photoreactivityc 

Intratracheal 

instillation 

Wistar rats, female No conclusiond (30 instillations at 0.5 mg/instillation) Pott and Roller (2005, 

157790) 

Intramuscular 

implantation 

Sprague-Dawley 

rats, male 

No increases in tumor at 

implantation sites 

(not specified) Hansen et al. (2006, 

090611) 

NMRI = Naval Medical Research Institute 

a

7.2 mg/m3 for 4 mo, followed by 14.8 mg/m3 for 4 mo and then 9.4 mg/m3 for 16 mo 

b

7.2 mg/m3 for 4 mo, followed by 14.8 mg/m3 for 4 mo and then 9.4 mg/m3 for 5.5 mo 

c

Nano-TiO2 particles not specified or have questionable identification 

d

Unexpected high acute toxicity; problem with ascertaining the identity of testing material 
Chapter 6. Summary 

This chapter briefly summarizes information from the preceding chapters, describing case 

studies of nano-TiO2 for arsenic removal in drinking water treatment and for topical sunscreens. It 

also highlights information gaps and research questions identified in these case studies as they might 

relate to future risk assessment efforts. 

The case studies were developed using the CEA framework, which, as described in Chapter 1, 

combines a product life-cycle perspective with the risk assessment paradigm to provide a more 

holistic examination of a material’s potential environmental impacts. However, the goal of this 

document is not to provide an actual CEA or to state conclusions regarding possible ecological or 

health risks related to nano-TiO2, but to provide a foundation for a process to identify and prioritize 

research directions to support future efforts, and, eventually, provide input to policy and regulatory 

decision-making. 

Given that the CEA is both a framework and a process, these case studies were used as a 

starting point for a formal collective judgement method known as a “nominal group technique” 

(NGT) to identify and prioritize research questions related to nano-TiO2. The NGT process was 

conducted at a workshop held on September 29-30, 2009, and a summary report is available that 

describes the workshop and summarizes the collective prioritization results (U.S. EPA, 2010, 

625483). A brief description of this process is provided in Section 6.2.1. Section 6.2.2 considers the 

information in the case studies in light of research themes identified in the U.S. EPA’s Nanomaterial 

Research Strategy Document (U.S.EPA, 2009, 625484) and discusses the potential for using this 

information together with collective priority ranking results to inform refined nanomaterial research 

strategies, not only within EPA but in the broader scientific community. Section 6.2.3 discusses ways 

in which information from the case studies, collective prioritization results, and emerging research 

can be integrated to support future assessment efforts for nanomaterials. 

6.1. Case Study Highlights 

This section summarizes what is known, as well as what is unknown, regarding life cycle 

stages (feedstocks, manufacturing, distribution, storage, use, and disposal/recycling), fate and 

transport in the environment, exposure and dose characterization for biota and humans, and 

ecological and health effects of two uses of nano-TiO2: (1) for arsenic removal from drinking water; 

and (2) in topical sunscreens. For each topic area, readers are referred back to the specific sections of 

the document where detailed discussion of the evidence and associated references are presented. In 

some cases, the findings and research questions are specific to nano-TiO2, while in others, they 

pertain to nanomaterials in general. These issues may be immediately useful to scientists engaged in 

ongoing nanomaterial research, as well as for those involved in shaping future research and 

assessment efforts. 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments. 

 6-1 
6.1.1. Analytical Methods 

Sensitive and accurate analytical methods for physicochemical characterization of 

nanomaterials are critical tools for CEA (Section 1.6). Measurement and characterization of 

nanomaterials, alone and in various media, are required for properly monitoring fate and transport, 

assessing exposure, conducting toxicological studies, estimating dose-response relationships, and 

understanding the behavior and effects of nanomaterials. Minimum characterization requirements for 

toxicological studies have been recommended to facilitate interpretation and comparison of studies. 

Physicochemical characterization of nano-TiO2, as with other nanomaterials, is extremely important 

at each stage of its life cycle, because these properties change depending on characteristics of the 

surrounding matrix (e.g., pH, ionic strength, presence of natural organic matter or large 

biomolecules, soil or sediment composition). In conducting laboratory studies, researchers must 

account for changes in the properties of the raw material from the point of production during their 

transport, storage, and preparation for testing. These physicochemical properties are also likely to 

change when nanomaterials enter the environment, making it difficult to predict their behavior based 

on laboratory studies. Additional challenges of characterizing nano-TiO2 in the environment include 

low expected concentrations and the difficulty of distinguishing naturally occurring materials in the 

nanoscale size range from manufactured nanomaterials. As a further complication, the critical 

properties or suite of properties that have the most influence on environmental behavior, exposure, 

and ecological and health effects are unknown, necessitating the use of multiple methods to 

characterize nanomaterials. This need for complex characterization distinguishes nano-TiO2 and 

other nanomaterials from chemical compounds typically studied in environmental assessments, 

which often may be characterized by mass concentration alone and maintain their chemical structure 

in different matrices. 

Properties of nano-TiO2 and other nanomaterials include particle size and size distribution, 

mass, surface area, particle number, crystal structure, chemical composition, and zeta potential. 

Concomitant measurements of relevant properties of liquid or solid media are also necessary for 

characterizing nanomaterials in those matrices, such as pH, salinity, or the presence of organic 

matter. Methods developed for measurements of aerosol properties are available for measuring 

nanomaterials in the air, including the condensation particle counter (CPC) for measuring particle 

number; the scanning mobility particle sizer (SMPS) and electrical low pressure impactor (ELPI) for 

measuring number, surface area, and mass; the optical particle counter (OPC) for number; direct 

number counts and size distribution by electron microscopy, which can be combined with EDS for 

elemental composition; and size-selective personal and static samplers for mass measurements of 

different size fractions. The BET method of gas adsorption to determine particle surface area has 

also been useful in characterizing nano-TiO2 and other nanomaterials. 

Liquid-phase techniques for particle size, as presented in Table 1-3 in Section 1.6.1, include 

dynamic light scattering (DLS) and static light scattering, size exclusion chromatography (SEC), 

acoustic techniques, SPM, and centrifugal sedimentation. Some of these techniques can also provide 

information on number and zeta potential (e.g., DLS) and mass or density (e.g., centrifugal 

sedimentation). Crystal structure has been measured by HRTEM and XRD. Field flow fractionation 

(FFF) to determine particle size has been combined with inductively coupled plasma atomic 

emission spectroscopy (ICP-AES) for detection and characterization of nano-TiO2 in commercial 

sunscreen, providing information on mass, size distribution, and Ti content of extracted nano-TiO2. 

Specific applications of other techniques to nano-TiO2 in water treatment processes and sunscreens 

have not been identified. 

Methods for characterizing nanomaterials in soil and sediment would ideally be performed in 

situ to avoid changes in nanoparticle properties caused by sample handling, but this requires portable 

instrumentation and techniques that can operate in complex environmental media. Therefore, liquidphase techniques are used following sample extraction. As presented in Table 1-4 in Section 1.6.2, 

 6-2 
these include centrifugation, ultrafiltration, FFF, and SEC for size fractionation; electron microscopy 

and DLS for size distribution; the BET method for surface area, and spectroscopic techniques for 

phase and crystalline structure. As with nanomaterials suspended in gas and liquid phases, the use of 

multiple techniques is recommended to provide more complete characterization. 

Although characterization of nanomaterials is recognized to be a critically important aspect of 

evaluating their fate and effects, no consensus has been reached on a critical set of properties. 

Remaining questions include standardization of methodology and terminology, the potential need for 

improvements to existing methods or entirely new methods, the influence of coatings and other 

formula components on nano-TiO2 properties and behavior in sunscreen, information on 

agglomeration of particles in drinking water treatment processes and sunscreens, and correlation 

among parameters measured in different phases. These questions were reflected in the collective 

prioritization results from the workshop (U.S. EPA, 2010, 625483); three of the four highest-priority 

topics were directly related to characterization, including the need for improved physico-chemical 

characterization throughout life cycle stages, method development and evaluation, and productspecific characterization needs. Research to address these questions may be an important part of 

future investigations into the ecological and health effects of nano-TiO2 and other nanomaterials. 

6.1.2. Life Cycle Characterization 

Feedstocks used in TiO2 production include ilmenite and rutile ores, with ilmenite accounting 

for approximately 90% of worldwide production of Ti minerals (Section 2.1). The presence of low 

levels of radioactive materials in these ores raises the question of the potential for risk associated 

with ore mining and processing. It is not clear at this time whether certain feedstocks are more 

suitable and widely used for production of nano-TiO2 compared to conventional TiO2. Information is 

also lacking on the nature and magnitude of contaminant release associated with mining and 

processing of Ti-containing ores. The production quantity of conventional TiO2 for use in pigments 

and other applications is far larger than production of nano-TiO2, with 2005 production of 

conventional TiO2 and nano-TiO2 estimated at 4.5 million metric tons and 2,000 metric tons, 

respectively. The production volume of nano-TiO2 is expected to grow rapidly over the next few 

decades, with widely varying estimates of the rate of growth. 

A variety of techniques are available for commercial production of nano-TiO2, many of which 

are adapted from manufacturing processes for conventional TiO2 (Section 2.2). These include CVD, 

flame hydrolysis, sol-gel, calcination, aerosol pyrolysis, and colloidal synthesis. One commercially 

available product (P25) is produced by flame hydrolysis using TiCl4 as a feedstock, resulting in 

agglomerated TiO2 particles with a mean diameter of approximately 3,600 nm, with the smallest 4% 

of particles having an average diameter of 160 nm (Section 2.2). This gas-phase chloride method 

generates hydrogen chloride as a by-product, with the potential for some residual chloride ions 

adsorbed onto the TiO2 particles. Specific information was not identified on processes for preparing 

or formulating nano-TiO2 for use in arsenic removal from drinking water, although information is 

available on nano-TiO2 formulations used in sunscreens. Rutile is a more photostable crystalline 

form of TiO2 than anatase, which should make it preferable for use in sunscreen applications, 

although anatase/rutile mixes are also common. The potential for photocatalytic action of nano-TiO2

is mitigated by surface coatings applied to the particles, such as silica, alumina, dimethicone, or 

other compounds; these coatings also improve particle behavior in formulation of sunscreen 

dispersions. Other components of these dispersions include emulsifiers, emollients, other physical or 

chemical UV blockers, and ingredients to improve characteristics such as spreadability, water 

resistance, and viscosity. Among the questions regarding the manufacturing component of the life 

cycles for these nano-TiO2 applications are the influence of different manufacturing techniques on 

physicochemical properties of nano-TiO2; the specificity of certain manufacturing techniques for 

 6-3 
nano-TiO2 used in either drinking water treatment or sunscreen, and the potential for new techniques 

to emerge; the potential for release of nanoscale and larger-sized waste products from nano-TiO2

manufacturing; and the potential for general population exposure to nano-TiO2 in the vicinity of 

manufacturing facilities. 

Limited information was identified specifically relating to nano-TiO2 distribution and storage 

(Section 2.3). Raw nano-TiO2 in powdered form is shipped in paper bags, in some cases lined with 

polyethylene film, and in glass bottles enclosed in sealed bags. Dispersions of nano-TiO2 are shipped 

in pails, drums, or totes. No specific information was identified on distribution and storage of nanoTiO2 formulated for drinking water treatment or sunscreen use. Some general information is 

available from the sunscreen industry on shipping and handling of topical sunscreens, with 

approximately two-thirds of sunscreen retail sales in the U.S. occurring in supermarkets, drugstores, 

and mass merchandise outlets. Accidental releases could occur to air, water, or soil at a variety of 

points along the distribution chain. Inclusion of distribution and storage information in life cycle and 

comprehensive assessments will require additional data regarding shipping modes, distances, and 

quantities for nano-TiO2 in various packaging, modes of storage prior to use in drinking water 

treatment and sunscreens, and estimates of releases under various scenarios of distribution and 

storage. 

The drinking water treatment case study considers only the application of nano-TiO2 for 

arsenic removal (Section 2.4.1), although it is possible that it can be used to remove other biological 

or chemical contaminants. Approximately 13 million people in the U.S. use water that is treated to 

remove arsenic. No information was identified on current use of nano-TiO2 in community water 

systems to remove arsenic, but future use could affect a substantial population. At least two 

commercial technologies are known to be capable of using nano-TiO2 in oxidative processes for 

water treatment, although currently they are not known to be used in this way. One process uses 

nano-TiO2 in a fixed membrane, while the other uses nano-TiO2 in a slurry. In bench-scale studies, 

slurry applications have been shown to produce higher arsenic oxidation rates compared to fixedmatrix nano-TiO2; however, immobilized applications presumably would result in less release to 

finished water than slurries, which require filtration. A bench-scale simulation of a conventional 

drinking water treatment process found that more than 20% of an initial 10 mg/L concentration of 

nano-TiO2 (15-40 nm particle size, 200-500 nm aggregate size) remained in water following 

coagulation, flocculation, and sedimentation (prior to filtration). Filtration through a 0.45 μm 

membrane reduced residual TiO2 to 1-8% of the initial concentration, although this level of filtration 

is not typical in drinking water treatment plants or whole-house filtration systems. Another factor 

that could be important, but for which information is lacking, is the effect of drinking water 

treatment chemicals on the solubility, particle size, and behavior of nano-TiO2. Information is also 

limited on the potential volume of nano-TiO2 required for arsenic removal in the U.S., details on 

different treatment processes and the likelihood of their use to serve populations of various sizes, 

release of nano-TiO2 to finished water or process waste, and the effect of nano-TiO2 on biofilms and 

corrosion in distribution systems. 

As discussed in Section 2.4.2, sunscreen use is substantial in the U.S., with most surveys 

reporting that 30-50% of respondents use sunscreen regularly. Parents report more frequent use of 

sunscreen on their children than on themselves. The amount and use of nano-TiO2 in sunscreens is 

unknown, in part because available survey data do not distinguish between conventional and nanoTiO2, although conventional TiO2 is likely to contain nanoscale TiO2. Production estimates of nanoTiO2 indicate that a substantial fraction (65%) is used in personal care products such as sunscreen 

and cosmetics. The lack of specific information on nano-TiO2 use in sunscreens represents an 

important gap in knowledge for life cycle and exposure assessment. Questions identified as priorities 

during the collective prioritization workshop (U.S. EPA, 2010, 625483) include the potential for 

release of nano-TiO2 to various media through different use patterns, the need for better 

characterization of nano-TiO2 as used in specific products, and the stability and behavior of surface 

 6-4 
treatments during sunscreen use. Removal of surface coatings may increase the photocatalytic 

activity of the nano-TiO2 particles and have implications for its effects on biota and humans. 

