United States __„ ,-„,_ _______
Environmental Protection EPA/600/R-09/057F
Agency
Nanomaterial Case Studies:
Nanoscale Titanium Dioxide in Water
Treatment and in Topical Sunscreen
November 2010
National Center for Environmental Assessment-RTF Division
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
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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.
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Table of Contents
List of Fiqures
List of Tables
Authors, Contributors, and Reviewers
Abbreviations
Foreword
Chapter 1. Introduction
1.1. Background
1.2. How to Read this Document
1.3. Terminology
1.4. Conventional Ti02
1.5. Nano-TiO?
1.5.1. Drinking Water Treatment
1.5.2. Sunscreen
1.6. Analytical Methods
1.6.1. Methods for Laboratory Research
1 .6.2. Methods and Instrumentation to Assess Environmental Occurrence
1 .6.3. Methods and Instrumentation to Assess Workplace Exposure
1.6.4. Summary of Analytic Methods
Chapter 2. Life Cycle Staqes
2.1. Feedstocks
2.2. Manufacturing
2.2.1 . Drinking Water Treatment
2.2.2. Sunscreen
2.3. Distribution and Storage
2.3.1 . Drinking Water Treatment
2.3.2. Sunscreen
2.4. Use
2.4.1 . Drinking Water Treatment
2.4.2. Sunscreen
2.5. Disposal
2.5.1 . Drinking Water Treatment
2.5.2. Sunscreen
Chapter 3. Fate and Transport
3.1. Water
3.1.1. Drinking Water Treatment
3.1.2. Sunscreen
3.2. Soil
3.2.1 . Drinking Water Treatment
3.2.2. Sunscreen
3.3. Air
vi
vii
viii
xiii
xix
1-1
1-1
1-3
1-4
1-7
1-8
1-12
1-13
1-14
1-15
1-16
1-18
1-19
2-1
2-1
2-2
2-3
2-3
2-3
2-4
2-4
2-4
2-4
2-6
2-7
2-7
2-7
3-1
3-2
3-4
3-5
3-5
3-7
3-7
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-Ti02 from Multiple Sources and Pathways 4-8
4.4. Cumulative Exposure to Nano-Ti02 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-Ti02 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-Ti02 5-1
5.1.1. Nano-Ti02 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-Ti02 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
Chapters. 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-Ti02 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-Ti02 in Sunscreen: Background Information A-1
REFERENCES A-8
Annex B. Nano-Ti02 in Sunscreen: Manufacturing Processes B-1
REFERENCES B-10
Annex C. Nano-Ti02 Exposure Control in the Workplace and Laboratory C-1
REFERENCES C-4
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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-Ti02 skin penetration. 4-19
Figure 5-1. The pulmonary effects of Ti02 or nano-Ti02 exposure through inhalation or instillation. 5-58
Figure B-1. Generic manufacturing process for nano-Ti02 for sunscreens. B-1
Figure B-2. Sulfate and chloride processes for Ti02 manufacture. B-2
Figure B-3. Nano-Ti02 manufacturing process used by Altair Nanotechnologies, Inc. B-3
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List of Tables
Table 1-1. Examples of nano-Ti02 physicochemical properties 1-9
Table 1-2. Characterization of three nano-TI02 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-Ti02 from sunscreen containing 5% nano-Ti02 for adults and 3-year-old children 4-5
Table 4-2. Tissue concentrations of various pollutants in fish after exposures to nano-Ti02 in water 4-13
Table 4-3. Nano-Ti02 disposition in animals after inhalation or intratracheal instillation 4-16
Table 4-4. Overview of Ti02 skin absorption/penetration studies 4-20
Table 4-5. Animal studies that measured Ti concentrations in brain after nano-Ti02 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-Ti02 ecological effects 5-11
Table 5-4. Summary of health effects of nano-Ti02 particles in mammalian animal models: dermal route 5-27
Table 5-5. Summary of health effects of nano-Ti02 particles in mammalian animal models: oral route 5-30
Table 5-6. Summary of health effects of nano-Ti02 particles in mammalian animal models: respiratory route 5-35
Table 5-7. Summary of health effects of nano-Ti02 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-Ti02 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-Ti02. 5-56
Table 5-10. Results of nano-Ti02 carcinogenicity studies in animals 5-59
Table A-1. Ti02 content in various sunscreen products. A-7
Table B-1. Selected list of nano-Ti02 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 Oberdorster—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.
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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—ARC ADI S
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 Gasman -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
Jay dee 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.
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
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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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EPA Workgroup1
Christian Andersen
Rochelle Araujo
Fred Arnold
Ayaad Assaad
Norman Birchfield
Deborah Burgin
Jim Caldwell
David Cleverly
Michele Conlon
Mary Ann Curran
Walter Cybulski*
J. Michael Davis*+
Jane Denne
Steve Diamond*
Jaimee Dong
Kevin Dreher*
Jeremiah Duncan
Brian Englert
Patricia Erickson
Cathy Fehrenbacher
Gina Ferreira
Kathryn Gallagher
Michael Gill
Michael Gonzalez
Maureen Gwinn+
Kathy Hart
Tala Henry
Ross Highsmith
Lee Hofmann
Marion Hoyer
Joe Jarvis
Bernine Khan
David Lai
Wen-Hsiung Lee
Laurence Libelo
Diana Locke
Jacqueline McQueen"
David Meyer
Gregory Miller
J. Vincent Nabholz
Nhan Nguyen
Carlos Nunez
David Olszyk
Martha Otto
Scott Prothero
Kim Rogers
Nora Savage
Phil Sayre
Rita Schoeny
Walter Schoepf
Najm Shamim
Deborah Smegal
Jose Solar
Neil Stiber
Timothy Taylor
Susan Thorneloe
Dennis Utterback
Amy Wang+
Eric Weber
Randy Wentsel
Doug Wolf
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
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Abbreviations
a-HBDH Alpha-hydroxybutyrate dehydrogenase
ACGIH American Conference of Governmental Industrial Hygienists
AFM Atomic force microscopy
A12(SO4)3-16H2O Alum
A12O3 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 fur 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
°C Degree(s) Celsius
C6o 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 fur 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
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EHS
ELISA
ELPI
EM
EN
EPA
EU
EWG
"F
F344
FDA
FE-SEM
FeTiO3
FFF
FHD
yH2AX
g
g/kg
GFAP
GGT
GSD
GSH
GSH-Px
GST
H202
H2S04
BBSS
HC1
HEPA
HPLC
hprt
HRTEM
Hz
i.p.
i.v.
IAEA
IARC
IC20, IC25
ICP
ICP-AES
ICP-MS
IEP
IFN-y
IL-10
Environmental health and safety
Enzyme-linked immunosorbent assay
Electrical low pressure impactor
Electron microscopy
European Norm
U.S. Environmental Protection Agency
European Union
Environmental Working Group
Degree(s) Fahrenheit
Fischer 344 (Rat strain)
U.S. Food and Drug Administration
Field emission-type scanning electron microscopy
Ilmenite
Field flow fractionation
Flame hydrolysis deposition
Phosphorylated form of histone H2AX (phosphorylation of H2AX at serine 139)
Gram(s)
Gram(s) per kilogram
Glial fibrillary acidic protein
y-Glutamyltransferase
Geometric standard deviation
Reduced glutathione
Glutathione peroxidase
Glutathione-S-transferase
Hydrogen peroxide
Sulfuric acid
Hank's Basic Salt Solution
Hydrochloric acid
High efficiency paniculate air
High performance liquid chromatography
Hypoxanthine-guanine phosphoribosyltransferase (gene)
High resolution transmission electron microscopy
Hertz
Intraperitoneal
Intravenous
International Atomic Energy Agency
International Agency for Research on Cancer
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
Inductively coupled plasma
Inductively coupled plasma atomic emission spectrometry
Inductively coupled plasma-mass spectrometry
Isoelectric point
Interferon-gamma
Interleukin-10
XIV
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IL-lp Interleukin-lp
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
jig Microgram(s)
jig/g Microgram(s) per gram
jig/kg Microgram(s) per kilogram
jig/L Microgram(s) per liter
jiL Microliter(s)
jim Micron(s), Micrometer(s)
jim2/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"1" 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
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MPPS
mSv
MT
MTC
MTP
Na+
NaCl
NAG
Nano-TiO2
Nano-TiO2 F-1R
NCEA
Nano-TiO2
ng/mL
NHEERL
NIOSH
nm
NMR
NMRI
NOEC
NOM
NOSH
02
OC
OECD
OH
•OH
OM
•OOH
OPC
OPPT
ORD
ORISE
OSHA PEL
OSP
n
P
P25
P805
PAM
PBS
PEC
pH
pHpzc
PGF
PMN
PNEC
Maximum penetrating particle size
Milliseviert
Metric ton(s)
Microbial Toxic Concentration, in microbial array for risk assessment (MARA) assay
Microsomal triglyceride
Sodium cation
Sodium chloride
Nacetyl-p-glucosaminidase
Nanoscale titanium dioxide
Nanoscale titanium dioxide a formula containing nano-TiO2 that is 3% anatase and 97% rutile
(U.S. EPA) National Center for Environmental Assessment
Nanoscale titanium dioxide
Nanogram(s) per milliliter
(U.S. EPA) National Health and Environmental Effects Research Laboratory
National Institute for Occupational Safety and Health
Nanometer(s)
Nuclear magnetic resonance
Naval Medical Research Institute
No observed effect concentration
Natural organic matter
Nanoparticle Occupational Safety and Health (Consortium)
Superoxide radical anion
Octocrylene
Organization for Economic Co-operation and Development
Hydroxyl
Hydroxyl radical(s)
Octyl methoxycinnamate
Hydroperoxyl radical(s)
Optical particle counter
(U.S. EPA) Office of Pollution Prevention and Toxics
(U.S. EPA) Office of Research and Development
Oak Ridge Institute for Science and Education
Occupational Safety and Health Administration permissible exposure limit
(U.S. EPA) Office of Science Policy
Pi, approximately equal to 3.14159
Pink-eyed dilution (gene)
Degussa Aeroxide® P25 (uncoated nano-TiO2)
Degussa Aeroxide® P805 (hydrophobic nano-TiO2)
Pulse amplitude modulation
Phosphate buffered saline
Predicted environmental concentration
Measure of acidity or alkalinity of a solution
pH at the point of zero charge
Placenta growth factor
Polymorphonuclear neutrophil
Predicted no-effect concentration
XVI
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ppb
PPE
ppm
PTFE
Pt
PTM
pun
RLE-TN
ROS
rPTM
RT-PCR
°g
s.c.