Disposal of nano-TiO2 after use is likely to vary between drinking water treatment processes 

and sunscreen (Section 2.5). Some fraction of nano-TiO2 used in drinking water treatment is likely to 

be associated with sludge, which may be taken to a landfill or applied to agricultural lands 

(U.S. EPA, 2010, 635678). To the extent that nano-TiO2 reaches finished water, it would then be 

expected to enter wastewater treatment facilities. Disposal of used sunscreen containers, presumably 

with some residual product, would also result in introduction of nano-TiO2 to municipal landfills or 

incinerators. Recycling of sunscreen containers is also possible, potentially introducing nano-TiO2

into recycled materials. Remaining disposal issues for which little information is available include 

disposal procedures for packaging containing nano-TiO2 and substandard product at manufacturing 

facilities, the behavior of nano-TiO2 in landfills, the exact nature of waste streams from water 

treatment facilities, and the circumstances that might result in release of nano-TiO2 from discarded 

sunscreen products. 

6.1.3. Fate and Transport 

Information is currently unavailable regarding the transport and transformation of nano-TiO2

specifically from drinking water treatment processes and sunscreens in air, water, ground water, soil, 

or sediment (Chapter 3). As mentioned in Chapter 1 and Section 6.1.1, physicochemical properties 

are likely to change when nanomaterials enter the environment, making it difficult to predict their 

behavior based on laboratory studies. One aspect of nano-TiO2 that is heavily influenced by local 

conditions is agglomeration of particles to form larger clusters. These agglomerates would tend to 

behave differently in the environment than individual particles. Degree of agglomeration is affected 

by ionic strength and presence of organic matter in water, as demonstrated in laboratory studies. 

Agglomeration is also pH-dependent, with minimum particle attraction at the pHpzc. Researchers 

have also demonstrated in bench-scale studies that free particles or agglomerates with diameters less 

than 100 nm can be present even when the predominant form of nano-TiO2 is larger clusters 

(Chapter 3), a finding that could have implications for agglomeration in natural waters. Surface 

modifications to maintain dispersion may also contribute to the presence of nanoscale particles under 

conditions normally considered to promote agglomeration. 

Nano-TiO2 that reaches wastewater treatment plants, such as through washing off of 

sunscreens, has the potential to pass through the facility in the liquid phase and reach receiving 

waters. Studies of other metal oxide nanomaterials in model wastewater treatment plants indicated 

that surfactants stabilized dispersions even at high ionic strength, although most nanoparticles were 

removed by agglomeration with microorganisms and subsequent sedimentation. The low solubility 

of nano-TiO2 compared with other metal and metal oxide nanoparticles suggests that it is likely to 

remain in the solid phase, although researchers have found that crystalline form, morphology, 

manufacturing method, and particle size can influence saturation concentration. Another aspect of 

nano-TiO2’s behavior in aqueous media that should be kept in mind is its photocatalytic generation 

of ROS in the presence of UV light, which may be a factor in surface waters exposed to sunlight. 

The lack of information on environmental behavior specific to nano-TiO2 used in drinking 

water treatment processes and sunscreens represents an open question. Substantial transformation of 

nano-TiO2 is not expected due to its physical and chemical stability, so the processes likely to be 

relevant are transport and accumulation in various environmental compartments. Chapter 3 describes 

scenarios through which nano-TiO2 used for arsenic removal could enter the finished water 

distribution system and thereby end up in surface water or the subsurface via leaks, or it could 

become part of sedimented sludge and enter the subsurface through landfilling. Likewise, nano-TiO2

from sunscreens could be released to natural bodies of waters through wear-off, and to wastewater 

 6-5 
via bathing or laundry following sunscreen use. This nano-TiO2 could migrate to sediment through 

agglomeration with natural organic matter or microorganisms or could remain in the water column. 

Studies have observed other sunscreen constituents in natural waters, providing plausibility for this 

scenario. However, no studies were identified that have documented the occurrence of nano-TiO2

specifically from sunscreens in wastewater or natural waters. 

Laboratory studies have measured transport of nano-TiO2 in synthetic porous media or model 

soil columns, and found nano-TiO2 to be mobile in these model systems, where large soil particles 

and low ionic strength favored increased mobility, while high clay content, dissolved organic carbon, 

and salinity favored retention (Section 3.2). Particle size also affected mobility, with smaller particles 

passing through the columns to a greater extent than larger particles. However, pH had an 

unanticipated effect in one model system, with high transport observed at pH values for which 

retention was expected. This indicates that current transport theory may not accurately predict 

transport of nanomaterials and agglomerates. Specific information was not identified regarding 

pathways for nano-TiO2 from arsenic removal or sunscreen use to end up in soil, although one 

possible scenario is accumulation in wastewater treatment sludge which is then applied to 

agricultural land as a soil amendment. 

Information is not available on the fate of nano-TiO2 that may be emitted to the atmosphere by 

manufacturing or distribution facilities (Section 3.3). It is unclear whether its atmospheric fate and 

transport behavior would be similar to that of ultrafine particulate matter emitted from combustion 

sources, which tends to agglomerate rapidly and undergo phase change (condensation/volatilization) 

near the source, due to the differing physicochemical characteristics of nano-TiO2 from combustion 

emissions. 

The collective prioritization process at the workshop identified three priority topics with fate 

and transport aspects (U.S. EPA, 2010, 625483). These include: (1) the role of physicochemical 

properties, including surface treatments and agglomeration, in environmental behavior of specific 

nano-TiO2 products; (2) identification of pathways that pose the greatest exposure potential to nanoTiO2; and (3) the spatial and temporal distribution of nano-TiO2 in the environment. Other questions 

relating to fate and transport of nano-TiO2 from drinking water treatment and sunscreen use include: 

what the effect is of environmental factors (e.g., pH, natural organic matter type and concentration) 

on nano-TiO2 mobility and fate; whether existing theory and models of particle transport are 

applicable to nano-TiO2 and other nanomaterials; the extent to which knowledge regarding fate and 

transport of other nanomaterials may be applicable to nano-TiO2; the potential for nano-TiO2 to 

influence the behavior of other water and soil constituents and to bioaccumulate; whether 

photocatalytic activity of nano-TiO2 is of concern in water treatment, wastewater treatment, or the 

environment; and the effect of co-occurring sunscreen constituents on nano-TiO2 fate and transport. 

6.1.4. Exposure and Dose Characterization 

6.1.4.1. Exposure Characterization 

The term exposure refers to contact between an individual and a pollutant, combining 

information on activity patterns and time spent in various microenvironments with concentration 

data in multiple environmental media. Biota and humans may be directly exposed to nano-TiO2 used 

in drinking water treatment or sunscreen, or may receive indirect exposure through contact with 

nano-TiO2 in air, water, soil, or sediment. Transfer of nano-TiO2 between these media is also likely 

to occur. As described in Chapter 4, limited evidence is currently available regarding environmental 

exposures of biota and humans to nano-TiO2, although some information is available on 

occupational exposures associated with nano-TiO2 manufacturing. 

 6-6 
Aquatic organisms may be exposed to nano-TiO2 present in the water column or in sediment, 

with exposure depending on the distribution of nano-TiO2 between water and sediment as well as the 

tendency of the organism to feed or otherwise spend time near the bottom of water bodies. The 

propensity of nano-TiO2 to agglomerate may also result in deposition to the surfaces of aquatic 

organisms, including the gills of fish. This could increase the concentration of nano-TiO2 relative to 

the water and may result in surface toxicity even when uptake is not observed (Section 4.1.1). For 

terrestrial organisms, exposure scenarios may include contact between material spilled during 

shipping or storage and microbial, invertebrate, and vertebrate species, as well as potential contact 

with nano-TiO2 in environmental media. No specific evidence has been identified regarding actual 

exposures of biota to nano-TiO2 in the environment. 

Human exposure to nano-TiO2 may occur either in occupational settings or among the general 

population (Section 4.2). The general population may be exposed through use of nano-TiO2

sunscreens or by drinking water with residual nano-TiO2, as well as through contact with nano-TiO2

from these applications that ends up in environmental media. The use of nano-TiO2 for arsenic 

removal in drinking water appears to be limited at present, although implementation of this 

technology could result in substantial exposure given the sizeable population receiving finished 

water that has been treated for arsenic. If nano-TiO2 were present in potable water, exposures could 

involve pathways other than ingestion, such as dermal contact and inhalation of droplets during 

bathing and showering. Potential exposures may be of greater concern for infants and children, who 

consume more water per body weight than adults. Sunscreen-related exposure to nano-TiO2 may 

occur through skin contact, although dermal uptake has been found to be relatively low; other 

potential pathways include inhalation of spray products and ingestion via hand-to-mouth contact 

(particularly for children). Based on one series of assumptions, the amount of applied nano-TiO2 per 

sunscreen application was estimated to range from 12 to 55 mg/kg body weight for a three-year-old 

child and 8-37 mg/kg for adults (Table 4-2). This higher exposure combined with parent reports of 

greater sunscreen use on their children than on themselves could indicate an important role for 

exposure to nano-TiO2 and related sunscreen constituents in children. 

Nearly every stage of the life cycle for the applications considered in these two case studies 

presents some potential for occupational nano-TiO2 exposure. Most available information pertains to 

manufacture of nano-TiO2 rather than product formulation, shipping, or use by operators of drinking 

water treatment facilities (Section 4.2.2). Manufacturers have reported workplace inhalation 

exposures of less than 0.3-0.5 mg/m3

, although concentrations in some areas were higher. As a frame 

of reference, the NIOSH has proposed a draft occupational exposure limit of 1.5 mg/m3

 for fine TiO2

and 0.1 mg/m3

 for ultrafine TiO2 based on relative surface area. Independent measurements in a 

facility producing nano-TiO2 found lower mass concentrations, ranging from 0.004 to 0.042 mg/m3

; 

personal sampling in this facility found a concentration of 0.010 mg/m3

. Number concentrations in 

the facility ranged from 15,000 to 29,000 particles/cm3

, with 60% of the particles in the 20 to 30 nm 

size range; airborne TiO2 concentrations outside the plant were measured at 13,000 particles/cm3

. 

Surface area concentrations were 13-50 μm2

/cm3. A modeling study using these and other data 

estimated that packers were exposed to 0.39 m2

 TiO2 per 300 m3 air, while surface treatment workers 

had lower exposure at 0.17 m2

 per 300 m3. These preliminary data indicate that there is a wide range 

of concentrations and exposures among nano-TiO2 manufacturing and handling facilities and that 

exposure may vary by occupation. In addition, dermal exposure may be relevant depending on the 

type and usage rate of personal protective equipment. 

As described in Chapter 4, both aggregate exposures (representing exposure to nano-TiO2

through multiple routes, such as dermal, ingestion, and inhalation) and cumulative exposures 

(representing exposures to multiple substances associated with the use of nano-TiO2 in drinking 

water treatment and sunscreen) are relevant to the consideration of nano-TiO2 exposure. However, 

limited evidence specific to nano-TiO2 is currently available. Cumulative exposure may involve 

other sunscreen constituents, transformation products of reactions catalyzed by TiO2 (in drinking 

water or sunscreen), or pollutants adsorbed to nano-TiO2 and carried into the body as co-

 6-7 
contaminants. This latter phenomenon has been observed for arsenic and cadmium, as summarized 

in the following section on dose characterization. Although models have not been specifically 

developed for characterizing exposure to nano-TiO2, EPA has various models that have been used in 

assessments of other chemicals to provide screening-level estimates of aquatic exposure, general 

population exposure, and consumer exposure. Adaptation of these models for use with nanomaterials 

would likely be necessary prior to their quantitative use for nano-TiO2. Researchers in Switzerland 

have developed models to predict environmental concentrations of nano-TiO2 and compared their 

estimates to no-effect concentrations in aquatic toxicity studies, although an explicit exposure 

component was not included. 

The collective priority ranking process identified several exposure-related questions 

(U.S. EPA, 2010, 625483), including: 

• which properties of nano-TiO2 are most relevant for exposure characterization, and 

whether available methods are adequate to characterize exposure in air, water, soil, and 

other media; 

• which pathways pose the greatest exposure for biota and human exposure to nano-TiO2

used in drinking water treatment and sunscreens; and 

• whether certain populations of biota and humans have greater exposure potential. 

Other questions include how exposure models can be developed or adapted to estimate nano-TiO2

exposure, and the degree of exposure to secondary contaminants associated with nano-TiO2 used in 

drinking water treatment and sunscreens. Occupational exposure can be further characterized as 

well, leading to questions such as: 

• the size of the potentially exposed population; 

• additional information on concentrations and durations of exposure in various job 

classifications; 

• which monitoring methods and properties are appropriate for measuring workplace 

exposure; and 

• which management practices and protective equipment are appropriate for controlling 

exposure by various routes. 

6.1.4.2. Dose Characterization 

Dose is defined as the amount of a substance that enters an organism by crossing a biological 

barrier. Various exposure routes have been investigated for uptake of nano-TiO2 in studies of fish, 

laboratory animals, and humans (Section 4.6). Two studies investigating accumulation of nano-TiO2

in fish following multi-day exposures have found mixed results, possibly due to differences in 

species (bottom feeder versus pelagic) and other aspects of study design. Two additional fish studies 

have indicated the potential for nano-TiO2 to increase uptake of arsenic and cadmium, presumably 

by adsorption and facilitated transport. In addition to this example of cumulative dose to multiple 

pollutants, bioaccumulation in the food web is also a possibility, although no studies were identified 

that demonstrated multi-species bioaccumulation of nano-TiO2. 

In animal toxicological studies relevant to terrestrial biota and humans, various exposure 

routes have been evaluated to determine the uptake of nano-TiO2 and other nanomaterials, including 

respiratory (inhalation and instillation), dermal, and ingestion. Animal studies have shown that 

inhaled or inspired nano-TiO2 can translocate into the interstitium of the lung, the lymph nodes, 

blood, and the brain (Section 4.6.2). Deposition patterns in the respiratory tract depend on several 

factors, including particle size and breathing pattern. Model results of human lung deposition 

 6-8 
indicate that very few nanoparticles reach the alveolar region, having been removed by diffusive 

deposition in the upper airways or tracheobronchial region. Studies in rats indicate that the retention 

half-life of inhaled nano-TiO2 was approximately three times as long as that of fine TiO2. 

Dermal uptake of nano-TiO2 is particularly relevant for sunscreens containing nano-TiO2, and 

both human and animal studies are available (Section 4.6.3). These studies predominantly indicate 

that nano-TiO2 does not penetrate beyond the stratum corneum or hair follicles into living cells of 

healthy skin. In a study comparing psoriatic and healthy skin, nano-TiO2 in a sunscreen formulation 

penetrated into deeper areas of the stratum corneum of psoriatic skin, but still did not reach living 

cells. No studies have been identified that evaluated nano-TiO2 penetration in damaged skin (e.g., 

from sunburn), although preliminary results indicate greater penetration of quantum dots and nanosilver in damaged skin compared to healthy skin. The extent and duration of nano-TiO2 accumulation 

on the skin via reapplication of sunscreen and the ultimate fate of nano-TiO2 from sloughed skin 

cells are both open questions at this time. 

Evidence for accumulation of nano-TiO2 following ingestion is extremely limited, with a 

single study reporting elevated concentrations in the liver, spleen, kidney, lung, and brain of female 

mice following oral gavage (Section 4.6.4). 

The potential for nanoparticles to cross the blood-brain barrier (BBB) has been investigated 

for medical applications, where in many cases the particle surfaces have been modified to enhance 

translocation (Section 4.6.5). Mixed evidence is available for translocation of nano-TiO2 across the 

BBB following injection or gavage, with some studies finding increased Ti concentrations in the 

brain and others finding no evidence of an increase. A recent study showed TiO2 particles and 

pathological changes in the brain of mouse offspring following maternal exposure during gestation, 

although it is not clear whether nano-TiO2 crossed the placenta or entered the milk to result in 

lactational exposure. 

Various metrics are possible for characterizing nanoparticle dose, such as mass, surface area, 

or particle number, as well as crystalline form, shape, and surface modifications (Section 4.6.6). 

Studies comparing mass and surface area to evaluate dose-response curves provide mixed results, 

with some evidence indicating that surface area provides a more consistent dose-response 

relationship for both fine and nano-TiO2. Composite metrics of two or more properties may also be 

useful, as suggested by a study indicating that separate surface-area-based dose-response curves for 

anatase and rutile TiO2 would better fit the data than a single dose-response curve. 

Questions highlighted during the collective prioritization process (U.S. EPA, 2010, 625483) 

for future research on dosimetry of nanomaterials in general and nano-TiO2 in particular include: 

• whether certain populations (e.g., children) may be particularly susceptible to receiving 

high doses of nano-TiO2 from its use in drinking water or sunscreens; and 

• which dose metrics are most relevant for characterizing nano- TiO2 dosimetry. 

Other questions include: 

• what modifications need to be made to physiologically-based pharmacokinetic models 

so that they are appropriate for understanding absorption, distribution, metabolism, and 

excretion of TiO2; 

• how to extrapolate received dose from animal toxicological studies to humans; the extent 

to which nano-TiO2 may bioaccumulate and biomagnify in food webs; and 

• whether increased uptake of copollutants in the presence of nano-TiO2 indicates the need 

for consideration of other substances in nano-TiO2 monitoring and exposure studies. 

 6-9 
6.1.5. Ecological and Health Effects 

Several factors influence the ecological and health effects of nano-TiO2, including 

physicochemical characteristics, experimental conditions, and environmental conditions 

(Section 5.1). The need for thorough characterization of nanomaterials used in toxicity studies is now 

well recognized. Important properties considered part of a minimum set of characteristics include: 

particle size, size distribution, and aggregation status; chemical composition and crystal structure; 

surface chemistry and charge; specific surface area; particle shape; and production method. Studies 

have found these variables to be important in determining the chemical and biological behavior of 

nanomaterials. Experimental conditions also modify the effects of nano-TiO2 and are therefore 

important to measure and report in detail. For example, skin penetration of nano-TiO2 increased for 

an oily dispersion compared with an aqueous dispersion, although nano-TiO2 did not reach living 

skin cells. Suspension media used in laboratory studies, such as deionized water, tap water, saline 

solutions, and BAL fluid, each lead to different states of agglomeration which can affect the uptake 

and effects of nano-TiO2. Different levels of in vitro OH radical production have been observed in 

different sunscreen formulations containing similar nano-TiO2, indicating that the other components 

of the mixture can affect the observed results. Similar issues exist for environmental conditions, such 

as differential effects due to changes in the aquatic chemistry of surface or ground water or the 

presence of natural organic matter. UV radiation is well known to increase the toxicity of nano-TiO2; 

in addition, it may make the skin more permeable by causing sunburn. Other issues that are 

potentially important include the influence of temperature and water saturation on skin penetration, 

but no studies were identified that have investigated these parameters. 

6.1.5.1. Ecological Effects 

Ecological effects on microorganisms, aquatic species, and terrestrial species are discussed in 

Section 5.2, and key studies are summarized in Table 5-3. Most studies have tested photocatalytic 

nano-TiO2 as would be used in water treatment, with only a few studies evaluating photostable nanoTiO2 intended for use in sunscreen. However, coatings used to increase the photostability of nanoTiO2 could be removed in the environment through weathering or biotransformation, yielding nanoTiO2 with photocatalytic properties. Studies of acute effects in microorganisms and higher aquatic 

species generally provide little evidence of toxicity at concentrations below 10 mg/L, with several 

studies finding no effects at concentrations of 100 mg/L or higher. However, a longer-term (14-day) 

study found respiratory toxicity, injury to the gill and intestine, and evidence of oxidative stress in 

the gill and intestine in juvenile rainbow trout following exposure to photocatalytic nano-TiO2 at 

concentrations as low as 0.1 mg/L. Studies evaluating terrestrial invertebrates found no effect on 

behavior or mortality for P. scaber and decreased reproduction without change in body length for 

C. elegans. Spinach growth was enhanced by nano-TiO2 in several studies, possibly due to increases 

in the activity of enzymes responsible for photosynthesis, nitrogen metabolism, and oxidative stress 

response. Incorporation of nano-TiO2 into sediment was not found to increase toxicity of sediment or 

elutriate, even at a 1:1 ratio. In general, the focus of these studies on growth and mortality provides 

little information on mode of action of nano-TiO2 ecotoxicity. 

One of the highest priority areas identified in the collective prioritization results from the 

workshop (U.S. EPA, 2010, 625483) included the question of whether standard ecotoxicity tests are 

appropriate for nanomaterials in general and nano-TiO2 in particular. Changes in nano-TiO2

properties in different matrices (raw materials, products containing nano-TiO2, environmental media, 

and biological systems) may lead to differing behavior and make extrapolation of test results 

difficult. It is not currently clear whether a suite of physicochemical properties can be used for a 

structure-activity relationship to predict biological effects. In addition, the interplay between 

physicochemical properties of nano-TiO2 and changes in environmental variables (e.g., pH, oxygen 

 6-10 
level) is not well understood and could result in changes to both the ecotoxicity of nano-TiO2 and 

underlying soil or aquatic chemistry. Other issues potentially relevant to changes in physicochemical 

properties include the effect of in vivo biochemical processes on nano-TiO2 and the potential for 

interaction between nano-TiO2 and associated substances resulting in increased uptake and effects of 

either nano-TiO2 or copollutants. The collective priority ranking results also included questions 

surrounding the mechanism or mode of action of nano-TiO2 and whether different modes of action 

are important at low and high concentrations. 

6.1.5.2. Health Effects 

Health effects of nano-TiO2 are discussed in Section 5.3. Both noncarcinogenic and 

carcinogenic effects have been examined. Noncarcinogenic effects have been investigated in animal 

toxicological studies for several exposure routes, including dermal, oral, and respiratory; however, 

no epidemiologic studies or case reports were identified pertaining specifically to nano-TiO2. 

Limited evidence from acute in vivo dermal exposure studies does not indicate skin irritation or skin 

cell toxicity following exposure to photocatalytic nano-TiO2; as discussed in Section 6.1.4, uptake of 

nano-TiO2 through healthy skin was not observed. No studies were identified that evaluated either 

effects in flexed or abraded skin or long-term effects of any kind, which would be relevant to typical 

sunscreen usage patterns. Of the three animal studies identified that evaluated toxicity following oral 

intake of nano-TiO2, two studies found no evidence of lethality or obvious acute toxicity following a 

single dose of 5,000 mg/kg, although brain morphological changes were observed in one of these 

studies. The third study found DNA damage in mice in both mothers and offspring following 

exposure to 60-600 μg/mL nano-TiO2 in drinking water for 5 days; this is also relevant to 

carcinogenic effects. A larger group of studies focused on respiratory effects following inhalation or 

instillation, and found pulmonary inflammation and impaired particle clearance, with effects 

generally most severe in rats, followed by mice, and least severe in hamsters. Nano-TiO2 effects 

were often more severe or persistent than conventional TiO2 at the same doses. Preliminary evidence 

has also been observed for systemic effects outside the lung following respiratory exposure, 

including changes in inflammatory cell and platelet counts in the blood, renal pathology, potential 

hepatic toxicity, and changes in brain morphology and neurotransmitter levels. Carcinogenic effects 

of nano-TiO2 have been examined in several studies due to the classification of TiO2 (size 

unspecified) as a possible human carcinogen by IARC. The evidence indicates that inhalation or 

instillation of photocatalytic nano-TiO2 increases lung tumor incidence in rats, but not mice. This 

raises the question of the human health relevance of rat-specific nano-TiO2 carcinogenicity due to 

increased susceptibility to pulmonary inflammation and poor particle clearance in the rat strains 

studied. No carcinogenic effects were observed following intraperitoneal or intramuscular 

administration of photocatalytic nano-TiO2. 

Another of the highest priority areas identified in the collective prioritization results from the 

workshop (U.S. EPA, 2010, 625483) included the question of whether current EPA test guidelines 

and assays are appropriate for determining the health effects of nano-TiO2, and, if not, which 

modifications, additional assays and standard reference materials would be useful. Additional 

priority questions from the workshop included: what the fundamental biological responses are for 

nano-TiO2interactions at the cellular level; what the important modes and mechanisms of action are 

for nano-TiO2 effects and whether the mode of action differs for different types of nano-TiO2 (e.g., 

photocatalytic versus photostable) or different organs (e.g., lung versus brain); and what the longterm effects of nano-TiO2 may be. Other potentially relevant issues included: which properties are 

necessary and desirable for proper characterization of nano-TiO2 during assays; whether nano-TiO2

has the potential to penetrate compromised skin; and whether nano-TiO2 has reproductive, 

developmental, or carcinogenic effects. 

 6-11 
6.2. Role of Case Studies in Research Planning and 

Assessment Efforts 

These two case studies are designed to benefit ongoing nanomaterial research efforts, the 

research planning process, and potential future assessment efforts on the environmental (ecological 

and health) effects of nano-TiO2 and other nanomaterials. The currently available information 

presented here, along with gaps in knowledge that have been identified, should be useful in the 

interpretation of newly available data as well as planning for future research and assessments. As 

stated previously, the case studies are not intended to represent completed assessments or to serve as 

the basis for near-term risk management decisions regarding the use of nano-TiO2 in drinking water 

treatment or sunscreen. In addition, other scenarios for potential use of nano-TiO2, such as in 

coatings or as a component of a solid matrix, may involve separate issues not considered in this 

document. This section describes how the case studies may be used in informing ongoing and 

planned research on nanomaterials and in developing information useful for future assessments. It 

also highlights some of the information gaps identified through the case studies and the associated 

workshop on research priorities, described in Section 6.2.1. 

6.2.1. Workshop on Research Priorities for Nano-TiO2

As part of the process of identifying areas where additional knowledge may be useful, NCEA 

held a workshop to identify and prioritize research directions. The workshop used a formal group 

decision method known as the “nominal group technique (NGT),” which is a process for a group of 

selected individuals to identify and rank a series of choices. Each individual presents a brief 

statement outlining the rationale for assigning a high priority to a particular choice. The group then 

discusses the priorities, with the opportunity to consolidate similar choices, and votes for the highest 

priority items. The result is a rank order of priorities based on the collective judgment of the 

individual participants. Research questions identified in the nano-TiO2 case studies were prioritized 

using this technique. 

A summary report (U.S. EPA, 2010, 625483) describes the workshop and summarizes the 

main outcomes of the ranking process. The reader is referred to this summary report, which provides 

more detailed information regarding the specific questions used to develop research priorities and the 

rationale for prioritizing each of the research needs; this information is not repeated here. The NGT 

process identified several high-priority topic areas, with the top-ranked priorities addressing whether 

existing human and ecological toxicity test protocols are appropriate for use with nano-TiO2, as well 

as questions regarding characterization of the physicochemical properties of nano-TiO2 at each stage 

of the product life cycle, in the environment, and in biological systems. Other priority topics 

included: determining what effect surface coatings and product formulations have on physicchemical properties and biological activity of nano-TiO2; evaluating exposure pathways and 

populations of greatest concern, and whether available methods are appropriate for characterizing 

exposure to nano-TiO2; developing a database of information on environmental concentrations of 

nano-TiO2 in various media, including biological systems; research into the mode of action of nanoTiO2, both at high and low doses; and determining the effects of long-term exposure to nano-TiO2. 

Many, if not all, of these topics are relevant to other nanoparticles, although separate lines of 

research may be useful in characterizing diverse particle types (e.g., metal oxides and carbon-based 

nanoparticles). 

 6-12 
6.2.2. Implications for Research Planning 

The U.S. EPA’s Nanomaterial Research Strategy document (U.S.EPA, 2009, 625484) outlined 

research themes and science questions relating to sources, fate, transport, and exposure to 

nanomaterials; human health and ecological effects, and nanomaterial risk assessment and risk 

management. These science questions are guiding ongoing research in EPA’s ORD and will form the 

basis for research planning efforts within ORD. The findings of these case studies are consistent with 

the themes emphasized in the Nanomaterial Research Strategy and should further inform 

interpretation of current results and planning for future research, both within EPA and among the 

broader scientific community. For example, the case studies and workshop highlighted the question 

of the appropriateness of health and ecological toxicity testing protocols for use with nano-TiO2. 