SAXS/WAXS
SCCNFP
SCCP
scro
SEC
SEM
Si02
SMPS
SOD
SPF
SPM
St-Cn
SWCNT
T805
TEC
TEM
TEOM®
TFF
TGA
TGF-P
THF
Ti
TiCL,
TiO2
TiOSO4
TLV
TNF-a
TRAIL
TS
TUNEL
USP
uv
Part(s) per billion
Personal protective equipment
Part(s) per million
Polytetrafluoroethylene
Platinum
Particle tracking model
Pink-eyed unstable (locus)
Rat alveolar type II epithelial cell line
Reactive oxygen species
Radius particle tracking model
Reverse transcription polymerase chain reaction
Geometric standard deviation
Subcutaneous
Small- and wide- angle X-ray scattering
(European Commission) Scientific Committee on Cosmetic Products and Non-Food Products
Intended for Consumers
(European Commission) Scientific Committee on Consumer Products
Severe combined immunodeficiency
Size exclusion chromatography
Scanning electron microscopy
Silicon dioxide
Scanning mobility particle sizer
Superoxide dismutase
Sunburn protection factor
Scanning probe microscopy
Sunscreen standard C from the Japan Cosmetic Industry
Single-walled carbon nanotube(s)
Degussa Aeroxide® T805 (hydrophobic nano-TiO2)
Threshold effect concentration
Transmission electron microscopy
Tampered element oscillating microbalance
Tangential-flow ultrafiltration
(Australian) Therapeutic Goods Administration
Transforming growth factor-beta
Tetrahydrofuran
Titanium
Titanium tetrachloride
Titanium dioxide
Titanyl sulfate
Threshold limit value
Tumor necrosis factor-alpha
Tumor necrosis factor-related apoptosis-inducing ligand
Technical Specification
Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling
U.S. Pharmacopeia
Ultraviolet (light/radiation), wavelengths in the range of 10 to 400 nm
XVII
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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
XVIII
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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.
XIX
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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.
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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).
/-k.
o<
Life Cycle
Stages
Feedstocks
Manufacture
Distribution
Storage
Use
Disposal
I
jmprenensive tnviionineniai Assessmeni
Environmental Fate & Exposure -
_ ,. _ _ Effects
Pathways Transport Dose
\
Air } Primary 1 "|
contaminants \ \ Ecosystems
f Water C Human
Secondary „,,,«„.. Human Health
Soil J contaminants) P°Pulatlons J
I I I I
Analytical methods development and application
Source: Adapted from Davis and Thomas (2006, 089638) and Davis (2007,
Figure ^-^. 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.
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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 nanomaterial s 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.
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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 decision-
making.
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
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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-Ti02
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 Ti02
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 "ultrafme," "PM-0.1" (which means particulate matter up to 0.1 micrometer [|im] 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.
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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 um mass median aerodynamic diameter [MMAD]
nano; 0.78 um MMAD fine) (Oberdorster, 2000, 036303: Oberdorster 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 "[aggregates
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
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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 nano-
TiO2 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 Ti02
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).
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1.5. Nano-Ti02
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 7i 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 n 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,
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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-TiCh 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
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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 (im, 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 (im, 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-um 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 x 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-Lite™ SF (manufactured by BASF company, Germany) had a
mainly rutile TiO2 core, coated with A1(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 A1(OH)3 outer layer was more stable.
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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 ultrafme 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-Ti02 particle types
Particle BET Surface /«i.«~.i««ir««~.~«»iti«- Chemical
Type Area(m2/g) Chemical Composition ReaBljvjtl
Uf-A 18.2
Uf-B 35.7
Uf-C 38.5
98% Ti02 (100% rutile), 2% alumina 10.1
88% Ti02 (1 00% rutile), 5% alumina, 1 .2
7% silica
92% Ti02 (79% rutile; 21 % anatase), 0.9
7% alumina, 1 % silica
Median Particle Size and u .
Size Range' gHm^
in Water0 in PBS Water
136nm±35% 1,990nm±25% 5.64
149.4 nm± 50% 2,669 nm± 25% 7.14
140nm±44% — 4.80
BET - Brunauer, Emmett, Teller method of calculating surface area
PBS - Phosphate buffered saline
"Chemical reactivity was tested using a Vitamin C (antioxidant) yellowing assay.
bAfter sonication for 15 min at 60 Hertz (Hz).
C0.1% 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, Kolaf 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
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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 nano-
TiO2 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 full-
scale 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; Purifies, 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
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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 photo catalysis, 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.
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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-l, 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 um, 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 anano-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
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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 (Kolar 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; video-
enhanced differential interference contrast (VEDIC) microscopy for uptake and localization
(Marquis et al., 2009, 193539); and scanning probe microscopy (SPM) for size and three-
dimensional 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.
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Table 1-3. Analytical methods for characterizing nanomaterials in aerosol and in liquid
Metric
Number
Surface area3
Mass
Size
Method
Condensation particle counter (CPC)
Scanning mobility particle sizer (SMPS)
Electrical low pressure impactor (ELPI)
Optical particle counter (OPC)
Electron microscopy (EM)
Scanning mobility particle sizer (SMPS)
Electrical low pressure impactor (ELPI)
SMPS and ELPI used in parallel
Diffusion charger
Specific surface area (BET, titration, diffusion charging)
Size selective personal sampler
Size selective static sampler
Tapered element oscillating microbalance (TEOM®)
Scanning mobility particle sizer (SMPS)
Electrical low pressure impactor (ELPI)
Dynamic light scattering (DLS)
Centrifugal sedimentation
Laser diffraction/static light scattering
Low pressure impactor and electrical low pressure impactor (ELPI)
Scanning/differential mobility analysis
Field flow fractionation (FFF)
Size exclusion chromatography (SEC)
Acoustic techniques
Electron microscopy (EM)
Scanning probe microscopy (SPM)
Time of flight mass spectroscopy
Atomic force microscopy (AFM)
Aerosol
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Maybe
No
Yes
Yes
Yes
No
No
No
No
Maybe
Yes
No
Liquid
No
No
No
Maybe
Yes
No
No
No
No
Titration techniques only
No
No
No
No
No
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Possible with
cryotechniques
Yes
No
Maybe
"For some particle shapes, electron microscopy can be used to estimate surface area (LeBlanc, 2009, 6252091. 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, 6252091.
Source: Modified with permission from Informa Healthcare, Maynard and Aitken (2007, 090674): Used with permission from Oxford University Press, Powers et al. (2006,
0887831: Used with permission from Informa Healthcare, Powers et al. (2007, 0906791: and data from Nanosafe (2008, 5948681
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 Valcarcel, 2009, 193648).
Also, many analytical methods require sample treatment and extraction (Englert, 2007, 193367).
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which may include solvent evaporation, and consequently could cause nanoparticle aggregation and
salt precipitation (Simonet and Valcarcel, 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 Valcarcel, 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; Hassellov et al., 2008, 157559; Stone et al., 2010, 633898; Tiede et al.,
2008, 196278; Tiede et al., 2009, 633895; Tiede et al., 2009, 193680).
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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 (IFF) 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 Only nanomaterials with a regular or pseudo-
(AFM) (height) measurements, and particle nanocrystalline geometries regular geometry and without significant
porosity
Phase and crystalline Electron diffraction
structure
X-ray diffraction (XRD)
X-ray absorption spectroscopy (XAS)
Raman spectroscopy
Source: Data from U.S. EPA (2008,1574801.
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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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.
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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.
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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
(HC1) 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 urn, 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 HC1 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 post-
manufacturing 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).
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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 Purifies) that can be used for drinking water, ground water, and wastewater
treatment (NSF International, 2009, 196092: Pichat, 2003, 196037: Purifies, 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
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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 retail-
sized 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.,
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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 (A12(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 (im (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, 1577991
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 Purifies, 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 (Purifies, 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 Purifies system was pilot-tested for two months in a community drinking water treatment
facility (Purifies, 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; Purifies, 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
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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.
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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 (floe) 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.
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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 nano-
TiO2, 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.
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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, 2016041
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 steady-
state, 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 nano-
TiO2 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 (ig/L
in tertiary effluent from wastewater treatment. Another investigator studied the effects of nano-TiO2
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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 poly disperse 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
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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 um membrane as a final process was able to leave
only 1% to 8 % of the total TiO2 mass (in floes 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 floe 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. EPAs 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
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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; Pent 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
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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.
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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 (floe) containing
nano-TiO2 would likely be created in the process of using nano-TiO2 to treat drinking water. If
nano-TiO2 settles with floe 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.
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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.
Dose2 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
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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-TiO2on 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.
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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).
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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-l 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 water-
resistant 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).
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Table 4-1. Estimated dermal exposure to nano-Ti02 from sunscreen containing 5% nano-Ti02 for
adults and 3-year-old children
Subject skirffcm^ °
3-yr-old child, total body surface 6,640
(50th percentile)
3-yr-old child, total body surface 7,640
(95th percentile)
Adult, body surface area 20,000
subjected to water contact in
swimming (50th percentile)
Adult, body surface area 23,000
subjected to water contact in
swimming (95th percentile)
AoDlied sunscreen APPlied sunscreen Applied1"
enrfo^ ^neitw amount nano-Ti02
(ma/cm^f (mg/person/ (mg/person/
' 9 ' application) application)
0.5
1.5
2
0.5
1.5
2
0.5
1.5
2
0.5
1.5
2
3,320
9,960
13,280
3,820
11,460
15,280
10,000
30,000
40,000
11,500
34,500
46,000
166
498
664
191
573
764
500
1,500
2,000
575
1,725
2,300
Applied nano-TiC>2
(mg/kg BW/
application)
12.0
35.9
47.9
13.8
41.3
55.1
8.0
24.0
32.1
9.2
27.6
36.9
"Body surface area values are based on Tables 6-6 and 6-16 of U.S. EPA (1997,;
bActual concentrations of nano-TI02 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 (2000,1579821, 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-to-
mouth 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 (im) 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.