This concept is included in the research strategy document as background for the key science 

questions on health and ecological effects of nanomaterials, and may form an overarching theme that 

can be used to integrate results from individual experiments using different study designs, protocols, 

and endpoints. Development of methods for physicochemical characterization of nanomaterials 

under controlled conditions, in environmental matrices, and in biological systems is also a common 

priority. This highlights the potential for integrated transdisciplinary research, a focus area for ORD, 

to utilize contributions from materials science, engineering, and biology to fully understand 

nanomaterial properties and effects. 

One area of missing information identified in the case studies was research into the long-term 

effects of nano-TiO2. Consideration of longer-term chronic effects in the context of existing research 

strategy themes and science questions would help address this gap in knowledge. Research into 

modes of action of nano-TiO2, both at low and high doses, is another priority area identified by the 

case studies and workshop. The research strategy references the importance of mode of action 

information, and specifically discusses the potential utility of determining the physical and chemical 

properties responsible for biological effects of nanomaterials. Integration of critical properties with 

their associated modes of action could help create a more complete understanding of the health and 

ecological effects of nanomaterials, as well as differences between nanoscale and conventional 

materials and effects unique to nanomaterials. Linkages such as these between the case study 

findings and research planning efforts may result in more focused and effective nanomaterial 

research, as well as providing information useful for future risk assessments of nano-TiO2 and other 

nanomaterials. 

6.2.3. Implications for Future Assessment Efforts 

The Nanomaterial Research Strategy (U.S.EPA, 2009, 625484) and EPA’s Nanotechnology 

White Paper (U.S. EPA, 2007, 090564) both highlight the importance of improved information and 

research results to support future assessment efforts. Their recommendations on research directions 

to support risk assessment for environmental fate and transport, as well as health and ecological 

effects, resonate with the priorities identified by the case studies and workshop. A variety of 

information will likely figure into future risk assessments, including characterization of nano-TiO2 in 

multiple matrices; information on the magnitude of potential releases, environmental concentrations, 

and exposure pathways for nano-TiO2; appropriateness of methods for evaluating human and 

ecological toxicity; information on the interaction between physicochemical properties, dose, and 

mode of action; and both short-term and long-term health and ecological effects of nano-TiO2. 

Information on the life cycle of nano-TiO2 and products incorporating nano-TiO2, including their fate 

and transport in the environment, can be combined with these data to support a CEA of nano-TiO2. 

These case studies have summarized what is known on these and other topics, as well as what 

remains unknown, and the accompanying workshop has presented a set of priorities that can be used 

to guide future research and assessment efforts. 

 6-13 
 6-14 

An important function of environmental assessments is the integration and synthesis of 

information from multiple lines of evidence. This is difficult at this stage for nano-TiO2 or other 

nanomaterials due to the limited and somewhat scattered evidence in many areas along with the 

near-total lack of evidence in other areas. Risk assessments for human health effects of 

environmental chemicals typically bring together evidence from toxicological, epidemiological, and 

controlled human exposure studies, which is then integrated to evaluate the likelihood of a causal 

relationship between the pollutant and a particular category of health effects. The CEA framework 

expands upon this by considering the impact of a material’s life cycle and environmental fate on 

health and ecological effects. CEA is particularly appropriate for engineered nanomaterials because 

it facilitates the transdisciplinary integration of information from materials science, engineering, and 

biology that is required to fully understand nanomaterial effects. CEA also can consider the multiple 

impacts expected to result from introduction of a new technology, compare those impacts with those 

from conventional technologies, and provide information relevant for evaluating the sustainability of 

new nanomaterials. Much work remains before this will be possible for nano-TiO2 to the same 

degree as it has been accomplished for other pollutants. The information presented in these case 

studies of nano-TiO2 in drinking water treatment and sunscreen provides a starting point for this 

important work. 
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193744
Annex A. Nano-TiO2 in Sunscreen: Background 

Information 

Nano-TiO2 has been used in topical sunscreen products since approximately 1990 (EWG, 

2008, 196343). Between 1995 and 2002, the market for inorganic sunscreen ingredients (both 

nanoscale and non-nanoscale) increased from a value of roughly $30 million to a value of 

approximately $38 million, and has maintained approximately a 20% share of the sunscreen 

ingredient market as a whole (Dransfield, 2005, 157809). Dransfield (2005, 157809) projected that 

the market for inorganic active ingredients in sunscreens would grow to approximately $75 million 

by 2010 which would account for one-third of the total market for sunscreen active ingredients. 

Dransfield (2005, 157809) suggested that the projected increase in the popularity of inorganics can 

be attributed to improved transparency in the products, which would imply particularly rapid growth 

in the market for nanoscale inorganics. In 2006, the Australian Therapeutic Goods Administration 

(TGA) estimated that 70% of Ti sunscreens and 30% of zinc sunscreens in Australia were formulated 

with nanoparticles (TGA, 2006, 089202). 

The U.S. topical sunscreen market in 2000 was approximately $553 million (65%) of the 

$853 million “sun-care” market (a category that includes self-tanning products, after-sun products, 

etc.) (Packaged Facts, 2001, 196053). The size of the U.S. sunscreen market had apparently not 

changed substantially since 1993, when retail sales were reportedly in the range of $550 to 

$575 million (Davis, 1994, 157946). The total U.S. sun-care market reached $1.1 billion in 2005, 

and is projected to reach $1.2 billion by 2010 (Jeffries, 2007, 157682). If sunscreens continue to 

account for 65% of the U.S. sun-care market, that would translate to $715 million in sunscreen sales 

in 2005, and a projected $780 million in sunscreen sales in 2010. Globally, sales of sun protection 

products that presumably include topical sunscreens and cosmeceuticals were expected to exceed 

$820 million in 2006 (Newman, 2006, 157745). As a “mature” market in the U.S., sun protection 

products are expected to have a growth rate of only approximately 2% per year (Jeffries, 2007, 

157682). Between 2005 and 2010, however, growth in the sun-care market was expected to be much 

faster abroad than in the U.S. (Jeffries, 2007, 157682). If the growth in cosmeceuticals has 

dampened demand for conventional sunscreen, this growth has led to even greater demand for 

sunscreen active ingredients, including micronized TiO2 (Davis, 1994, 157946). 

A.1. Sunscreen Chemistry, and the Role and Properties of 

Nano-TiO2

UV radiation is classified by wavelength into three types: UV-A (320-400 nm), UV-B 

(290-320 nm), and UV-C (200-290 nm). The shorter the wavelength, the more energy the UV 

radiation transmits. Consequently, the shorter wavelength rays can cause more damage to skin than 

the longer wavelength rays. Approximately 10% of the solar radiation that reaches Earth’s surface is 

UV, and approximately 95% of that is UV-A. The long wavelengths of UV-A contribute to skin 

aging, skin wrinkling, and skin cancer. UV-B is in the middle range of UV, and contributes to 

burning and tanning, skin aging, and skin cancer. Although UV-C has the shortest wavelength and 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments. 

A-1 
can be dangerous, it is blocked by ozone in the atmosphere and does not reach Earth’s surface 

(Jeffries, 2007, 157682; Shao and Schlossman, 1999, 093301). 

The traditional SPF rating system measures protection against UV-B radiation only. The U.S. 

FDA proposed an official rating system that also takes UV-A radiation into account, awarding 

sunscreens between one and four stars based on their UV-A protection (72 FR 49070). This system 

was expected to go into effect in November 2008 or later but has not been finalized as of April 2010. 

Various other UV-A protection ratings systems are in use or have been proposed in Australia, New 

Zealand, Europe, Japan, China, and Korea (Moyal, 2008, 193559). 

A.1.1. Size of Nano-TiO2 Particles (Mean and Distribution) 

The composition of nano-TiO2–based sunscreens is determined or constrained by several 

factors, including unique properties of nano-TiO2, general principles of sunscreen chemistry, and 

aesthetic and other concerns. The size of nano-TiO2 particles (both the primary particle size and the 

effective particle size of aggregates and agglomerates) affects protection against UV-A and UV-B 

radiation, the opacity of the sunscreen, and the stability of the dispersions. In most cases, a range of 

nano-TiO2 sizes is present due to various primary particle sizes and aggregation. 

The size of nano-TiO2 particles affects how much UV-A and UV-B the particles transmit and 

scatter, and therefore, the degree of protection the particles provide against UV-A and UV-B 

radiation. Shao and Schlossman (1999, 093301) found that a nano-TiO2 dispersion with a primary 

particle size of approximately 15 nm transmitted less UV-B and more UV-A and visible light than 

did dispersions with primary particle sizes of 35, 100, and 200 nm. (The particles were present in 

aggregates of mean sizes 125.3, 154.1, 251.1, and 263.4 nm, respectively.) The results of this study 

indicated that smaller nano-TiO2 particles are better for UV-B protection, and larger nano-TiO2

particles are better for UV-A protection. Dransfield (2005, 157809) presented data indicating that 

TiO2 particles (not specifying whether they were primary or secondary particles) at 100 nm diameter 

provide the best UV-A protection but also significant visible light attenuation (i.e., leaving a white 

hue on skin if such particles are used in sunscreens), and particles in the range of 40 to 60 nm 

provided the best UV-B protection. In addition, 20-nm nano-TiO2 did not provide sufficient 

protection against UV-A or UV-B. 200-nm TiO2 particles provide poor UV-A and UV-B protection 

and high attenuation of visible light (Dransfield, 2005, 157809). According to Hewitt (2002, 

093307), theoretical calculations suggest that the optimal mean TiO2 primary particle size for good 

UV-B and UV-A protection is approximately 50 nm. Chaudhuri and Majewski (1998, 093308) noted 

that nano-TiO2 with a primary crystal size of 10-20 nm and an effective particle size of 

approximately 100 nm is expected to have a “very high UV scattering effect.” 

Particle size also determines the opacity of nano-TiO2 formulations. Larger primary particles 

transmit less visible light (Shao and Schlossman, 1999, 093301). Aggregation will also make a 

formulation more opaque (Chaudhuri and Majewski, 1998, 093308). TiO2 particles larger than 

200 nm in sunscreen or cosmetics leave a white hue on the skin and are considered aesthetically 

unacceptable in many applications. Nano-TiO2 particles smaller than 100 nm are generally not 

visible, and the sunscreen appears transparent when applied. A presentation by Schlossman et al. 

(2006, 093309) included pictures demonstrating the opacity of formulations with different particle 

sizes when applied to skin. Formulations with an effective agglomerated particle size of 100-120 nm 

(primary particle size of 10 nm) or 120-150 nm (primary particle size of 15 nm) were transparent or 

nearly transparent. Schlossman et al. (2006, 093309) noted that, in addition to particle size, two other 

factors affected the opacity/transparency of formulations: the difference between the refractive index 

of the particle and that of the media, and the uniformity of particle dispersion. 

Chaudhuri and Majewski (1998, 093308) noted that particle size also affects the stability of 

sunscreen dispersion. The reason for this was not made clear in the article, but in a discussion of 

pigmentary particles in paints, Himics and Pineiro (2008, 155626) explained that smaller pigmentary 

particles produce a better dispersion because the larger surface area creates a higher viscosity, which 

 A-2 
prevents settling and clumping. The phenomenon that Chaudhuri and Majewski (1998, 093308) 

noted could have a similar explanation. 

A range of particle sizes provides a range of UV protection, but too wide a range could pose a 

risk of opacity or of compromising the stability of the dispersion (e.g., if too many particles are too 

large). In the past, controlling the range of particle sizes produced by manufacturing processes was 

difficult, and distributions with a mean particle size of 50 nm included particles in the visible range. 

As technology has improved, creating particles of desired size and size distributions with much 

greater accuracy (Hewitt, 2002, 093307) has become possible. 

A.1.2. Active Ingredient Purity 

The U.S. Pharmacopeia (USP) sets reference standards for TiO2 and other active ingredients in 

over-the-counter and prescription drugs. The 2006 edition of the USP national formulary 

monographs, USP-NF 30 (U.S. Pharmacopeia, 2006, 155639), declares that TiO2 “contains not less 

than 99.0% and not more than 100.5% of TiO2.” USP specifies tests for water-soluble impurities, 

acid-soluble impurities, arsenic, and organic volatile impurities, and notes that FDA also has set 

limits on acceptable lead, antimony, and mercury contamination. USP also specifies that the material 

must be stored in well-closed containers, and that it be properly labeled as attenuation grade (with 

names and amounts of added coatings, stabilizers, and other treatments listed) if intended for 

UV-attenuation. 

A.1.3. Photostability and Surface Coating/Doping 

Nano-TiO2 is a natural semiconductor with photocatalytic properties. Its electrons can easily 

become excited by energy absorbed from UV radiation. When the electrons return to ground state, 

longer wavelength radiation is emitted. Alternatively, if the energized electrons escape from the 

particle, they can catalyze chemical reactions (oxidation/reduction processes) in nearby molecules. 

These reactions can create free radicals, which can damage skin cells or degrade other sunscreen 

ingredients. The choice of nano-TiO2 crystal affects photostability. In particular, rutile is much more 

photostable than anatase (Chaudhuri and Majewski, 1998, 093308; Maynard, 2008, 157522). 

Although anatase is less photostable, it appears to be in common use. Barker and Branch (2008, 

180141) studied five TiO2 sunscreens purchased over the counter and found that one was pure rutile 

and the other four were anatase/rutile mixes in which anatase predominated. 

To increase TiO2 and nano-TiO2 photostability (i.e., to reduce the likelihood that excited 

electrons will escape), the crystals are commonly given a surface coating. Coating TiO2 with silicon 

dioxide and alumina (3.5% by weight) can reduce photocatalytic activity by 99% (SCCNFP, 2000, 

092740). Other TiO2 or nano-TiO2 surface coatings mentioned in the literature include inorganic 

oxides (Bird, 2002, 093306), simethicone (Chaudhuri and Majewski, 1998, 093308), methicone, 

lecithin (Schlossman et al., 2006, 093309), stearic acid, glycerol, silica, aluminum stearate, 

dimethicone (SCCNFP, 2000, 092740), metal soap, isopropyl titanium triisostearate (ITT), triethoxy 

caprylylsilane, and C9-15 fluoroalcohol phosphate (Schlossman et al., 2006, 093309). Alumina is 

often used in combination with other coating materials. The amount of surface coating applied varies 

substantially from product to product. For examples of common coating concentrations and 

combinations, see Appendix B, Table B-1. 