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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 um, see below for details) and 0.1 mg/m3 for
ultrafine TiO2 (primary particle <0.1 um, 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 um, with some collection of particles up to 10 um).
"Ultrafine" particles were defined as the fraction of respirable particles with primary particle
diameter <0.1 um(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 DuPont™ 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).
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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 |im]; respirable refers to particles that penetrate to the alveolar
[pulmonary] region with a mass median aerosol diameter [MMAD] of approximately 4 urn1 (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 um2/cm3 for "alveolar deposited" particles and 50 um2/cm3 for
"tracheobronchial deposited" particles. Under normal operating conditions, the particle surface area
concentrations were 50 um2/cm3 for the alveolar deposited particles and 13 um2/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 urn 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 um (F.J. Miller, personal
communication (2009, 625211)).
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(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-Ti02 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 fafades
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
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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-Ti02 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 nanop articles, 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,
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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 ug/L ("realistic
scenario") or 16 (ig/L ("high scenario"), compared to a PNEC of <1 (ig/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 (ig/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,
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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 14°C for rainbow trout and at 23°C 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
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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 Daphnid), 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 (BMP) 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-Ti02 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 (ig/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 (ig/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 x arsenic concentration in fish (ug/g dry weight)/arsenic concentration in water (ug/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.
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Table 4-2. Tissue concentrations of various pollutants in fish after exposures to nano-Ti02 in water
Test Species
Fish (carp, Cyprinus
carpio)
Fish (carp, Cyprinus
carpio)
Fish (carp, Cyprinus
carpio)
Fish (rainbow trout,
Oncorhynchus
mykiss)
Material
21 -nm primary
particle, 50- to
200-nm aggregates
in water (P25)
(photocatalytic)
21 -nm primary
particle, 40- to
500-nm aggregates
in water (P25)
(photocatalytic)
21 -nm primary
particle, BET 50 m2/g
(P25) (photocatalytic)
21-nm, 75%rutile:
25% anatase,
sonicated (P25)
(photocatalytic)
Protocol
(no UV illumination,
unless specified)
Up to 25-day exposure to 3 and
10 mg/L nano-Ti02 (water changed
daily, Ti02 concentrations in water ~2
and ~7 mg/L, respectively, after the first
few hr)
Up to 25-day exposure to 10 mg/L
nano-Ti02 with and without 200 ug/L
arsenate
Up to 25-day exposure to ~97 ug/L
cadmium alone, cadmium with 10 mg/L
nano-Ti02, or cadmium with 1 0 mg/L
natural sediment particles
0-, 7-, or 14-day exposure to 0, 0.1 ,
0.5, or 1 .0 mg/L nano-Ti02
Study Outcome
Ti02 accumulated in internal organs > gills
> skin and scales > muscle
Bioconcentration factors were higher at
3 mg/L than at 10 mg/L
Arsenate adsorbed onto nano-Ti02
Higher arsenic concentrations in tissues
(skin and scales; muscle; gills; liver;
stomach; intestine) with arsenate plus
nano-Ti02 exposure, compared to arsenate
exposure alone
Cadmium adsorbed onto nano-Ti02
Higher cadmium concentrations in tissues
( skin and scale; muscle; gills; viscera;
whole body) with cadmium plus nano-Ti02
exposure, compared to cadmium exposure
alone, or cadmium plus natural sediment
particles
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.
Respiratory distress, organ pathologies, and
oxidative stress at concentrations as low as
0.1 mg/L.
Reference
Zhang et al. (2006,
157722)
Sun et al. (2007,
193662)
Zhang et al. (2007,
090114)
Federici et al. (2007,
091222)
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 (ig/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 (ig/g in the cadmium-only group and 22.3 (ig/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 (im) (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
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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 by-
product (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;
Oberdorster et al., 1992, 045110; Oberdorster 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; Oberdorster et al., 1994, 046203) [upper particle size limit: 200 nm (Elder et al.,
2006, 089253)1; (2) the circulating blood and then crossing the blood-brain barrier (Oberdorster et
al., 2004, 055639); and (3) the olfactory mucosa and through the ethmoid bone into cerebrospinal
fluid (Ilium, 2000, 157897). One of the most visually convincing demonstrations of olfactory nerve
transport, as mentioned in Oberdorster 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 Oberdorster 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;
Oberdorster 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 (im, aerodynamic deposition predominates. Between 0.1 and 1 (im, the
combined effects of aerodynamic and diffusive deposition are important.
Oberdorster 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
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regions. Since these simulations are based on the International Commission on Radiological
Protection (ICRP) model, which was designed for larger particles (supplement of Oberdorster 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 (Oberdorster 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 (Oberdorster et al.,
1994. Q46203).
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).
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Table 4-3. Nano-Ti02 disposition in animals after inhalation or intratracheal instillation
Species/Strain
Aerosol
Study Protocol
Observations
Reference
Fischer 344 rats,
females (6 wk)
B3C3F1 mice,
females (6 wk)
Hamsters,
females (6 wk)
Ti02:1.29-1.44 urn
MMAD
(og = 2.46-3.65),
21 -nm primary particles
Animals exposed via inhalation
6 hr/day, 5 days/wk, for 13 wk to
0.5,2,and10mg/m .
Control animals exposed to filtered
air.
Animals sacrificed at 0,4,13, 26,
and 56 days (49 for hamsters)
postexposure.
Groups of 25 animals per species
and time point.
Ti02 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 Ti02 in
lymph nodes increase with time postexposure in mid-
and 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 x 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.
Bermudez et al.
(2004, 056707)
Wistar rats,
20 adult males,
250±10g
Ti02(22-nmCMD,
og = 1.7)
Spark generated
0.11 mg/m3
7.3 x 106 particles/cm3
(SD 0.5x106
particles/cm3)
Rats exposed 1 hr via endotracheal
tube while anesthetized and
ventilated at constant rate
Lungs fixed at 1 or 24 hr
postexposure
Distributions of particles among lung compartments Geiser et al. (2005,
(airspace, epithelium/endothelium, connective tissue, 087362)
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.
Re-evaluation of the data from Geiser et al. (2005,
087362) with a new statistical method in which a
relative deposition index (RDI) was calculated for
each compartment. When RDM, the particle number
is the same as one would expect for the size of the
compartment, if the particle distribution is random.
RDM suggests a preferential distribution. The new
analysis suggested that at 1 hr postexposure,
connective tissue was the preferential target for the
nano-Ti02, 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.
Muhlfeld (2007,
091106)
WKY/NCrl Ti02(22-nmCMD,
(Charles River) og = 1.7)
Lats' ., » Spark generated
5 young adult
males,
250±10g
Rats exposed 1 hr via endotracheal
tube while anesthetized and
ventilated at constant rate
Lungs fixed immediately
postexposure
Of particles in tissues, 72% were aggregates of 2 or
more particles; 93% of aggregates were round or
oval; 7% were needle-like. The size distribution of
particles in lung tissues (29 nm CMD, og = 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
Kapp et al. (2004,
156624)
CMD - Count median diameter; MMAD - Mass median aerosol diameter; og - 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.
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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 um
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
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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
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Stratum corneum
Stratum lucidum s^—rj
Stratum granulosum
Stratum spinosum
Stratum basale
Sebaceous gland
Hair follicle
Apocrine sweat gland
Matrix of hair follicle
Blood vessels
- Stratum corneum
==— Stratum lucidum
- Stratum granulosum
- Stratum spinosum
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, 6251971: Used with permission from Informa Healthcare, Nohynek et al. (2007, 0906191
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-TiOa 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
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Table 4-4. Overview of Ti02 skin absorption/penetration studies
Test Material
Skin Model"
(Sampling Technique)
Results
Reference
Sunscreen Formulations Containing Nano-Ti02
Nano-Ti02 in a
sunscreen formulation
Sunscreen that
contains nano-Ti02
Nano-Ti02 in a
sunscreen formulation
Sunscreen that
contains nano-Ti02
Sunscreen that
contains nano-Ti02
Nano-Ti02 in
sunscreen
formulation/sunscreen
that contains
nano-Ti02
Various Ti02 in
sunscreen
formulations
Primary particle 17 nm
(Kemira, 2000, 157896). rutile,
AI203/stearic acid coated,
aggregates 150-170 nm (UV-
Titan M 160) in an oil-in-water
emulsion, provided by L'Oreal
(Clichy, France)
Not specified
20-nm nano-Ti02, coated with
silicone
A commercially available
sunscreen, hydrophobic
emulsion containing nano-Ti02
(AntheliosXLSPF60, La
Roche Posay, France)
A commercially available
sunscreen, hydrophobic
emulsion containing nano-Ti02
(AntheliosXLSPF60, La
Roche Posay, France)
50-100 nm, mixture of anatase
and rutile, no coating
information
Sunscreen base formulation
containing no Ti02 or 5% of
one of three types Ti02:
Micro-sized Ti02
Human forearm, repeated
application for 4 days (tape
stripping, biopsy)
Human skin (healthy and
psoriatic), in vivo, 2 hr (biopsy)
Human skin, in vitro, and human
skin, in vivo (skin stripping)
Human foreskin grafts
transplanted onto SCIDb mice;
Ti02 emulsion on the graft in
occlusion for 1,24, or 48 hr
Human foreskin grafts
transplanted onto SCID mice;
Ti02 emulsion on the graft at
2 mg/cm2 in occlusion for 24 hr
Human abdominal skin, in vitro
Female Yucatan minipigs (in
vivo), 2-mg emulsion/cm skin,
5 days/wk for 4 wk (necropsy)
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.
No penetration into living skin.
Deeper nano-Ti02 penetration in psoriatic
skin than in healthy skin.
No penetration beyond stratum corneum in
both psoriatic and healthy skin.
Penetration limited to upper layers of stratum
corneum. Nanoparticles in skin furrows or
follicular opening could be mistaken to be in
the epidermal compartment.
Ti02 in the corneocyte layers of stratum
corneum.
In two cases, penetration through the stratum
corneum, to the stratum granulosum was
observed.
Ti02 in stratum corneum, not in deeper layers
of the skin.
Penetration limited to upper layers of stratum
corneum.
Increased Ti levels in epidermis in all Ti02-
treated groups.
No penetration of Ti02 particles into the
dermis.
Lademann et al.
(1999.090591)
Pinheiro et al.
(2007, 180160)
Mavon et al.
(2007, 090587)
Kertesz et al.
(2005, 180334)
Kiss et al. (2008,
157547)
Dussert and
Gooris (1997,
193359)
Sadrieh et al.
(2010.594511)
Nano-Ti02, uncoated
Nano-Ti02, coated with
aluminum hydroxide and
dimethicone/methicone
copolymer
No increases in Ti levels in lymph nodes or
liver of any treated animals.
Photostable
nano-Ti02 in various
formulations
Photostable nano-Ti02,
needle-like shape, 45-150 nm
* 17-35 nm, coated with
alumina and silica (Loden et
al., 2006,157757). in the
following formulations: (1)
Eucerin® Micropigment
Creme 15: commercial
sunscreen, 5% Ti02
concentration (Beiersdorf
company); (2) a liposome
dispersion:! 8% Ti02,
containing Phospholipon 90 G
and Tioveil AQ-N (Tioxide
Specialties Ltd., Billingham,
UK); (3) formula SG110:4.5%
Ti02, containing Tioveil AQ-N;
and(4) pure predispersion
Tioveil AQ-N: 40% Ti02
Pig skin, in vitro
Particles on/in the stratum corneum; minimal Menzel et al.
penetration into stratum granulosum. (2004,180361)
No penetration into living skin.
4-20
-------
Test Material
Skin Model"
(Sampling Technique)
Results
Reference
Photostable
nano-Ti02 in
sunscreen
formulations
(1) T-Lite SF-S: rutile, coated Pig skin, in vitro, up to 24 hr
with Si02 and methicone; and (tape stripping)
(2) T-Lite SF: rutile, coated
with methicone
Both primary particles are
needle-like: 30-60 nm x
10 nm. Aggregates and
agglomerates in water phase,
mostly up to 200 nm
Both are oil/water emulsions
containing 10%Ti02
No penetration beyond stratum corneum.
Receptor solution recoveries of 0.8-1 .4% of
applied dose.
Gamer etal.
(2006, 090588)
Other Nano-Ti02 Formulations
T 805 Degussa
Various nano-Ti02 in
oil-in-water emulsions
Nano-Ti02
Various Ti02 and
nano-Ti02
Coated with Pig, in vivo
trimethyloctylsilane; ~20 nm in
diameter
Emulsions contained 4% Human forearm, in vivo, 6 hr
nano-Ti02, only differed in (biopsy)
nano-Ti02 types: (1) 20-nm
cubic primary particle, coated
with trimethyl octylsilane,
hydrophobic surface (T805,
Degussa); (2) 10-1 5 nm
primary particle, aggregated
into ~100-nm needles, coated
with AI203 and Si02,
amphiphilic surface (Eusolex
T-2000, Merck); and (3)100-
nm needles, coated with
alumina and silica, hydrophilic
surface (Tioveil AQ-10P, in
dispersion, Solaveil)
10-100 nm, coated with Si02~, Human, in vivo (biopsy)
AI203", AI203,/Si02
14 nm-200 urn, anatase and Pig and human skin, in vivo and
rutile, coated and uncoated in vitro (skin stripping or biopsy)
materials
Ti02 found exclusively in the outermost
stratum corneum layer. Traces of Ti02 were
found in the upper part of the follicle, with no
evidence of uptake into the follicular
epithelium.
Penetration of particles into the upper layers
of stratum corneum.
No penetration into living skin.
Particles on or in the outmost surface of the
stratum corneum.
No penetration into living skin.
No penetration beyond the stratum corneum
in any study.
Pfluckeretal.
(1999.644132)
Pfluckeretal.
(2001.157887)
and Schulz et al.
(2002, 157872)
Schulz etal.
(2002, 157872)
SCCNFP (2000,
092740)
4-21
-------
Test Material
Skin Model"
(Sampling Technique)
Results
Reference
Various nano-Ti02
100%anatase, uncoated,
nano-Ti02 (Zhejiang Wanjin
Material Technology Co., Ltd.):
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
160m2/g
75% anatase/25% rutile,
uncoated nano-Ti02 (P25 from
Degussa, Germany):
21 nm, hydrophilic surface,
surface area 50 m2/g
100% rutile, uncoated,
nano-Ti02 (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
(A) Porcine skin, in vitro,
isolated pig ear skin (without
and with tape stripping) on a
modified Franz equipment,
nano-Ti02 suspension (4,10,
25,60, or 90 nm) on the skin for
up to 24 hr
(B) Porcine skin, in vivo, shaved
pig ear starting at age of 4 wk,
approximately 24 mg of test
formulation containing 5%
nano-Ti02 (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
(C) BALB/c hairless mice skin,
in vivo, starting at age of 7-8 wk.
Test formulation containing 5 %
nano-Ti02 (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 urn
nano-Ti02) per cm 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.
(A) No penetration beyond the stratum Wu et al. (2009,
corneum 193721)
(B) After 30 days, nano-Ti02 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-Ti02 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-Ti02 exposure. Increased Ti
in the lung may be significant in the 21- and
60-nm groups.
Ti02
Mixed particle sizes, mostly
less than 10 urn in aqueous
solution (range from <2 urn to
>20um), no coating
information, 20% Ti02 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 Lansdown and
and outer hair follicles. Taylor (1997,
157928)
No penetration into living skin.
Uptake of Ti02 affected by the vehicle: in
caster oil>in water>in polyethylene glycol.
Nano-Ti02 in various
gels
For ion microscopy study: 20-
nm xioo-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-Ti02 particles
Porcine and human skins, for
30 min to 48 hr (biopsy)
After wash with water, nano-Ti02 remains on Lekki et al. (2007,
skin, with most in stratum corneum and some 180280)
in hair follicles.
Nano-Ti02 observed seen in hair follicles as
deep as 400 urn, but not in living cells
surrounding the follicles.
For autoradiography study:
proton-irradiated 20-nm Ti02,
rutile (R-HD2, Huntsman),
coated with alumina
(Huntsman, 2008,157555)
4-22
-------
Test Material
Skin Model"
(Sampling Technique)
Results
Reference
Ti02/Nano-Ti02 Particles of Unknown Size
Sunscreen that
contains Ti02
Ti02
Sunscreen that
contains Ti02
Various "microfine"
Ti02
Not specified
Not specified
Sunscreen containing 8%
microfine Ti02 (size, crystal
form, and coating were not
specified)
Commercial microfine Ti02
dispersions in octyl palmitate
and in water (Tioxide
Specialties Ltd, Billingham,
U.K.)
Human (tape stripping)
Mouse, pig, and human skin,
in vitro
Human skin (13 patients,
59-82 yr old), in vivo, applied
Ti02 sunscreen daily for
9-31 days until 2 days prior to
surgical removal of the skin
(tape stripping)
Human abdominal skin, in vitro,
and skin equivalents
(keratinocytes and fibroblasts of
native human origin), in vivo
Particles on or in the outmost layers of the
stratum corneum. No penetration into living
skin.
Ti02 detected in the intercellular spaced
between corneocytes of the outermost layers
of the stratum corneum. No penetration into
living skin.
Ti concentration in the dermis of patients
exposed to sunscreen was higher than
concentration in cadavers (controls), after
exclusion of one control outlier.
No correlation between the duration of
sunscreen application and Ti concentration.
Microfine Ti02 penetrates into the human
stratum corneum probably via sebum lipids of
the hair follicles.
Gottbath and
Mueller-Goymann
(2003, 193401)
Gontier et al.
(2004, 193398)
Tan etal. (1996,
157933)
Bennat and
Muller-Goymann
(2000, 157403)
'Topical application unless specified.
bSCID = 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-TiO2used 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. Kertesz 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; Pfliicker 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)1. 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-Ti02 exposures
through injection or oral gavage
Nano-Ti02
Study design
Findings in the brain
Reference
Nano-Ti02,25 nm and 80 nm,
rutile, uncoated (from Hangzhou
Dayang Nanotechnology Co. Ltd.
FineTi02,155±33nmTi02,
anatase, uncoated, >10 wt% at
<100nm(fromZhonglina
Chemical Medicine Co. (Chen,
personal communication, 2008,
157588)
Single oral gavage at 5,000 mg/kg to male and
female CD-1 (ICR) mice
Ti content was measured 2 wk after gavage by
ICP-MS with a detection limit of 0.074 ng/mL
Ti concentrations in brain were increased in
all three Ti02 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.
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.
Wang et al. (2007,
090290)
Nano-Ti02,20-30 nm, 17%
anatase, 30% rutile, uncoated,
BET surface area 48.6 m2/g
Single i.v. injection at 5 mg/kg BW through the tail
vein of male Wistar rats
Ti02 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 urn/organ
Ti02 was not detected in the brain at any Fabian et al. (2008,
tested time points. 157576)
Nano-Ti02,15 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
Ti concentrations in brain were measured at
5 min, 72 hr, and 1 mo after injection by ICP-MS
with an unspecified detection limit
No increase of Ti in the brain of treated mice Sugibayashi et al.
was observed compared to negative controls (2008,157489)
at any tested time points.
Nano-Ti02,5 nm, anatase
Conventional Ti02
Both types of Ti02 were made
from controlled hydrolysis of
titanium tetrabutoxide.
Multiple i.p. injection to female CD-1 (ICR) mice
once per day for 14 days with nano-Ti02 at 5,10,
50,100, and 150 mg/kg BW or conventional Ti02
at 150 mg/kg BW
Ti concentration was measured 14 days after the
treatment began by ICP-MS with a detection limit
of 0.076 ng/mL
Ti concentrations in the brain increased with
increasing nano-Ti02 doses. All Ti02
treatments increased Ti concentration in the
brain, as compared to negative controls. At
150 mg/kg, brain Ti concentration was higher
in the nano-Ti02 group than in the
conventional Ti02 group.