Another technique for increasing photostability is “doping” the TiO2 or nano-TiO2 particles by 

embedding within them minute amounts of metals such as manganese, vanadium, chromium, and 

iron. Doping rutile nano-TiO2 with manganese is reported to increase UV-A absorption, reduce free 

radical generation, and increase free radical scavenging behavior (Reisch, 2005, 155634; Wakefield 

et al., 2004, 193693). Doped TiO2 is colored instead of white, which can have desirable cosmetic 

effects in products such as skin lighteners (Park et al., 2006, 193593). 

 A-3 
Recent research by Barker and Branch (2008, 180141) has found that the surface coatings on 

nano-TiO2 in many sunscreens might not be stable or effective. The investigators studied the 

weathering of paint in contact with sunscreen. Out of five nano-TiO2 sunscreens tested, four released 

photocatalytically generated hydroxyl radicals that accelerated the weathering of the paint. All four 

of those sunscreens used an anatase/rutile mix. The one nano-TiO2 sunscreen that showed no 

appreciable effect on paint weathering was Oxonica’s OptisolTM, which is 100% rutile, and is doped 

with manganese rather than surface-coated. It is not known whether nano-TiO2 sunscreens generate 

hydroxyl radicals when applied to skin or whether such hydroxyl radicals would penetrate the skin 

and pose a threat to the health of the sunscreen user (Brausch and Smith, 2009, 193297; Maynard, 

2008, 157522). 

A.1.4. Dispersion and pH Considerations 

Nano-TiO2 can exist as a dry powder, but most sunscreen applications require the particles to 

be suspended in a fluid medium. This liquid is called a “dispersion” because special care must be 

taken to ensure that nano-TiO2 will be distributed evenly and to minimize further aggregation and 

agglomeration (which could negatively impact properties such as UV scattering performance and 

transparency by increasing the effective particle size). Sunscreen manufacturers can purchase 

nano-TiO2 powder and formulate their own dispersion, or they can purchase ready-made 

“predispersions.” 

In an effective dispersion, suspended particles are attracted to the dispersion medium and repel 

each other. Surface coatings influence the interaction of nano-TiO2 with the dispersion medium, 

which can be water-based (aqueous), oil-based, or silicone-based. Early TiO2 dispersions were 

generally oil-based (Bird, 2002, 093306). Surface coatings that make TiO2 dispersible in nonaqueous 

media can be lipophilic (e.g., metal soap, ITT, lecithin); hydrophobic (e.g., methicone, dimethicone, 

triethoxy caprylylsilane); or both (e.g., C9-15 fluoroalcohol phosphate) (Shao and Schlossman, 

1999, 093301). For methicone and C9-15 fluoroalcohol phosphate, silicone might be the preferred 

medium (Shao and Schlossman, 1999, 093301). Bird (2002, 093306) states that coatings have been 

developed to enable TiO2 to be dispersed effectively in aqueous media as well, but provides no 

examples. Chaudhuri and Majewski (1998, 093308) describe one product, an “amphiphilic” powder 

(Eusolex® T-2000) containing approximately 80% USP-grade rutile coated with alumina and 

simethicone, that is easily dispersible in both water and oil. 

Two related concepts that are useful in discussing the dispersion of particles are the pHpzc, 

which is the pH point at which the surface charge density of a particle is zero, and the isoelectric 

point (IEP), which is the pH at which the net surface electric charge of a particle is zero. In situations 

where no ions other than H+ and OH- are adsorbed at the particle surface, pHpzc is identical to the 

IEP. 

At most pH values, nano-TiO2 particles suspended in a dispersion have a positive electrical 

charge or a negative electrical charge and repel each other. At the pHpzc/IEP, however, there is no 

electrostatic repulsion, and particles tend to agglomerate (Hewitt, 1995, 157939). To maintain 

electrostatic repulsion and prevent agglomeration, the dispersed product must be maintained at a pH 

other than the IEP (usually at a lower pH) at every stage of production and storage. 

Surface coating can affect a particle’s pHpzc/IEP and can potentially extend the pH range at 

which the dispersion can be handled. For example, uncoated nano-TiO2 has an IEP of pH 6, and 

nano-TiO2 coated with alumina and simethicone has an IEP of pH 9 (Chaudhuri and Majewski, 

1998, 093308). Bird (2002, 093306) cites lecithin as another coating that is advantageous for 

electrostatic reasons. 

Experimental tests show additional pH considerations. Nano-TiO2 performance can be 

adversely affected by strongly acidic formulations (effects include more agglomeration, lower SPF, 

and greater opacity), unless special formulating techniques are used (Hewitt, 1995, 157939). 

 A-4 
Additional compounds can be added to the dispersion as “dispersants.” “[The] proper 

dispersant can help particles to disperse into [the] vehicle so as to shorten the dispersion time and 

increase the degree of dispersion. It can reduce the viscosity and yet stabilize the dispersion by either 

electrostatic or steric repellency” (Shao and Schlossman, 1999, 093301). Different dispersants are 

used in water- and oil- (or silicone-) based formulations. PEG-10 dimethicone is used as a dispersant 

for nano-TiO2 in a cyclopentasiloxane carrier in the predispersion CM3K25VM made by Kobo 

Products, Inc. manufactures. Polyhydroxystearic acid is used as a dispersant in a C12-15 alkyl 

benzoate carrier in Kobo’s TNP40TPPS predispersion (Shao and Schlossman, 2004, 157825). 

Mitchnick and O’Lenick (1996, 157935) mention lecithin and phosphate esters as potential 

“dispersing aids” for TiO2 dispersions, but they also use language suggesting that they might actually 

mean surface coatings. 

A.1.5. Distribution of Active Ingredient in Emulsion 

Most sunscreens are emulsions – mixtures of two fluids (called “phases”) that are immiscible 

(do not combine easily). For instance, water and oil, two immiscible fluids, may be mixed in an 

emulsion by an energetic process such as stirring or shaking. In some cases, the two fluids tend to 

quickly separate again. To prevent separation, an emulsifier (typically a surfactant or a polymer) can 

be added. In an emulsion containing two types of liquids, generally, droplets of one fluid are 

dispersed in a larger amount of the other fluid. The two fluids are referred to as the “dispersed 

phase” and the “continuous phase,” respectively. 

Types of emulsions used in sunscreens and other cosmetic products include oil in water (in 

which an oil phase is dispersed in a water phase, abbreviated “o/w”); water in oil (w/o); water in 

water (w/w); and occasionally water in oil in water (w/o/w). In “oil-free” formulations, oil is 

substituted by silicones (w/Si, Si/w) (Hewitt, 2000, 157898). As noted above, nano-TiO2 is most 

easily dispersed in oil, but emulsions can be formulated with nano-TiO2 in a water phase, an oil 

phase, or a silicone phase. The nano-TiO2 can be present in the dispersed phase or the continuous 

phase of a sunscreen emulsion (Dransfield, 2005, 157809). 

The emulsifiers used to keep the two phases from separating are typically partially hydrophilic 

and partially hydrophobic. By gathering on the interface between the dispersed phase and the 

continuous phase, emulsifiers bind the two phases (this is the principle behind soaps, shampoos, and 

detergents, which enable water to wash away oils and other normally hydrophobic particles), or at 

least prevent the two phases from repelling each other. Emulsifiers used in sunscreen emulsions 

include glyceryl stearate, PEG-100 stearate, and polyglyceryl-3-methyl glucose distearate (Oxonica, 

2005, 157793). 

A.1.6. Other Ingredients – Active and Inactive 

Nano-TiO2 can be combined with other physical UV blockers, such as ZnO (which can also be 

micronized), or with chemical UV filters to improve the UV protection the sunscreen provides. The 

sunscreen formula can also include a diverse array of inactive compounds for a variety of purposes. 

TiO2 and ZnO can form agglomerates. This attribute presents an obstacle to using TiO2 and 

ZnO in the same sunscreen. A solution is to put one active ingredient in the oil phase of the emulsion 

and the other in the water phase (Hewitt, 1995, 157939). 

Combining nano-TiO2 with chemical UV filters often provides better UV-B protection than 

expected, based on the SPF of each ingredient. The improved protection is probably due to the 

scattering the physical UV blocker provides, which increases the optical path length of the radiation 

and creates more opportunities for absorption by the chemical filter (Bird, 2002, 093306; Chaudhuri 

and Majewski, 1998, 093308). 

Emollients are often included in sunscreens to make the products feel more pleasing on the 

skin or to moisturize. In excessive quantities, emollients could break down the dispersion 

 A-5 
microstructure. This effect can be counteracted by using suitable surfactants or polymers (Hewitt, 

1996, 157936). 

Increasingly, nano-TiO2 is found in “cosmeceuticals,” products that combine a variety of 

active ingredients to perform multiple health and beauty functions. These products include 

moisturizers and color cosmetics (see below for more on cosmeceuticals). The manganese added to 

some nano-TiO2 formulations to prevent formation of free radicals during UV exposure can also help 

scavenge free radicals generated by other means, thus providing extra skin-protection benefits. 

Inert ingredients can be added to achieve the right viscosity or liquidity, spray-ability, color or 

transparency, pH, water-resistance, or spreadability. Silicones and related compounds can be added 

to impart water-resistance, improve skin feel, serve as emulsifiers in various formulations, and 

enhance the SPF of oil-based dispersions (Hewitt, 2000, 157898). 

A.2. Some Sunscreens with Nano-TiO2 or Micronized TiO2

as Active Ingredient 

Table A-1 was compiled from information contained in the Environmental Working Group’s 

cosmetic database “Skin Deep” (EWG, 2008, 196343) and from on-line shopping sources. Products 

labeled as containing TiO2 of unspecified particle size were excluded. The list of products provided 

in Table A-1 is likely not exhaustive. Also, product formulations and labels could change over time. 

 A-6 
 A-7 

Table A-1. TiO2 content in various sunscreen products. 

Brand/ Manufacturer Product Percentage TiO2

Abella Solar Shade, SPF 45 N/A 

Alba Botanica Chemical Free Sunscreen, SPF 18 7.0% 

B. Kamins Chemist Bio-Maple Sunbar Sunscreen, SPF 30 Fragrance-Free 2.04% 

BABOR High Protection Lotion, SPF 30 N/A 

BABOR Moderate Protection Sun Cream, SPF 20 4.5% 

BENEV Pure TiO2 N/A 

Bliss Oil-free Sunban Lotion for the Face, SPF 30 6% 

California Baby SPF 30 & Fragrance Free Sunscreen; also available as Sunblock Stick, SPF 30 4.5% 

California Baby Sunscreen SPF 30+ - Everyday Year Round; also available as Sunblock Stick 4.5% 

California Baby Water Resistant, Hypo-Allergenic Sunscreen, SPF 30 N/A 

Cellex-C Sunscreen, SPF 15 2% 

Cellex-C Water Resistant Sunscreen, SPF 30 2% 

Cellex-C Sun Care Broad Spectrum UV-A, UV-B Sunblock & Moisturizer, SPF 15 N/A 

Cellex-C Sun Care, SPF 30 2% 

Colorescience SPF 30 All Clear Sparkles Shaker Jar; SPF 30 Perfectly Clear Sparkles Shaker Jar; SPF 

30 Almost Clear Sparkles Shaker Jar; these variations also available in trial size, 

brushable, and rock and roller ball forms 

12% 

Dermalogica Oil Free Matte Block, SPF 20 4% 

Dermalogica Ultra Sensitive FaceBlock, SPF 25 14% 

EmerginC Sun 30 (and tinted version) N/A 

Fallene/Total Block Total Block Clear, SPF 65 4% 

Fallene/Total Block CoTZ, SPF 58 10% 

Fallene/Total Block Total Block Cover-Up/Make-Up, SPF 60 10% 

Fallene/Total Block Total Block Tinted, SPF 60 10% 

Jan Marini Bioglycolic Facial Lotion, SPF 15 5.5% 

June Jacobs Micronized Sheer, SPF 30 14.5% 

Lancôme Soleil High Protection Face Cream – Gel, SPF 30 4.5% 

Lancôme Soleil Soft-Touch Moisturizing Sun Lotion, SPF 15 4.5% 

Peter Thomas Roth Instant Mineral, SPF 30 15% 

Pevonia Botanica Pevonia Soleil Sun Block, SPF 15 N/A 

ProCyte TiSilc Sheer, SPF 45 N/A 

ProCyte TiSilc Sheer, SPF 45 (tinted) 3.5% 

ProCyte TiSilc Sunblock, SPF 60+ 8% 

ProCyte TiSilc Untinted, SPF 45 3.5% 

ProCyte Z-Silc Plus Sunblock, SPF 30+ 4.0% 

Total Skin Care LLC pH Advantage Basics Sun Blocker, SPF 15 N/A 

Wilma Schumann Wilma Schumann Sunscreen, SPF 20 N/A 

N/A – Not available. 