Liu et al. (2009,
193516)
Nano-Ti02,25-70 nm anatase,
surface area 20-25 m /g, purity
99.9% (from Sigma-Aldrich)
s.c. injections of 100 uLof 1 mg/mL nano-Ti02
(i.e., 0.1 mg nano-Ti02) each time per pregnant
Sic:ICP mice once per day at 3, 7,10 and 14 days
post-mating.
Presence of nano-Ti02 in the brain was assessed
in the male offspring at age of 4 days and 6 wk by
FE-SEM/ EDS
Nano-Ti02 particles were seen in the brain
(olfactory bulb and the cerebral cortex -
frontal and temporal lobes) of the 6-wk-old
mice from nano-Ti02-exposed dams. (Results
from 4-day-old mice were not reported.)
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-Ti02-exposed dams.
Takeda et al. (2009,
193667)
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 X- i.v. - Intravenous
ray spectrometry 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
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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; Oberdorster 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 urn in diameter (Oberdorster 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 (Oberdorster 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
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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.
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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 by-
products (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-TI02
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: Hassellov 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.
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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-Ti02 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 (Oberdorster 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 (Oberdorster, 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 (Oberdorster, 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
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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
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hydrophilic P25, although the authors concluded that silanization1 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 off-
line measurements/aspects for nanomaterial characterization for toxicological studies (Table 5-2)
(Oberdorster 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.
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Table 5-1. Published recommendations for measuring nanomaterial parameters for exposure
during characterization inhalation studies
Recommendation
Metric Measurement
Off-line (Discrete)3 On-line (Continuous)'5
Mass E (coupled with on-line)
Size distribution E E/D
Surface area 0 0
Particle Number N E
"Off-line: Collected and analyzed later.
bOn-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.
0 - 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, Oberdorster et al. (2005, 090087).
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Table 5-2. Published recommendations for off-line nanomaterial characterization using
noncontinuous techniques fortoxicological studies
Toxicity Screening Studies
Characterization
Size distribution (primary particles)
Shape
Surface area
Composition
Surface chemistry
Surface contamination
Surface charge -suspension/solution
Surface charge - powder (use bio fluid surrogate)
Crystal structure
Particle physicochemical structure
Agglomeration state3
Porosity
Method of production
Preparation process
Heterogeneity11
Prior storage" of material
Concentration
Human Exposure
E (combine with
agglomeration state)
E
D
E
D
D
0
0
0
E
E
D
E
--
D
E
E
Supplied Material
E
E
E
E
E
N
E
E
E
E
N
D
E
--
E
E
--
Material as
Administered
D
0
D
0
D
D
E
N
0
D
E
N
--
E
E
E
E
Material after
Administration
(in vivo/in vitro)
D
0
0
0
D/0
N
0
0
0
D
D
N
--
--
D
--
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.
0 -These characterizations are considered to provide valuable but nonessential information.
N-These characterizations are not considered to be of significant value to screening studies.
aAs 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.
bTime and conditions, including temperature, humidity, exposure to light and atmosphere composition.
"Ratios of different components.
Source: Reprinted with permission from BioMed Central, Oberdorster et al. (2005, 0900871.
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" (Oberdorster 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
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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 um 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 nano-
sized particles, including TiO2, in several suspension media, including: PBS; rat and mouse BAL
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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 (Ceo) studies. C6o toxicity in daphnids and fishes was higher when the C6o 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 C6o 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
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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
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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-TiO? 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: Hassellov 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: Hassellov 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-Ti02 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 TiO2in 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
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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 A1OOH 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-Ti02 ecological effects
Test Species
(Reference)
Material
Protocol
(No UV illumination, unless
specified)
Study Outcome
Acute Exposure to Microorganisms
Bacteria (Escherichia 66-nm powder, -35% rutile:65% anatase,
co// and Bacillus subtilis) average 330-nm in water (Sigma product
634662) (Lyon, personal communication, 2008,
157524)
(Adams et al., 2006,
157782)
6-hr exposure to: (1) 50,100,500,1,000,
2,000, 5,000 ppm in medium3, in direct
sunlight; or (2) 1,000 ppm in medium3, in
dark
In dark, similar growth inhibition
for both bacteria
In light, B. subtilis: 0%, 75%, and
99% growth inhibition at 500,
1,000, and 2,000 ppm,
respectively
£. co//: 0%, 15%, 44%, and 46%
inhibition at 100, 500,1,000, and
2,000 ppm, respectively
Bacterium (Vibrio
fischeri)
(Blaiseetal.,2008,
157592)
<100-nm powder (Sigma product 634662,
Canada or France)
15-min exposure, measure the reduction of IC25 >100 mg/L
light output from bioluminescent marine
bacterium, Vibrio fischeri (Microtox® toxicity
test) as an indicator of growth inhibition,
tested concentrations not specified
Mix in a 1:1 ratio with certified reference
material sediment, measure light output
(Microtox® toxicity test) (indirect
toxicity/interaction)
Nano-Ti02 did not affect the
toxicity of certified reference
material sediment
Bacterium (Vibrio
fischeri)
(Heinlaan et al., 2008,
193414)
25- to 70-nm powder mixture of anatase and
rutile, ratio not disclosed (Sigma product 13463-
67-7, Estonia) (Heinlaan, personal
communication, 2008,157558)
Conventional Ti02: size and crystal form not
disclosed (Sigma product 14027, Estonia; a
former Riedel-de Hae'n product) (Heinlaan,
personal communication, 2008,157558)
30-min exposure for up to 20,000 mg/L
nano-Ti02 and conventional Ti02, 8-hr
exposure to 20,000 mg/L conventional Ti02
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-Ti02 (30-min
exposure) did not decrease
bacterial growth
The highest concentration tested:
20,000 mg/L conventional Ti02
(30-min and 8-hr exposure) did
not decrease bacterial growth
5-11
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Test Species
(Reference)
Material
Protocol
(No UV illumination, unless
specified)
Study Outcome
Bacterium (Vibrio
fischeri)
(Velzeboeretal.,2008,
157476)
<75-nm (primary particle) nano-Ti02 in water
suspension (Sigma product 643017, the
Netherlands), mixture of rutile and anatase, ratio
not reported (Velzeboer et al., 2008,157476)
15 min, 1,10,100 mg/L, measure reduction EC50 >100 mg/L
of light output from bioluminescent bacteria
(Microtox® method, which could be
affected by turbidity of 100 mg/L Ti02
suspension)11
Bacteria (from a soil
sample, species not
identified)
(Velzeboeretal.,2008,
157476)
<75-nm (primary particle) nano-Ti02 in water
suspension (Sigma product 643017, the
Netherlands), mixture of rutile and anatase, ratio
not reported (Velzeboer et al., 2008,157476)
7 days (Biolog®test, gram positive), EC50 >100 mg/L
100 mg/Lb
Bacteria and yeast
(proprietary information)
(Blaiseetal.,2008,
157592: Dando.
personal
communication, 2008,
157582)
<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
MT0100 mg/L
18-hr exposure to the filtered elutriate from
certified reference material sediment with
and without nano-Ti02 mixed in a 1:1 ratio
(MARAassay) (indirect toxicity/interaction),
tested concentrations not specified
Nano-Ti02 did not affect the
toxicity of the elutriate of certified
reference material sediment
Acute Exposure to Aquatic Organisms
Alga (green alga,
Desmodesmus
subpicatus)
(Hund-Rinke and
Simon. 2006. 090607)
25-nm primary particle, 20% rutile:80% anatase
(Degussa P25) (Baun et al, 2008,157598)
(photocatalytic)(Product 1)
100-nm primary particle, 100% anatase;
(Hombikat UV100) (Baun et al, 2008.157598):
photocatalytic (Mehrvar et al, 2002,193541)
(Product 2)
72-hr growth inhibition, following the
guidelines for EU standard algal assay (DIN
38412-33,1991.667415: ISA8692,2004,
- 667212: OECD 201, 2006,199838) with
modifications to include pre-illumination of
nano-Ti02 dispersion with simulated
sunlight (wavelength 300-800 nm) at 250
watts for 30 min; illumination alone did not
affect D. subpicatus growth
Algal growth (without pre-illumination): 0,
3.1,6.2,12.5,25, 50 mg/L (products 1
[P25]and2[UV100])
Shading effect: 0,12.5,25,50 mg/L
Algal growth (with pre-illumination): 12.5,
25, 50 mg/L (Product 1[P25])
EC5o and effects of additional
particle cleaning:
Product 1 (P25): EC50 was not
different between nano-Ti02
washed once as manufacturer
recommendation (32 mg/L) and
nano-Ti02 with an additional
wash (44 mg/L), suggesting
toxicity was not from
contaminants
Product 2 (UV100) :EC50
>50 mg/L, both nano-Ti02 with
and without the additional wash
(at up to 50 mg/L) caused less
than 40% decrease in growth
No shading effect: when
nano-Ti02 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-Ti02 effects was not due to
lowered light intensity, but due to
a toxicity of nano-Ti02
Pre-illumination of nano-Ti02
(Product 1 [P25]) did affect
nano-Ti02 effects on algal growth
Alga (green alga,
Pseudokirchneriella
subcapitata)
(Velzeboeretal.,2008,
157476)
<75nm (primary particle) nano-Ti02 in water
suspension (Sigma product 643017, the
Netherlands), mixture of rutile and anatase, ratio
not reported (Velzeboer et al, 2008,157476)
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 Ti02 suspension
EC50>100mg/Lb
Alga (green alga, 140 nm in water, 79% rutile: 21% anatase,
Pseudokirchneriella coated (90-wt % Ti02,7% alumina, and 1 %
subcapitata) amorphous silica) (DuPont uf-C Ti02) (photo-
(Warheit et al, 2007, passivative/ photo-stable) (Warheit, 2008,
091075) 157470)
OECD 201 (2006,199838) (72-hr growth),
with light3
0.01,0.1,1,10, and 100 mg/L (uf-C Ti02
and fine Ti02)
EC50 21 mg/L (based on
decreases in healthy cell
number)
EC50 87 mg/L (based on inhibition
of growth rate)
Fine Ti02:380 nm in water, rutile, coated (-99%
Ti02 and ~1 % alumina)
EC5016 mg/L (based on
decreases in healthy cell
number)
EC50 61 mg/L (based on inhibition
of growth rate)
5-12
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Test Species
(Reference)
Material
Protocol
(No UV illumination, unless
specified)
Study Outcome
Alga (green alga,
Pseudokirchneriella
subcapatitata)
(Huang et al., 2005,
157801)
Photocatalytic nano-TI02: 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 Ti02 particles on their
surface
Cellular weight increased by
>130%
Ala (greenalga,)
(Aruojaetal.,2009,
193254)
Nano-Ti02
Conventional (Bulk) Ti02
OECD 201 (2006,199838) (algal growth
inhibition test) Algal cell culture; formation
of agglomerates.