Source: Used with permission from the Environmental Working Group, for their Skin Deep Database (EWG, 2008, 196343). 
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62: 313-320. http://dx.doi.org/10.1016/j.porgcoat.2008.01.008 180141

Bird S (2002). Sense and stability. Soap, Perfum Cosmet, 75, 42-44. 093306

Brausch JM; Smith PN (2009). Pesticide resistance from historical agricultural chemical exposure in Thamnocephalus 

platyurus (Crustacea: Anostraca). Environ Pollut, 157: 481-487. 193297

Chaudhuri RK; Majewski G (1998). Amphiphilic microfine titanium dioxide: Its properties and application in sunscreen 

formulations. Drug Cosmet Ind, 162: 24-31. 093308

Davis DA (1994). Sunscreen oddities. Drug Cosmet Ind, 155: 20-24. 157946

Dransfield G (2005). Manufacture of novel, transparent TiO2 based sunscreens. Retrieved May 09, 2008 from 

http://www.wun.ac.uk/nanomanufacturing/archive/05_06_series/documents/dransfield.pdf. 157809

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http://www.cosmeticsdatabase.com/special/sunscreens2008/. 196343

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Hewitt JP (1995). Formulating with physical sunscreens: Control of emulsion pH. Drug Cosmet Ind, 157: 28-32. 157939

Hewitt JP (1996). The influence of emollients on dispersion of physical sunscreens. Drug Cosmet Ind, 159: 62-65. 157936

Hewitt JP (2000). Partners in protection. Soap, Perfum Cosmet, 73, 85-86. 157898

Himics R; Pineiro R (2008). The Importance of Particle Size in Liquid Coatings - Coating problems and solutions 

associated with particle size reduction. Prod Finish, 63: 46-53. http://www.pfonline.com/articles/the-importance-ofparticle-size-in-liquid-coatings. 155626

Jeffries N (2007, February). SPF, efficacy and innovation. GCI Magazine. 

http://www.gcimagazine.com/marketstrends/segments/suncare/27627099.html. 157682

Maynard AD (2008). Living with nanoparticles. Nano Today, 3: 64. 157522

Mitchnick M; O'Lenick AJ Jr (1996). Patent No. 5565591 (class: 556/10). United States: United States Patent Office. 

http://www.freepatentsonline.com/5565591.html. 157935

Moyal D (2008). How to measure UVA protection afforded by sunscreen products. Expert Review of Dermatology, 3: 307-

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Reisch MS (2005). New-wave sunscreens: active ingredient makers are frustrated by the long list of sunscreens and UV-A 

testing protocols that are still awaiting FDA decisions. Chem Eng News, 83: 18-22. 

http://pubs.acs.org/cen/coverstory/83/8315sunscreens.html. 155634

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concerning titanium dioxide Colipa n S75 (Report No. SCCNFP/0005/98). Brussels, Belgium: Scientific 

Committee on Cosmetic Products and Non-Food Products Intended for Consumers. 

http://ec.europa.eu/health/ph_risk/committees/sccp/documents/out135_en.pdf. 092740

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Schlossman D; Shao Y; Detrieu P (2006). Perspectives on supplying attenuation grades of titanium dioxide and zinc oxide 

for sunscreen applications. Retrieved October 28, 2007 from 

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Shao Y; Schlossman D (1999). Effect of particle size on performance of physical sunscreen formulas. Talk presented at 

PCIA Conference, Shanghai, China. http://www.koboproductsinc.com/Downloads/PCIA99-Sunscreen.pdf. 093301

Shao Y; Schlossman D (2004). Discovering an optimum small micropigment for high UV shielding and low skin 

whitening. Talk presented at 23d IFSCC Congress, Orlando, FL. 

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radical generation of micronised titanium dioxide and its consequences for the photostability of UVA absorbing 

organic sunscreen components. Photochem Photobiol Sci, 3: 648-652. 

http://www.rsc.org/delivery/_ArticleLinking/DisplayArticleForFree.cfm?doi=b403697b&JournalCode=PP. 193693
Annex B. Nano-TiO2 in Sunscreen: 

Manufacturing Processes 

B.1. Overview of Nano-TiO2 Manufacturing Process 

A generic manufacturing process for nano-TiO2 for sunscreen applications is outlined in 

Figure B-1. 

Source: Dransfield (2005, 157809) 

Figure B-1. Generic manufacturing process for nano-TiO2 for sunscreens. 

B.1.1. Titanium Dioxide Nuclei Synthesis 

Commercial-scale TiO2 synthesis is mostly by sulfate or chloride processes. In this section, a 

sulfate process, chloride process, and patented Altair process are described. These three processes 

can be used to synthesize both conventional (or pigmentary) and nanoscale TiO2. There are many 

new processes being developed in the laboratory, but it is outside the scope of this Appendix to cover 

them; see review of nano-TiO2 synthesis by Chen and Mao (2007, 193313). The sulfate process and 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments. 

B-1 
the chloride process, illustrated in Figure B-2, are two common methods used to produce TiO2 in a 

variety of grades for many different applications. 

Source: Millennium Inorganic Chemicals (2007, 195899). 

Figure B-2. Sulfate and chloride processes for TiO2 manufacture. 

The sulfate process, a wet process for creating pigmentary TiO2, dates from around 1930, and 

it was the dominant method used to produce TiO2 until the chloride process was developed in the 

1950s (Hext et al., 2005, 090567). The chloride process now accounts for approximately 60% of 

worldwide TiO2 pigment production (Hext et al., 2005, 090567). The chloride process, a gas-phase 

process, is more energy efficient than the wet-phase sulfate process; it can produce finer particles 

and particles with specific morphologies (Osterwalder et al., 2006, 157743). The sulfate process is 

used primarily to create pigmentary particles. Because attenuation-grade TiO2 can be produced using 

“the same processes as larger pigmentary grades”1

 (Schlossman et al., 2006, 093309), the sulfate 

process and the chloride process are considered in this document as possible manufacturing 

techniques for nano-TiO2 in sunscreen. 

The sulfate process and the chloride process differ in the feedstock and techniques for nuclei 

synthesis. In both processes, particles are milled and surface-treated to prepare them for the intended 

application. The “surface treatment” step in Figure B-2 corresponds to the “coating” step in 

Figure B-1. 

The Altair process, a patented, spray-hydrolysis-based process, is illustrated in Figure B-3. 

This process is used by Altair Nanotechnologies, Inc. to produce not only coated nano-TiO2 for 

sunscreen applications, but also uncoated and larger TiO2 particles and several ceramic oxides 

(Verhulst et al., 2003, 157854). The feedstock for this process is titanium oxychloride. This patented 

process is comparable in many respects to the sulfate process. What makes it unique, according to 

 

1 Pigment-grade refers to a classification of particles of size 200 nm or larger. However, any grade of particles will contain a range of 

particle sizes, and “[a]lthough pigment-grades of TiO2 are usually considered to consist of micron sized particles, particles below 100 nm 

may be present in such grades” (Scientific Committee, 2007, 157639).

 B-2 
Verhulst et al. (2003, 157854), is the spray hydrolysis step, which eliminates the aqueous filtration 

step. 

Source: Reprinted with permission for Verhulst et al. (2003, 157854) 

Figure B-3. Nano-TiO2 manufacturing process used by Altair Nanotechnologies, 

Inc. 

Details of the sulfate process, chloride process, and the Altair Process (derived from spray 

hydrolysis) are provided in the following paragraphs. The steps unique to each process are presented 

first, followed by steps shared in these processes. Additionally, processes specific to manufacturing 

nano-TiO2 include an additional gas-phase process (TiCl4 + 2H2O → TiO2 + 4HCl) and three 

additional wet processes (TiOCl2 + 2NaOH → TiO2 + 2NaCl + H2O ; Na2TiO3 + 2HCl → TiO2 + 

2NaCl + H2O ; and Ti(OR)4 + 2H2O → TiO2 + 4ROH) (Dransfield, 2005, 157809). The gas-phase 

process is similar to the chloride method except that the titanium tetrachloride is hydrolyzed rather 

than oxidized. It is also similar in some aspects to the Altair method. These three wet processes rely 

on feedstocks that are not found in nature, and thus require some additional, unspecified preparatory 

steps. Waste products from the various processes include hydrochloric acid, salt, water, and 

compounds formed from impurities. 

Specific Steps in the Sulfate Process 

The sulfate process begins with ilmenite ore (FeTiO3), which is dried, ground, and treated with 

concentrated sulfuric acid (H2SO4) in an exothermic digestion reaction, producing a cake of titanyl 

sulfate (TiOSO4) and other metal sulfates. This cake is then dissolved in water or a weak acid. After 

chemical flocculation, a clear solution and an insoluble mud are produced. The clear solution is 

cooled to crystallize ferrous sulfate heptahydrate (FeSO4 · 7H2O, known as “copperas”). The ferrous 

sulfate heptahydrate is separated and sold as a by-product (Millennium Inorganic Chemicals, 2007, 

195899). 

 B-3 
The insoluble mud is washed, filtered, and evaporated to produce a concentrated TiOSO4

liquor. The liquor is hydrolyzed to produce a suspension or “pulp” that consists mainly of colloidal 

hydrous titanium oxide clusters (Millennium Inorganic Chemicals, 2007, 195899). 

The TiO2 is precipitated from the suspension, which is typically facilitated by a seeding 

technique to control particle size (no description of the seeding technique was provided). After 

further washing, heat is applied to crystallize the particles in a process known as calcination, which 

is also used in other processes. Either anatase or rutile crystals can be produced, depending on the 

additives applied before calcination (Millennium Inorganic Chemicals, 2007, 195899). 

The following equations represent the chemical processes involved in the sulfate process 

(Dransfield, 2005, 157809): 

FeTiO3 + 2H2SO4 → TiOSO4 + FeSO4 + 2H2O 

TiOSO4 + H2O → TiO2 + H2SO4

Specific Steps in the Chloride Process 

Natural or synthetic rutile is the feedstock material for the chloride process. During the 

chlorination step, rutile is added to chlorine and a source of carbon in a fluidized bed at 900oC. The 

exothermic reaction produces titanium tetrachloride (TiCl4) plus a variety of impurities. As the gas 

cools, low-volatile impurities (e.g., iron, manganese, and chromium chlorides) condense out. A 

stable, very pure liquid TiCl4 is achieved following condensation and fractional distillation 

(Millennium Inorganic Chemicals, 2007, 195899). 

The pure TiCl4 is then oxidized to TiO2 in a second exothermic reaction. Temperature and 

other reaction parameters determine the mean particle size, size distribution, and crystal type of the 

resulting TiO2. The TiO2 is cooled, and impurities are removed. Chlorine released by the oxidation 

reaction is recycled for reuse (Millennium Inorganic Chemicals, 2007, 195899). 

The following equations represent the chemical processes involved in the chloride process 

(Dransfield, 2005, 157809): 

TiO2 (impure) + 2Cl2 + C → TiCl4 + CO2

TiCl4 + O2 → TiO2 + 2Cl2

Specific Steps in the Altair Process – Spray Hydrolysis 

The patented Altair process (Verhulst et al., 2003, 157854) was derived from a spray 

hydrolysis method for TiO2 synthesis. The feed is a titanium oxychloride aqueous solution. The feed 

solution can be produced by hydrating liquid TiCl4 in a dilute hydrogen chloride (HCl) solution. In 

spray hydrolysis, heat (from hot air or a hot receiving surface) causes rapid and complete 

evaporation of the water in the feed solution as the solution is sprayed. An amorphous, 

homogeneous, dense, thin film remains on the receiving surface. The film is composed of dry, 

hollow, almost completely amorphous, TiO2 particles containing some free or hydration water and 

some HCl (Verhulst et al., 2003, 157854). 

Calcination for Sulfate and Altair Processes 

Calcination is the process of heating a solid material to a temperature high enough to change 

its chemical composition (though generally not high enough to liquefy it). In wet processes like the 

sulfate and Altair processes, calcination generally occurs after the hydrolysis step. Verhulst et al. 

(2003, 157854) describe the calcined product as a porous crystalline structure of nanoparticles. The 

crystalline structure retains the shape of the original droplets from the hydrolysis step and will 

eventually be broken down by milling. The duration and temperature of calcination and the additives 

 B-4 
introduced during calcination directly influence the structure, particle size, and particle-size 

distribution of the calcined product. For example, the anatase structure can be stabilized by adding 

phosphates during calcination (Verhulst et al., 2003, 157854). 

Milling and Micronizing for Sulfate, Chloride, and Altair Processes 

1 Milling breaks apart the hollow crystalline lattice structure produced in the calcination step, 

but has to be mild enough not to break the individual crystallites (Verhulst et al., 2003, 157854). 

Milling also breaks down agglomerates or aggregates into smaller particles. 

Both a wet media mill (e.g., with zirconia beads) and ultrasonic milling can be effective 

(Verhulst et al., 2003, 157854). After spray drying, the milled particles (“loosely agglomerated 

balls”) can be “further micronized to produce a dispersed powder.” While both micronizing and 

milling decrease the agglomerates, they are different processes. 

In micronizing, agglomerates collide with each other in a circulating stream of air or steam, 

and the collision breaks down the agglomerated particles. In milling, an external grinding agent is 

used to decrease the size of agglomerates. For instance, agglomerates in a liquid medium are fed into 

a mill containing small ceramic beads, and the impact from the beads on the agglomerates during 

mixing break the agglomerated particles. 

B.1.2. Surface Treatments and Doping 

Some, but not all, nano-TiO2 particles used for sunscreen undergo surface treatment to prevent 

the creation of free radicals, which could degrade the sunscreen or damage the skin (DuPont, 2007, 

157699; Schlossman et al., 2006, 093309; Wakefield et al., 2004, 193693). Surface coatings for 

nano-TiO2 in sunscreen can include combinations of inorganic oxides, simethicone, methicone, 

lecithin, stearic acid, glycerol, silica, aluminum stearate, dimethicone, metal soap, isopropyl titanium 

triisostearate (ITT), triethoxy caprylylsilane, and C9-15 fluoroalcohol phosphate. 

In a patent they hold, Mitchnik and O’Lenick (1996, 157935) describe a sample protocol for 

applying a silicone surface treatment to TiO2 for sunscreen. The patent does not specify the size of 

the TiO2 particles. A quantity of silicone compound (generally between 0.1% and 25% by weight of 

the total formulation) is combined with TiO2 powder. The mixture is heated to 40-100oC for 

2-10 hours, or long enough to remove 97% of the alcohol produced in the reaction. The patent 

holders claim that the resultant coated particles provide superior performance because the coating 

“preserves the structure of the TiO2 crystals, eliminates the reactivity in water, and makes them 

hydrophobic.” 

Nano-TiO2 particles can also be doped with various metals such as manganese, vanadium, 

chromium, and iron. Park et al. (2006, 193593) listed examples of doping methods, including: 

(1) combining particles of a host TiO2 lattice with a second component in solution or suspension, and 

then baking at no lower than 300oC. The second component is typically a salt, such as a chloride, or 

an oxygen-containing anion, such as a perchlorate or a nitrate; (2) mixing solutions of the dopant salt 

and of a titanium alkoxide, and then heating the solution to convert the alkoxide to the oxide and 

precipitate out the doped material; and (3) flame pyrolysis 2

 or plasma routes (no additional detail 

provided). 

 

1 Lattice is the geometrical arrangement of atoms in a crystal. 

2 Flame pyrolysis is a synthesis method in which flame heat is applied to vaporize stock material (gas phase precursors) and to initiate 

chemical reaction for particle (including nanoparticles) production. 

 B-5 
B.2. Nano-TiO2 Particles and Products Used in 

Sunscreens 

Several commercially-available nano-TiO2 particles intended for sunscreen application and 

some of their characteristics are summarized in Table B1 (SCCNFP, 2000, 092740), and an 

additional list is on the internet (EWG, 2009, 625314). Although these nano-TiO2 particles were 

selected for their applicability to the European market, they are likely to be fairly representative of 

nano-TiO2 active ingredients used in the U.S. 

Table B-1. Selected list of nano-TiO2 particles used in sunscreen 

Average 

crystal size Particle name Manufacturer Crystal type Coating materials and concentrations 

T805 Degussa20/80 RU/AN Degussa rutile/ anatase 21 nm silicone dioxide <2.5% 

T817 Degussa79/12/2 RU/AN/Fe Degussa rutile/ anatase 21 nm silicone dioxide <2.5% (also doped with di-iron trioxide 2%)

UV-Titan M160 Kemira rutile 17-20 nm alumina 5.5-7.5%, stearic acid 10% 

UV-Titan M212 Kemira rutile 20 nm alumina 5-6.5%, glycerol 1% 

UV-Titan X161 Kemira rutile 15 nm alumina 8.5-11.5%, stearic acid 10% 

UV-Titan X200 Kemira rutile 20 nm none 

Eusolex T-2000 Merck unknown 14 nm alumina 8-11%, simethicone 1-3% 

TTO 51A Merck rutile 35 nm alumina 11%, silica 1-7% 

TTO 51C Merck rutile 35 nm alumina 11%, silica 1-7%, stearic acid 3-7% 

MT-100 AQ Mitsubishi/Tayca rutile 15 nm alumina 4-8%, silica 7-11% 

MT-100 AR Mitsubishi/Tayca unknown 15 nm alumina 4-8%, silica 7-10% 

MT-100 T-L-1 Mitsubishi/Tayca rutile 15 nm alumina 3.3-7.3%, stearic acid 5-11% 

MT-100SA Mitsubishi/Tayca rutile 15 nm alumina 4-7.5%, silica 2-4% 

MT100TV (or MT-100TV) Mitsubishi/Tayca rutile 15 nm alumina 1-15% or 3-8%; aluminum stearate 1-13% or 

1-15% or stearic acid 5-11% 

MT100Z (or MT-100Z) Mitsubishi/Tayca rutile 15 nm alumina 6-10%, stearic acid 10-16% 

MT-500SA Mitsubishi/Tayca rutile 35 nm alumina 1-2.5%, silica 4-7% 

Mirasun TiW60 Rhodia anatase 60 nm alumina 3-7%, silica 12-18% 

UV-Titan M262 Rhodia and Kemira rutile 20 nm alumina 5-6.5%, dimethicone 1-4% 

Tioveil dispersions Uniqema rutile 10-28 nm alumina 10.5-12.5% or 5-15% and silica 3.5-5.5%; alumina 

5-15% and aluminum stearate 5-15% 

Source: Used with permission of the European Union, SSCNFP (Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers) (SCCNFP, 2000, 092740).

Three manufacturers of USP-grade nano-TiO2 for sunscreen applications provided information 

on their products and processes: Kobo Products Inc., which specializes in powders and dispersions; 

Oxonica, a European nanomaterials group; and Uniqema, a manufacturing company specializing in 

oleochemicals 1

 and specialty chemicals for cosmetics and personal care products. Uniqema was 

acquired by Croda in 2006 (Croda, 2006, 193851). 

Kobo manufactures a line of 26 attenuation-grade TiO2 dispersions containing nano-TiO2. The 

primary particle sizes are mostly 10-35 nm in 25 of 26 dispersions; one dispersion contains 90 nm 

 

1 Oleochemicals, e.g., fatty acids, fatty alcohols, and fatty esters, are derived from biological oils or fats. 

 B-6 
primary TiO2 particles. The nano-TiO2 aggregate sizes in dispersions (measured by dynamic light 

scattering [DLS]) are mostly 103-165 nm in 25 of 26 dispersions, including the dispersion with 

90 nm primary particles; one dispersion contains 230 nm aggregates (Kobo Products Inc, 2009, 

196045). One of the Kobo TiO2 dispersions called TNP40VTTS contains nano-TiO2 particles coated 

with alumina and an isopropyl titanium tri-isostearate/triethyl caprylysilane crosspolymer 

(Kobo Products Inc, 2009, 196045; Shao and Schlossman, 1999, 093301). Polyhydroxystearic acid is 

used to disperse the product in the solvent/carrier, C12-15 alkyl benzoate, which is an ester 

(Kobo Products Inc, 2009, 196045; Shao and Schlossman, 1999, 093301). The particles in another 

dispersion, CM3K40T4, are surface-treated with alumina and methicone and are dispersed in the 

cyclopentasiloxane carrier with the help of PEG-10 dimethicone (Kobo Products Inc, 2009, 196045; 

Shao and Schlossman, 1999, 093301). 

OptisolTM UV Absorber, a nano-TiO2 product, is the first commercial product from Oxonica 

Materials (a branch of Oxonica), and the first commercial health product from Oxonica. OptisolTM is 

a powder composed of uncoated rutile nano-TiO2 (size not specified) with approximately 0.67% 

manganese in the crystal lattice (Kobo Products Inc, 2009, 196045; Shao and Schlossman, 1999, 

093301). Doping with manganese gives the sunscreen the advantages of increased UV-A absorption, 

reduced free radical generation, and increased free radical scavenging behavior (Reisch, 2005, 

155634; Umicore, 2008, 193688). 

1 Uniqema/Croda manufactures several TiO2 sunscreens, including a line of SolaveilTM Clarus 

using nano-TiO2 (Chandler, 2006, 193834). Solaveil CT-100 and Solaveil CT-200, two of the 

products in the Solaveil Clarus line, are discussed here as examples. Solaveil CT-100 has more than 

50% C12-C15 alkyl benzoate, 25-50% nano-TiO2, and 1-5% each of aluminum stearate, 

polyhydroxysteric acid, and alumina (Croda, 2007, 193875). Solaveil CT-200 has 15-40% 

nano-TiO2, 10-30% isohexadecane, 10-30% glycerol tri(2-ethylhexanoate), 3-7% aluminum stearate, 

and 1-5% each of polyhydroxysteric acid and aluminum oxide (Croda, 2008, 193878). The TiO2

particle size distribution is very narrow, with the vast majority of particles falling in the nano-range 

(Croda, 2008, 193878). Uniqema (2004, 155637) recommended using CT-200 at a concentration of 

2-30%. The dispersion can be included in the oil phase in an oil-in-water (o/w) emulsion, or in the 

water phase in water-in-oil (w/o) emulsion, or added separately to a w/o emulsion after 

emulsification (Uniqema, 2004, 155637). 

B.3. Formulations for Sunscreen Containing Nano-TiO2

Sunscreen formulations that major manufacturers use are proprietary. Companies that produce 

sunscreen ingredients, however, promote their products by publicizing suggested formulations. 

These suggested formulations indicate the types of ingredients and processes that might be typical in 

sunscreen formulation. Two such suggested formulations are discussed here. 

Generally, compatible ingredients are combined into a number of fluid phases. These phases 

are then energetically mixed in a particular sequence (sometimes at specified temperatures) to form 

an emulsion. Formulators have to take care not to allow the pH of the mixture to reach the isoelectric 

point (IEP) of the nano-TiO2 or any other dispersed ingredient. 

Table B-2 shows a sample formulation using Croda Solaveil CT-10W and Solaveil CT-200 

(Croda, 2009, 193880). Table B-3 lists a sample formulation that uses nano-TiO2 from Kobo for 

SPF 35 sunscreen that appears transparent when applied on skin (Kobo Products Inc, 2009, 196045). 

 

1

 Croda acquired Uniqema in 2006 (Croda, 2006, 193851). In this Appendix, information sources are cited as it was presented at the time 

of publication. 

 B-7 
Table B-2. Formula SC-383-1 for “Weightless Morning Dew with Sun Protection” 

Ingredients % 

Part A 

Water QS 

Hydroxypropyl starch phosphatea 1.00 

Arlatone V-150 [steareth-100 (and) steareth-2 (and) mannan (and) xanthan gum] 0.50 

Arlatone LC 2.00 

PricerineTM 9088 (glycerin) 4.00 

Solaveil CT-10W [water (and) TiO2 (and) isodeceth-6 (and) oleth-10 (and) aluminum stearate (and) 

alumina (and) simethicone] 

5.00 

Part B 

Solaveil CT-200 [TiO2 (and) isohexadecane (and) 2.00 

triethylhexanoin (and) aluminum stearate (and) alumina (and) polyhydroxystearic acid] 

Ethyl methoxycinnamateb 4.00 

BRIJTM 721 (steareth-21) 2.00 

Arlamol PS15E (PPG-15 stearyl ester) 5.00 

Part C 

Phenoxyethanol (and) methylparaben (and) ethylparaben (and) 1.00 

propylparabenc

pH: 6.75 ± 0.5; viscosity: 223.5 ± 10% (centipoise) cps 

Procedure: 

Disperse Arlatone V-150 in water. Then disperse the preservative. Add Pricerine 9088 and heat to 60°C and add Arlatone LC. 

Continue heating to 80°C and add Solaveil CT-10W. Combine and heat Part B to 80°C. Add Part B to Part A. Homogenize for 

2 minutes. Return to stirring and cool to 40°C. Add Part C. Stir to room temperature. 

Note: QS means a sufficient quantity. 

a

Structure XL, National Starch 

b

Eusolex 2292, Merck KGaA 

c

Phenonip XB, Clariant 

Source: Croda (2009, 193880). 

 B-8 
 B-9 

Table B-3. Formula KSL-17 for High SPF Transparent Sunscreen 

Ingredients % 

Part 1 

Rose Talc-MS2 – Kobo Products : Talc (and) Methicone 1.00 

Velvesil 125 – Momentive/Kobo Products : Cyclopentasiloxane (and) C30-45 Alkyl Cetearyl Dimethicone Crosspolymer 3.00 

Net-WO – Barnet : Cyclopentasiloxane (and) PEG-10 Dimethicone (and) Disteardimonium Hectorite 0.20 

CM3K40T4 – Kobo Products : Cyclopentasiloxane (and) TiO2 (and) PEG-10 Dimethicone (and) Alumina (and) Methicone 35.00 

Uvinul MC80 – BASF : Ethylhexyl Methoxycinnamate 7.00 

Salacos 99 – Nisshin Oil : Isononyl Isonanoate 5.00 

Lexol EHP – Inolex Chemical : Ethylhexyl Palmitate 4.00 

Squalane – Fitoderm : Squalane 0.20 

Tocopherol – Cognis : Tocopherol 0.20 

SF96-350 – Momentive/Kobo Products : Dimethicone 1.00 

SF96-100 – Momentive/Kobo Products : Dimethicone 1.00 

SF1202 – Momentive/Kobo Products : Cyclopentasiloxane 27.10 

Propyl Paraben NF – International Sourcing : Propylparaben 0.10 

Part 2 

Sodium Citrate – Roche : Sodium Citrate (and) Water 2.00 

Net-DG – Barnet : Dipotassium Glycyrrhizinate 0.10 

Sodium Hyaluronate – Centerchem : Sodium Hyaluronate (and) Water 1.00 

Keltrol CG-T – CP Kelco : Xanthan Gum (and) Water 2.00 

Butylene Glycol – Ruger : Butylene Glycol 4.00 

Methyl Paraben NF – International Sourcing : Methylparaben 0.10 

Water 6.00 

Manufacturing Procedure: 

*Use explosion-proof mixers and equipment during batching process * 

Mix each Part separately. Make sure Net-WO is dispersed in Part 1. 

Heat both Parts to 40oC and add Part 2 to Part 1 while stirring with homogenizer at 3,000 rotations per minute (rpm). 

Increase the rotation to 5,000 rpm and continue to emulsify for 5 minutes. 

Cool down to room temperature with sweeping mixer. 

Source: Kobo Products Inc. (2009, 196045). 
REFERENCES 

Chandler M (2006). Innovative UV Protection. Creating your advantage in sunscreen products. Talk presented at Croda 

Educational eSeminar, Edison, NJ. 

http://www.cosmeticsandtoiletries.com/networking/eventcoverage/3729901.html. 193834

Chen X; Mao SS (2007). Titanium dioxide nanomaterials: Synthesis, properties, modifications, and applications. Chem 

Rev, 107: 2891-2959. 193313

Croda (2006). Croda invests in growth: Acquires Uniqema [brochure]. Cosmetics & Toiletries. 193851

Croda (2007). Product Trade Name: SOLAVEIL CT-100 [brochure]. 193875

Croda (2008). Chemical Composition of Solaveil CT-200 [brochure]. 193878

Croda (2009). Weightless Morning Dew with Sun Protection (SC-383-1) [brochure]. 193880

Dransfield G (2005). Manufacture of novel, transparent TiO2 based sunscreens. Retrieved May 09, 2008 from 

http://www.wun.ac.uk/nanomanufacturing/archive/05_06_series/documents/dransfield.pdf. 157809

DuPont (2007). Nanomaterial Risk Assessment Worksheet DuPont(TM) Light Stabilizer. Retrieved June 18, 2008 from 

http://www.edf.org/documents/6913_TiO2_Worksheet.pdf. 157699

EWG (2009). EWG's 2009 sunscreen investigation - Section 5: Impact of nanoparticles. Retrieved April 29, 2010 from 

http://www.ewg.org/cosmetics/report/sunscreen09/investigation/impact-of-nanoparticles. 625314

Hext PM; Tomenson JA; Thompson P (2005). Titanium dioxide: inhalation toxicology and epidemiology. Ann Occup Hyg, 

49: 461-472. 090567

Kobo Products Inc (2009). Attenuation grade TiO2 dispersions. Retrieved July 16, 2009 from 

http://www.koboproductsinc.com/Downloads/Kobo-TiO2Dispersions.pdf. 196045

Millennium Inorganic Chemicals (2007). Titanium dioxide manufacturing processes. Retrieved February 16, 2009 from 

http://www.millenniumchem.com/Products+and+Services/Products+by+Type/Titanium+Dioxide+-

+Paint+and+Coatings/r_TiO2+Fundamentals/Titanium+Dioxide+-

+Paint+and+Coatings+TiO2+Fundamentals_EN.htm. 195899

Mitchnick M; O'Lenick AJ Jr (1996). Patent No. 5565591 (class: 556/10). United States: United States Patent Office. 

http://www.freepatentsonline.com/5565591.html. 157935

Osterwalder N; Capello C; Hungerbühler K; Stark WJ (2006). Energy consumption during nanoparticle production: how 

economic is dry synthesis? J Nanopart Res, 8: 1-9. 157743

Park GB; Knowland JS; Flutter BR (2006). Patent No. 20060134026, class: 424/59. United States: U.S. Patent and 

Trademark Agency. http://www.freepatentsonline.com/y2006/0134026.html. 193593

Reisch MS (2005). New-wave sunscreens: active ingredient makers are frustrated by the long list of sunscreens and UV-A 

testing protocols that are still awaiting FDA decisions. Chem Eng News, 83: 18-22. 

http://pubs.acs.org/cen/coverstory/83/8315sunscreens.html. 155634

SCCNFP (2000). Opinion of the scientific committee on cosmetic products and non-food products intended for consumers 

concerning titanium dioxide Colipa n S75 (Report No. SCCNFP/0005/98). Brussels, Belgium: Scientific 

Committee on Cosmetic Products and Non-Food Products Intended for Consumers. 

http://ec.europa.eu/health/ph_risk/committees/sccp/documents/out135_en.pdf. 092740

Schlossman D; Shao Y; Detrieu P (2006). Perspectives on supplying attenuation grades of titanium dioxide and zinc oxide 

for sunscreen applications. Retrieved October 28, 2007 from 

http://www.fda.gov/nanotechnology/meetings/kobo_files/textmostly/index.htm. 093309

Scientific Committee on Computer Products (2007). Preliminary opinion on safety of nanomaterials in cosmetic products. 