Nano-Ti02 formed large
agglomerates with almost all
algal cells entrapped.
EC50 = 5.83 mg/L
NOEC = 0.98 mg/L
Conventional Ti02 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, 25-nm primary particle, 20% rutile:80% anatase ISO 6341 (1996, 667232). OECD 202
Daphnia magna) (Hund- (Degussa P25) (2008,157598) (photocatalytic);
Rinke and Simon, 2006, ultrasonic dispersion
090607)
(2004.667207) 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
noniiluminated nano-Ti02
0,1,1.5, 2,2.5,3 mg/L
100-nm primary particle, 100% anatase;
(Hombikat UV100) (2008,157598):
photocatalytic (Mehrvar et al., 2002,193541)
ultrasonic dispersion
Pre-illumination increased toxicity
compared to the same
concentration
No dose-response relationship
with either pre-illuminated or
noniiluminated nano-Ti02
Pre-illumination showed a trend
of increasing toxicity
No dose-response relationship
with either pre-illuminated or
noniiluminated nano-Ti02
Invertebrate (water flea,
Daphnia magna)
(Lovern and Klaper,
2006.088040)
Primary particle <25 nm (smallest 5-nm),
anatase, uncoated (photocatalytic) (Klaper,
personal communication, 2008.157546): filtered
through a 0.22-um nylaflo filter, secondary
particle 20-30 nm in deionized water
EPA 48-hr tox test (U.S. EPA standard
operating procedure 2024,1991, 667211)
(mortality)
Filtered nano-Ti02:0.2,1,2,5, 6,8, and
10 ppm
LC50 5.5 mg/L
LOEC 2.0 mg/L
NOEC 1.0 mg/L
Primary particle <25 nm (smallest 5 nm),
anatase, uncoated (photocatalytic) (Klaper,
personal communication, 2008,157546)
sonicated, unfiltered, secondary particle
100-500 nm in deionized water
Sonicated, unfiltered nano-Ti02:50, 200, LC50 >500 mg/L
250,300,400, and 500 ppm
Invertebrate (water flea,
Daphnia magna)
(Wiench et al., 2007,
090635)
20-30 nm, 80% anatase, 20% rutile, no surface
coating, BET surface area 48.6 m /g
50 nm x 10 nm, rutile, surface coating aluminum
hydroxide, dimethicone/methicone copolymer,
BET 100 m2/g (T-LiteTM SF) (photostable UV
filter)
50 nm x 10 nm, rutile, surface coating aluminum
hydroxide, hydrated silica,
dimethicone/methicone copolymer, BET 100
nf/g (T-LiteTM SF-S) (photostable UV filter)
50 nm x 10 nm, rutile, surface coating aluminum
hydroxide, hydrated silica,
dimethoxydiphenylsilane/triethoxycaprylsilane
crosspolymer, BET 100 m2/g (T-LiteTM MAX)
(photostable UV filter)
-300 nm, BET surface area 6 m2/g (pigment
grade)
OECD 202 (2004,667207). part 1 (48-hr
immobility), tested concentrations: 0
- (untreated control), 0.01, 0.1,1.0,10.0 and
100.0 mg/L
EC50>100mg/L
EC50>100mg/L
EC50>100mg/L
EC50>100mg/L
EC50>100mg/L
Invertebrate (water flea,
Daphnia magna)
(Lovern etal., 2007,
091069)
30 nm, anatase
1-hr exposure to 2.0 mg/L
No changes in heart rate or
behaviors
5-13
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Test Species
(Reference)
Material
Protocol
(No UV illumination, unless
specified)
Study Outcome
Invertebrate (water flea,
Daphnia magna)
(Warheit et al., 2007,
091075)
140 nm in water, 79% rutile:21 % anatase,
coated (90-wt % Ti02,7% alumina, and 1 %
amorphous silica) (DuPont uf-C Ti02) (photo-
passivative/ photo-stable) (Warheit, 2008,
157470)
Fine Ti02: ~380-nm in water (buffered), rutile,
BET surface area 5.8 m /g, coated with alumina
(-99% Ti02 and ~1 % alumina)
OECD 202 (2004,667207) (48-hr
immobility)
0.1,1.0,10, and 100 mg/L (uf-C and fine
Ti02)
EC50> 100 mg/L
(10% immobility at 100 mg/L)
EC50> 100 mg/L
(10% immobility at 10 mg/L, 0%
immobility at 100 mg/L)
Invertebrates (water
flea, Daphnia pulex and
Ceriodaphnia dubia)
(Griffittetal.,2008,
157565)
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)
48-hr mortality, 14:10 hr light:dark cycle, for
D. pulex adults and C. dubia neonates
(<24-hr old)
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
LC5o. However, the estimated LC50 was not
specified.)
LC50 >10 mg/L for both D. pulex
and C. dubia
Invertebrate (water flea,
Chydorus sphaericus)
(Velzeboeretal.,2008,
157476)
<75 nm (primary particle) nano-Ti02 in water
suspension (Sigma product 643017, the
Netherlands), mixture of rutile and anatase, ratio
not reported (Velzeboer et al., 2008,157476)
48-hr mortality, 17:7 hr light:dark cycle
(Chydotox test)b
EC50>100mg/Lb
Invertebrates (water
flea, Daphnia magna',
fairy shrimp,
Thamnocephalus
platyurus)
(Heinlaan et al., 2008,
193414)
25- to 70-nm powder mixture of anatase and
rutile, ratio not disclosed (Sigma product 13463-
67-7, Estonia) (Heinlaan, personal
communication, 2008,157558)
48-hr mortality for D. magna
24-hr immobilization for T. platyurus
NOEC>20,000 mg/L for T.
platyurus', not tested in D. magna
Conventional Ti02: size and crystal form not
disclosed (Sigma product 14027, Estonia; a
former Riedel-de Hae'n product) (Heinlaan,
personal communication, 2008,157558)
Up to 20,000 mg/L for both nano- and
conventional Ti02
NOEC>20,000 mg/L for!
platyurus', 60% mortality at
20,000 mg/L for D. magna
Invertebrate (fairy
shrimp,
Thamnocephalus
platyurus)
(Blaiseetal.,2008,
157592)
<100-nm powder (Sigma product 634662,
France), characteristics in water not reported
24-hr lethality (ThamnoToxkit assay), tested LC50 >100 mg/L
concentrations not specified
Invertebrate (freshwater
hydra, Hydra attenuate)
(Blaise et al., 2008,
157592)
<100-nm powder (Sigma product 634662,
France), characteristics in water not reported
96-hr morphological changes, tested
concentrations not specified
EC50 in 10 to 100 mg/L range
Fish cell (trout primary
hepatocytes)
(Blaiseetal.,2008,
157592)
<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
Fish (zebrafish, Danio
rerio), embryo and
larvae
(Zhuetal., 2008,
193742)
Nano-Ti02: 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
Conventional Ti02: 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
96-hr exposure to 0,1,10,50,100, or
500 mg/L nano-Ti02 or conventional Ti02 to
fish eggs (started within 1.5 hr post-
fertilization); 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
Neither nano-Ti02 nor
conventional Ti02 at the tested
condition caused changes in any
of the three endpoints measured.
Fish (zebrafish, Danio
rerio)
(Griffittetal.,2008,
157565)
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)
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
LC50>10 mg/L for both adults
and fry
5-14
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Test Species
(Reference)
Material
Protocol
(No UV illumination, unless
specified)
Study Outcome
Fish (rainbow trout,
Oncorhynchus mykiss)
(Warheit et al., 2007,
091075)
140 nm in water, 79% rutile:21 % anatase,
coated (90-wt % Ti02,7% alumina, and 1 %
amorphous silica) (DuPont uf-C Ti02) (photo-
passivative or photo-stable) (Warheit, 2008,
157470).
OECD 203 (1992,667208) (96 hr acute
toxicity)
0.1,1.0,10, and 100 mg/L (uf-C and fine
Ti02)
LC50>100mg/L
Chronic Exposure to Aquatic Organisms
Invertebrate (water flea,
Daphnia magna)
(Adams et al., 2006,
157782)
66-nm powder, -35% rutile:65% anatase,
average 330 nm in water, (Sigma product
634662) (photocatalytic) (Lyon, personal
communication, 2008,157524)
8-day exposure to suspension at 1,10 or 40% mortality at 20 mg/L
20 ppm (concentration over time was not
reported)
Invertebrate (water flea,
Daphnia magna)
(Wiench et al., 2007,
090635)
50 nm x 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 (2008.667210) (21-day
reproduction), test concentrations: 0.01,
0.03, 0.1,0.3,1,3,10,30,100 mg/L
NOEC 3 mg/L
LOEC10mg/L
OECD 202 (2004,667207) (Chronic LC0 = 30 mg/L
toxicity, 21-day immobility)
Fish (rainbow trout,
Oncorhynchus mykiss)
(Federici et al., 2007,
091222)
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 Ti02
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)
Respiratory distress, organ
pathologies, and oxidative stress
at as low as 0.1 mg/L; nano-Ti02
could be a surface acting toxicant
Acute Exposure to Terrestrial Organisms
Photosynthetic enzyme <100-nm powder (Sigma product 634662,
complexes isolated from Canada or France), characteristics in water not
spinach leaves reported
(Blaiseetal.,2008,
157592)
15 min, tested concentrations not specified, IC20 >100 mg/L
measure the decrease in chlorophyll
fluorescence emitted from the enzyme
complexes as an indicator of inhibition of
photosynthetic efficiency (Luminotox assay)
(Bellemareetal.. 2006.157779)
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-Ti02 did not affect the
toxicity of certified reference
material sediment
Plant (spinach, Spinacia
oleracea)
(Linglan et al., 2008,
157534)
Nano-Ti02:5 nm, anatase, not coated
Conventional Ti02
Soak the seeds in 0.25% nano-Ti02 or
conventional Ti02 for 48 hr, and spray
0.25% nano-Ti02 or conventional Ti02 onto
. the leaves from 2-leaf stage to 8-leaf stage
at 0.25%
Nano-Ti02:
Enhanced growth (size, single
plant fresh weight, single plant
dry weight)
Increased chlorophyll content
Increased net photosynthetic rate
Increased mRNA, protein
concentration, and activity of
Rubisco activase
Conventional Ti02:
No significant changes
Plant (spinach, Spinacia
oleracea)
(Zheng et al., 2005,
157784)
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)
Plant (willow trees)
(Seegeretal., 2009,
644124)
Nano-Ti02: 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
(Ti02) and 0,1,20, and 50 mg/L (Ti02) for
Degussa, and 100 mg/L (Ti02) for Hombikat
UV100.