Brussels, Belgium: European Commission, Health and Consumer Protection Directorate-General. 157639

Shao Y; Schlossman D (1999). Effect of particle size on performance of physical sunscreen formulas. Talk presented at 

PCIA Conference, Shanghai, China. http://www.koboproductsinc.com/Downloads/PCIA99-Sunscreen.pdf. 093301

B-10 
 B-11 

Umicore (2008). Nano-sized oxide powders for UV applications. Abstract from Innovation for Sustainable Production (iSUP2008), Bruges, Belgium. 193688

Uniqema (2004). Solaveil CT-200 (Report No. PC/E/03-03/GLOB/14.5/CT200). London: Uniqema. 155637

Verhulst D; Sabacky BJ; Spitler TM; Prochazka J (2003). Process for the production of nano-sized TiO2 and other ceramic 

oxides by spray hydrolysis. Reno, Nevada: Altair Nanomaterials Inc. 157854

Wakefield G; Lipscomb S; Holland E; Knowland J (2004). The effects of manganese doping on UVA absorption and free 

radical generation of micronised titanium dioxide and its consequences for the photostability of UVA absorbing 

organic sunscreen components. Photochem Photobiol Sci, 3: 648-652. 

http://www.rsc.org/delivery/_ArticleLinking/DisplayArticleForFree.cfm?doi=b403697b&JournalCode=PP. 193693 
Annex C. Nano-TiO2 Exposure Control in the 

Workplace and Laboratory 

C.1. Workplace Exposure Controls 

This section provides examples of strategies that are currently in place or recommended to 

decrease exposures to nanomaterials in the workplace (Nanosafe, 2008, 196066; NIOSH, 2009, 

196073) and to ensure the effectiveness of personal protective equipment (PPE) against nano-TiO2

(Golanski et al., 2008, 196048; Guizard and Tenegal, 2008, 196049) (Nanosafe, 2008, 196066). 

Other approaches to reduce worker exposure have been developed and are undergoing further 

refinement; the examples provided here are intended to be illustrative rather than exhaustive. While 

this section focuses on workplace practice of nanomaterial manufacturers, some of the principles and 

use of PPE are also applicable to laboratories and other settings. 

The NanoSafe Dissemination Report (Nanosafe, 2008, 196066) provided several tiers of 

approaches to decrease nanomaterial exposure in the workplace. During production, the first and 

preferred approach is to avoid potential exposure to free air flowing particles. If this avoidance is not 

possible, the process should be contained. If process containment is not possible, extended PPE 

(which includes double gloves of nitrile, a mask [FFP3 or powered respirators incorporating 

helmets], a protective suit, and safety shoes) and an effective local exhaust system, such as a high 

efficiency particulate air (HEPA) H14 filter, should be used. 

During loading and unloading of reactors, and while packing containers, exposure can be 

decreased by process containment (e.g., by using a glove box or emptying the reactor using an 

industrial vacuum with a HEPA filter through a liquid trap) (Nanosafe, 2008, 196066). Less 

preferred alternatives are to transfer nanoparticles within a laminar air-flow booth or extraction hood, 

or to conduct the transfer in an isolated area equipped with HEPA H14 filter. These alternative 

options would require the use of extended PPE (Nanosafe, 2008, 196066). 

During cleaning, special vacuums to avoid dust explosion can be used to trap nanoparticles. 

The vacuums should be cleaned in a room equipped with a HEPA H14 filter and a washer to clean 

the protective suites (Nanosafe, 2008, 196066). Alternatively, particles can be drawn into a powdercollection system using a variable-speed fan. Components should be cleaned in a hood equipped 

with a HEPA filter and an explosion vent panel. 

NIOSH has a nanotechnology program to increase safety and decrease potential exposures to 

nanomaterials in the workplace (NIOSH, 2009, 196073). In a NIOSH document for safe 

nanotechnology (NIOSH, 2009, 196073), occupational health surveillance and guidelines for 

working with engineered nanomaterials are discussed, among other topics. Some of these programs 

could also educate and encourage the general public to reduce environmental releases from the 

products into the environment. Some companies that manufacture nano-TiO2 have engineering 

safeguards and additional programs in place to reduce or eliminate occupational and environmental 

exposures (e.g., BASF, 2008, 193811; DuPont, 2007, 157699). Various production methods to 

decrease worker exposure are also being investigated [for nano-TiO2, see Guizard and Tenegal 

(2008, 196049)]. 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments. 

C-1 
With a goal toward managing nanotechnology safely and effectively within the industrial 

setting, the NOSH Consortium has investigated methods for monitoring workplace exposure and 

testing protective technologies. The NOSH Consortium has measured the effectiveness of standard 

respiratory filters with silicon dioxide (SiO2) aerosol nanoparticles. With the exception of prolonged 

exposure (400 minutes or longer), the filter efficiencies for both charged and re-neutralized SiO2

aerosol nanoparticles met the specifications of the filter type (Ostraat, 2009, 196077). The longest 

exposure time within which the N100 filter performed at or exceeded the efficiency specified by the 

filter ranking (>99.97% filtration efficiency) was 210 minutes (Ostraat, 2009, 196077). No PPE 

specifically designed for nanomaterials exists or is under development (Klaessig, personal 

communication, 2008, 196042). For filter efficacy against nano-TiO2 aerosol penetration tested by 

NanoSafe, see Section C.1.1. 

C.1.1. Personal Protective Equipment 

In this section, two types of PPE are briefly discussed in terms of their protection against 

nano-TiO2 aerosols: (1) filters for inhalation protection; and (2) protective clothing and gloves for 

skin protection. Eye-protective gear is available as a third type of PPE commonly used for protection 

against nano-TiO2 aerosols, but no information was found on this subject. 

Each type of nanomaterial is different, and the methods for testing PPE efficiency (such as 

using charged or neutralized particles) could greatly affect the measured barrier effectiveness. For 

example, fibrous filters often remove more charged aerosol nanoparticles than uncharged or 

neutralized aerosol nanoparticles (Kim et al., 2006, 193470; Ostraat, 2009, 196077). Other 

physicochemical properties of nanoparticles that affect filtration efficiency include size, chemical 

composition, and shape. The size of the particle that most effectively penetrates into a specific filter 

is called the maximum penetrating particle size (MPPS). For particles smaller than the MPPS, the 

particle penetrations decrease with decreasing particle size; for particles larger than the MPPS, the 

particle penetrations decrease with increasing particle size. Particles smaller than the pore size of the 

filter may be filtered out when the Brownian movement of the particles leads to collision of the 

particle and filter (McKeytta, 1984, 196036). 

Electrostatic filters are charged polypropylene fibers, classified as FPP3 – minimum filtration 

efficiency 99% – based on European Norm (EN) certification. When an electrostatic filter was tested 

with nano-TiO2 aerosols, for which size ranged from 16 nm to greater than 76 nm, the MPPS was 

approximately 35 nm, which was very similar to graphite MPPS (Golanski et al., 2008, 196048). At 

the MPPS, however, nano-TiO2 penetration was nearly five times higher than that for graphite. Near 

the MPPS, the differences between nano-TiO2 and graphite particle penetration increase by an order 

of magnitude. 

HEPA filters have a minimum filtration efficiency of 99.97%, are composed of glass fibers, 

and are classified as H12 for particles <1 μm. Like electrostatic filters, HEPA filters showed one 

order of magnitude higher penetration of nano-TiO2 (10-19 nm) than that of graphite (10-19 nm), 

with the highest penetration at approximately 0.2% for 19-nm TiO2 (Golanski et al., 2008, 196048). 

The penetration efficacy of platinum (Pt) particles through HEPA filters was only slightly lower than 

that of nano-TiO2 particles. Golanski et al. (2008, 196048) showed that particle size alone might not 

be a sufficient indicator of HEPA filter performance and suggested that nano-TiO2 might penetrate 

fibrous filters more efficiently than other nanomaterials, namely graphite and Pt. The exposure 

duration of the Golanski et al. (2008, 196048) study was not reported, and therefore, it could be 

possible that the filtration efficiency of HEPA filters for nano-TiO2 might decrease with prolonged 

exposure, as was found for the N100 filter for more than 400 minutes of exposure to SiO2 aerosol 

nanoparticles (Ostraat, 2009, 196077). 

The efficacy of protective clothing in preventing nano-TiO2 penetration by diffusion was 

higher for nonwoven fabric than woven cotton and polyester fabric (Golanski et al., 2008, 196048). 

Air-tight, nonwoven, polyethylene Tyvek (115 μm thick) was more effective against nanoparticle 

 C-2 
penetration than woven cotton (650 μm thick) and woven polyester (160 μm thick) for 10-nm 

nano-TiO2 (Golanski et al., 2008, 196048), 10-nm nano-Pt (Golanski et al., 2008, 196048), and 

40- and 80-nm graphite (Nanosafe, 2008, 196066). 

Nitrile, latex, and Neoprene gloves were reported to be effective against nano-TiO2 aerosol 

penetration via diffusion for a short exposure time (minutes). No penetration through gloves was 

detected when the gloves were exposed to aerosols of approximately 10-nm nano-TiO2 and 10-nm Pt 

(Golanski et al., 2008, 196048) or 20- to 100-nm graphite (Nanosafe, 2008, 196066). However, 

continuous flex of gloves could lead to cracks and holes in the gloves (Schwerin et al., 2002, 

193636), so changing gloves throughout the day is recommended (Harford et al., 2007, 196051). 

C.2. Manufacturer and Laboratory Practices 

In 2006, the University of California-Santa Barbara completed a study of nanomaterial 

manufacturers and laboratories for the International Council on Nanotechnology by surveying 

organizations about their manufacturing and laboratory practices. Survey results indicated that only 

36% of the 64 responding organizations stated that they monitored exposure to the nanomaterials in 

their workplace. Additionally, 38% of the organizations surveyed believed their nanomaterials posed 

no special risks, 40% had safety concerns, and 22% were unaware of whether or not the 

nanomaterials they worked with or manufactured pose safety risks (Gerritzen et al., 2006, 097620). 

Subsequently, the same research team published additional findings based on a larger sample 

size of 82 versus the original 64. Of the 82 responding firms and laboratories, 89% had a general 

environmental health and safety program, and 70% provided some type of special training on 

nanomaterial safety. Nanomaterial safety training was more prevalent in North American firms and 

laboratories (88%) than in European (64%) or Asian (61%) organizations. Nearly 82% of 

respondents made nano-specific PPE recommendations to employees. Those tended to be the same 

firms and laboratories that used advanced engineering controls (i.e., beyond fume hoods) to prevent 

exposure. Controls included exhaust filtration, air filtration, wet scrubbers, and automated or 

enclosed operations. Approximately 56% of North American respondents practiced workplace 

monitoring for nanoparticles, compared to 32% of all respondents. Waste-containing nanomaterials 

were disposed of as hazardous waste in 78% of North American organizations, compared to 60% of 

all respondents (Conti et al., 2008, 155619). 

A survey of 43 New England nanotechnology firms found that larger companies (with 500 or 

more employees) were more likely to recognize potential environmental health and safety (EHS) 

risks potentially posed by nanoparticles and had EHS measures in place. Many smaller firms either 

did not perceive risks or did not implement EHS measures (due both to staff and resource constraints 

and a lack of information on how to quantify nanoparticle risks) (Lindberg and Quinn, 2007, 

155629). 

 C-3 
 C-4 

REFERENCES 

BASF (2008). Guide to safe manufacture and for activities involving nanoparticles at workplaces in BASF AG. Germany: 

BASF. http://basf.com/group/corporate/en/function/conversions:/publish/content/sustainability/dialogue/indialogue-withpolitics/nanotechnology/images/BASF_Guide_to_safe_manufacture_and_for_activities_involving_nanoparticles.pd

f. 193811

Conti JA; Killpack K; Gerritzen G; Huang L; Mircheva M; Delmas M; Harthorn BH; Appelbaum RP; Holden PA (2008). 

Health and Safety Practices in the Nanomaterials Workplace: Results from an International Survey. Environ Sci 

Technol, 42: 3155-3162. 155619

DuPont (2007). Nanomaterial Risk Assessment Worksheet DuPont(TM) Light Stabilizer. Retrieved June 18, 2008 from 

http://www.edf.org/documents/6913_TiO2_Worksheet.pdf. 157699

Gerritzen MA; Lambooij E; Stegeman JA; Spruijt BM (2006). Slaughter of poultry during the epidemic of avian influenza 

in the Netherlands in 2003. Vet Rec, 159: 39-42. 097620

Golanski L; A. Guiot; Tardif F (2008). Experimental evaluation of individual protection devices against different types of 

nanoaerosols: graphite, TiO2 and Pt. Retrieved March 22, 2009 from 

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