Solutions shaken. Adsorption measured by
light microscopy; growth, transpiration, and
water use efficiency measured using
standard willow tree acute toxicity test
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.
5-15
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Test Species
(Reference)
Material
Protocol
(No UV illumination, unless
specified)
Study Outcome
Plant (maize, Zea mays
LjAsliand Neumann
(2009,193771)
Nano-Ti02: Degussa P25
(mean diameter 30 nm)
Bentonite clay (1-60nm)
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 .Og/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.
Invertebrate (isopod,
[woodlouse] Porcellio
scaber) (Jemecetal.,
2008.157552)
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
3-day dietary exposure to nonsonicated
nano-Ti02 at 0.1,0.5,1,10,100,1,000,
2,000, or 3,000 ug/g food or to sonicated
nano-Ti02 at 1,000, 2,000, or 3,000 ug/g
food (leaves soaked in nonsonicated or
sonicated nano-Ti02 dispersion and then
dried)
Decreased activities of catalase
and glutathione-S-transferase
(GST) in digestive glands at 0.5,
2,000, and 3,000 ug/g
nonsonicated nano-Ti02, but not
in middle doses of nonsonicated
nano-Ti02 or any doses of
sonicated nano-Ti02
No changes in feeding rate,
defecation rate, food assimilation
efficiency, weight, or mortality
were noted up to 3,000 ug/g
Invertebrate (nematode, Nano-Ti02, anatase, primary particle diameter
Caenorhabditis elegans) 50 nm, measured BET surface area 325 m2/g for
(Wang et al., 2009, primary particle, purity >99%, hydrodynamic
193696) 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)
Conventional Ti02, 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)
Expose synchronized worms in the L1
stage to nano-Ti02 or conventional Ti02 in
ultrapure water with pH adjusted to 7.0 with
HN03andNaOH
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
Lethality to the vermiform
nematode: 24-hr LC50 was
significantly lower for nano-Ti02
(79.9 mg/L) than for conventional
Ti02(135.8mg/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-Ti02 and
at 95.9 mg/L or higher
concentrations of conventional
Ti02
N/A-Not applicable
"Authors reported cloudy appearance or difficulty to dissolve nano-Ti02 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-Ti02 in medium were not
reported.
bAuthors reported cloudy appearance in 100 mg/L Ti02 suspension. After centrifugation, nano-Ti02 concentrations were no more than 10% of initial
concentrations. For example, 200 ug/L nano-Ti02 was added into pond water, and nano-Ti02 was only 1 ug/L after centrifugation.
BET- Surface area measured by Brunauer, Emmett, and Teller analysis
DIN - Deutsches Institut fiir Normung (German Institute for Standardization)
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
end points
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)
MTC - Microbial Toxic Concentration, calculated by
comparing the area under and above the growth curve
(Gabrielson et al., 2003,157862: 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 x LOECJ1/2
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
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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.
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For photostable nano-TiO2, EC50 values determined for 72-hour growth inhibition in green
alga (P seudokirchneriella subcapitatd) 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 dose-
response 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; Hund-
Rinke 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).
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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, Thamnocephcilus 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; Lohr 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-Lite™ SF, coated T-Lite™ SF-S,
and coated T-Lite™ 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 inD. 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
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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 (Oberdorster 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 A12O3 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.
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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 disrupter 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
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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 anti-
oxidative stress enzymes in the digestive gland (hepatopancreas) were measured. The activities of
both enzymes were decreased at 0.5, 2,000, and 3,000 (ig/g of nonsonicated nano-TiO2, but not at
middle concentrations (1, 10, 100, and 1,000 (ig/g) of nonsonicated nano-TiO2 or at any
concentration (1,000, 2,000, and 3,000 (ig/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 (ig/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 LI 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
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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 and the Luminotox solid phase assay2) 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 macro-
organisms, 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.
Luminotox assay measures the inhibition of photosynthetic efficiency of photosynthetic enzyme complexes isolated from spinach leaves.
For the Luminotox solid-phase assay, IC2o 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.
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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; Oberdorster 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; Maatta and
Arstila, 1975, 157979; Yamadori et al., 1986, 193728). as have Ti particles in the lymph nodes
(Maatta 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.
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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 ultrafme 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
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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.
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Table 5-4. Summary of health effects of nano-Ti02 particles in mammalian animal models:
dermal route
Animal Testing Material Treatment Conditions
Summary of Major Effects
Reference
Mouse
Female
hairless
CRLSKH1
In vitro
exposure
Single and
repeated
exposures
Commercially available
sunscreens, some of
which contained Ti02
(size not specified)
For testing indirect dermal effect
a) Commercially available sunscreens,
applied at 2 mg/cm 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.
b) Combination of Ti02with phenyl
trimethicone, ZnO, and octyl
methoxycinnamate (OM)
c) TiSilc untinted sunscreen, which
contains Ti02 was applied. 2,4-D was
also applied. Both were applied on skin,
and then again 4.5 hr after the first
application.
d) TiSilc untinted sunscreen and
pesticides: Paraquat, Malathion, and
Parathion
Some (not all) tested sunscreens increased
transdermal penetration of herbicide/pesticide.
Solvent, notTi02 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 Ti02 (and 1 out of 2 sunscreens that contain
no Ti02) increased transdermal penetration of
herbicide 2,4-D.
b) Formulation effects:Ti02 alone, Ti02 plus ZnO,
and Ti02 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) Sunscreen effect on transdermal penetration of
other pesticides: Absorption of pesticides
(Paraquat, Malathion, and Parathion) was also
increased in skin pretreated with sunscreen TiSilc.
Brand et al.
(2003.157866)
Human
foreskin grafts
on SCID mice
In vivo
exposure
Single
exposure
A commercially available
sunscreen, hydrophobic
emulsion containing
nano-Ti02 (Anthelios XL
SPF 60, La Roche
Posay, France)
For testing dermal effects
Sunscreen containing nano-Ti02 applied
to skin at 2 mg/cm in occlusion for 1, 24,
or48hr
Sacrificed after exposure time; punch
biopsy from the human skin graft area
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.
Tested sunscreen containing nano-Ti02 did not
affect viability, proliferation, apoptosis,
differentiation, or adhesive properties of skin cells.
(NanoDerm
(2007.157660)
Rabbit
New Zealand
White
In vivo
exposure
Single
exposure
Mouse
Female,
CBA/JHsd
In vivo
exposure
Repeated
exposure
Nano-Ti02 (identified as
uf-C, a pre-commercial
version of DuPont Light
Stabilizer 210), 79%
anatase/21 % rutile, not
coated, approximately 90
wt% Ti02,7% alumina,
and 1 % amorphous
silica, average particle
size 140.0 ±44 nmin
water, average BET
surface area 38.5 m /g
For testing acute dermal irritation
Doses-0 or 0.5 g
Single exposure for 4 hr (nano-Ti02 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
No dermal irritation effects, no clinical signs of Warheit et al.
toxicity, and no BW loss. (2007, 091075)
Not considered a skin irritant.
For testing dermal sensitization (local
lymph node assay)
0,5, 25, 50, or 100% nano-Ti02 on both
ears for 3 days
Positive control group: 25%
hexylcinnamaldehyde in 4:1 acetone:olive
oil for 3 days
(Vehicle of positive control) group: 4:1
acetone :olive oil for 3 days
Sacrifice on test day 5
Diluting vehicle: N,N-Dimethyl formamide
Increases in cell proliferation in the draining
auricular lymph node of the ears treated with 50%
and 100% nano-Ti02 compared to the vehicle
control group.
No dermal sensitization by nano-Ti02: 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-Ti02 treated groups. Consequently
the ECS value (the estimated concentration
required to induce a threshold positive response,
i.e., where stimulation index equals 3) for nano-Ti02
was not calculated.
Positive control group had a dermal sensitization
response.
Warheit et al.
(2007.091075)
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Animal Testing Material Treatment Conditions
Summary of Major Effects
Reference
Pig
Male
In vivo
exposure
Repeated
exposure
Mouse
Male and
female
BALB/c
(hairless)
In vivo
exposure
Repeated
exposure
100%anatase,
uncoated, nano-Ti02
(Zhejiang Wanjin
Material Technology Co.,
Ltd.):
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 nf/g
75% anatase/25% rutile,
uncoated nano-Ti02 (P25
from Degussa,
Germany):
21 nm, hydrophilic
surface, surface area 50
m2/g
100% rutile, uncoated,
nano-Ti02 (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 m /g
90 nm, hydrophobic
surface, measured
particle size 90 ± 10 nm,
surface area 40 m /g
Porcine skin, in vivo, shaved pig ear
starting at age of 4 wk, approximately
24 mg of test formulation containing 5%
nano-Ti02 (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
forTEM.
Pig: After 30 days of treatment
Nano-Ti02 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-Ti02 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).
Wu (2009,
193721)
BALB/c hairless mice skin, in vivo, dorsal
region starting at age of 7-8 wk. Test
formulation containing 5% nano-Ti02
(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 ug nano-Ti02) 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.
Hairless mice: After 60 days of treatment
Mice treated with 10, 21, and 25 nm nano-Ti02 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.
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).
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-Ti02
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
Brdll - Bromo-deoxy-uridine SOD - superoxide dismutase
ECS - Estimated concentration required to induce a threshold positive response, where TUNEL -Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling
stimulation index equals 3
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.
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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 (a-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 (ig/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 (pw) mutation through DNA deletions of duplicated pink-
eyed dilution (p) gene in the offspring of C57BL/6J/>un//?un 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. Mutageni city/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.
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Table 5-5. Summary of health effects of nano-Ti02 particles in mammalian animal models: oral
route
Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Rat Nano-Ti02 (identified as uf-C, a
Female, strain/stock pre-commercial version of
not specified DuPont Light Stabilizer 210),
79% anatase/21% rutile, not
coated, approximately 90 wt%
Ti02,7% alumina, and 1 %
amorphous silica, average
particle size 140.0 ± 44 nm in
water, average BET surface
area 38.5 mig
For testing acute effects
Doses-175,550,1,750,
or5,000mg/kg(3rats per
dose)
Single oral gavage
Observation for 14 days
postexposure
No mortality, no biologically important BW losses, and no
gross lesions present in the rats at necropsy.
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).
Oral IDs, >5,000 mg/kg for female rats.
Warheitetal.
(2007.091075)
Mouse
Male and female
CD-1 (ICR)
Nano-Ti02 (Hangzhou Dayang
Nanotechnology Co. Ltd.),
rutile, uncoated, 25 nm
(measured average size
21.1 ±5.1 nm), surface area
43.0 m /g, column/spindle
shape, purity >99% (Chen,
personal communication, 2008,
157588)
Nano-Ti02 (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 Ti02 (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)
Single oral gavage (acute
effects)
Dose-5,000 mg/kg
10 female and 10 male
mice per Ti02 size group
Necropsy at 2 wk after the
gavage
Hepatic Toxicity:
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).3 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).'
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), a-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 a-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 Ti02. No pathological changes in any organs of mice
exposed to 25 nmTi02.
Distribution:
Ti02 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).
Wang et al.
(2007. 090290)
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Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Mouse
Wild-type and
C57BL/6Jpun/pun
Nano-Ti02 (P25),
photocatalytic, 80%
anatase/20% rutile, not coated
For testing genotoxicity in
two generations
Wild-type adult mice: 60,
120, 300 and 600 ug/mLin
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-Ti02 in
drinking water from
8.5-18.5 days post
conception. Offspring were
sacrificed at 20 days old.
Increased genomic instability (adult mice):
DMA damage was increased in cells in peripheral blood at
600 ug/mL DMA damage was measured by alkaline Comet
assay, which detects DMA single strand breaks, double
strand breaks, alkaline liable sites, and other lesions.
DMA double strand breaks (measured by yH2AX immuno-
staining) were increased in bone marrow at all tested doses.
Chromosomal damage (measured by micronucleus assay)
was increased in peripheral blood at 600 ug/mL
Oxidative DMA damage (measured by HPLC) was increased
in liver at 600 ug/mL
Increased genomic instability (offspring):
Increases in DMA deletions at the pink-eyed unstable (pun)
locus which result from homologous recombination or double
strand breaks between the DMA 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-a, IFN-y, and IL-8 (KC) (but not anti-inflammatory
markers, TGF-p, IL-10 or IL-4) in peripheral blood at
500 mg/kg as measured by real time RT-PCR.
Trouiller et al.
(2008.157484)
aLDH may be from heart, liver, kidney, skeletal muscle, brain, blood cells, and lungs. A test for LDH isotypescan 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).
a-HBDH -Alpha-hydroxybutyrate dehydrogenase
yH2AX - Phosphorylated form of histone H2AX (phosphorylation of H2AX at
serine 139)
ALT-Alanine aminotransferase
AST-Aspartate aminotransferase
BET- Brunauer, Emmett, Teller method of calculating surface area
BUN - Blood urea nitrogen
HPLC - High performance liquid chromatography
IFN-y - Interferon-gamma
IL-4 - lnterleukin-4
IL-8 (KC) - IL-8 stands for interleukin-8 and KC for chemokine (CXC motif)
ligand 1 (CXCL1)
IL-10-lnterleukin-10
LDH - Lactate dehydrogenase, a general marker of cell injury (Ma-Hock et al.,
2009)
LD50 - Lethal dose 50; the dosage that is lethal to 50% of the tested
population
RT-PCR - Reverse transcription polymerase chain reaction
TGF-p -Transforming growth factor-beta
TNF-a -Tumor necrosis factor-alpha
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
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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
Oberdorster 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
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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 macrophage-
mediated 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:
Oberdorster 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: Oberdorster 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,
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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-(im
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-(im 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 (Oberdorster 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 (Oberdorster 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 (ig
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-Ti02 particles in mammalian animal models:
respiratory route
Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Inhalation and Instillation in the same report
Mouse
Male C57BL/6
Nano-Ti02
(Nanostructured and
Amorphous Materials),
anatase, 5 nm, measured
BET surface area
219 ±3 m2/g, surface
functionalization: 0, 0-H,
H20. Aerosol size:
11 9 ±1. 56 nm (inhalation
high dose),
122.9±1.55nm
(inhalation low dose)
Nano-Ti02(Degussa),
Single inhalation exposure for
4hr
Particle concentration in
chamber:
5nmTi02:
Low: 0.77 mg/m3 (necropsy
immediately after exposure)
High: 7.22 mg/m3 (necropsy
immediately after exposure);
7.35 mg/m (necropsy 20 hr
after the end of exposure)
91 nmTin,-
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).
No changes in histology of the lung, total protein, LDH
activity, or neutrophil number in BAL fluid.
Nano-Ti02 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.
Calculated/estimated particle mass per mouse (ug) and
particle surface area (cm2):
Grassian et al.
(2007, 093170)
anatase/rutile, 21 nm,
BET surface area
41 ±1.1 m2/g, surface
functionalization: 0, 0-H,
H20. Aerosol size:
138.8±1.44m2/g
(inhalation high dose),
152.9±1.38m2/g
(inhalation low dose)
Low: 0.62 mg/m3 (necropsy
immediately after exposure)
High: 7.16 mg/m3 (necropsy
immediately after exposure);
7.03 mg/m (necropsy 20 hr
after the end of exposure)
5 nmTi02Low: 1.3 ug/mouse and 3.2cm (immediately
after exposure)
5 nmTi02 High: 12.5 ug/mouse and 30.3 cm2
(immediately after exposure) 12.7 ug/mouse and 30.7
cm2 (20 hr after exposure)
21 nmTi02 Low: 1.1 ug/mouse and 2.2 cm2
(immediately after exposure)
21 nmTi02 High: 12.4 ug/mouse and 24.8 cm2
(immediately after exposure) 12.2 ug/mouse and 24.4
cm (20 hr after exposure)
5-35
-------
Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Single intra-nasal instillation
Particle concentration in
instillation solutions:
5nmTi02:
Low: 0.1 mg/mL
Medium: 0.4 mg/mL
High: 0.6 mg/mL
21 nmTi02:
Low: 0.5 mg/mL
Medium: 2.0 mg/mL
High: 3.0 mg/mL
Necropsy 24 hr after instillation
21 nmTi02 induced more inflammation than 5 nmTi02:
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-1p
(21 nmhigh) in BALfluid.
No pathological changes in lung; no changes in TNF-a in
BAL fluid.
21 nm anatase/rutile Ti02 and 5 nm anatase Ti02 do not
share the same dose-response curve for neutrophil
concentration in BAL fluid as a function to either particle
mass or surface area.
Calculated/estimated particle mass per mouse (ug) and
particle surface area (cm2):
5 nmTi02 Low: 5 ug/mouse and 12.1 cm2
5 nm Ti02 Medium: 20 ug/mouse and 48.4 cm2
5 nmTi02 High: 30 ug/mouse and 72.6 cm2
21 nmTi02 Low: 25 ug/mouse and 12.5 cm2
21 nmTi02 Medium: 100 ug/mouse and 50 cm2
21 nmTi02 High: 150 ug/mouse and 75 cm2
Rats
Female F344
Fine T|02 (Fisher
Scientific), mean primary
particle size 250 nm,
anatase
Nano-Ti02(Degussa),
mean primary particle size
21 nm, anatase
Acute intratracheal instillation
and intratracheal inhalation
Intratracheal inhalation
exposure for 2 hr at 125 mg/m3
Intratracheal instillation
exposure to the equivalent
amount of Ti02 as in the lung at
day 0 of intratracheal inhalation
(500 ug fine Ti02 or 750 ug
nano-Ti02 in 0.2 mL saline)
Necropsy 0,1, 3 or 7 days
postexposure (3 rats per group)
Compared to fine Ti02, nano-Ti02 caused more
pulmonary responses and slightly higher (not significant)
lungTi02 burden.
Compared to intratracheal instillation, intratracheal
inhalation to Ti02 generally caused less severe and less
persistent pulmonary responses and slightly (not
significant) higher Ti02 lung burden.
Increases in polymorphonuclear leukocytes in BAL cell
pellet on day 1 after intratracheal inhalation of fine Ti02;
on days 1, 3, and 7 after intratracheal instillation of
nano-Ti02; and days 0 and 1 after intratracheal
inhalation of nano-Ti02.
Decreases in macrophage inflammatory protein-2 levels
in BAL supernatant on days 0,1, and 3 after
intratracheal inhalation of nano-Ti02; and day 1 after
intratracheal instillation of nano-Ti02. Increases in
macrophage inflammatory protein-2 levels in BAL cell
pellets on days 1,3, and 7 after intratracheal instillation
of nano-Ti02; and on days 0 and 1 after intratracheal
inhalation of nano-Ti02.
Increases in TNF-a 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-Ti02 and intratracheal inhalation of fine Ti02; and
at all time points after intratracheal inhalation of
nano-Ti02.
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-a protein
levels in BAL cell pellet or supernatant or in lung
sections stained immunocytochemically.
Osier etal.
(1997.086056)
Inhalation
Rats
Male F344
Nano-Ti02, -20 nm,
anatase (Degussa)
Fine Ti02, -250 nm,
anatase (Fisher Scientific)
Crystalline Si02, -800 nm
Subchronic inhalation
Nano-Ti02:23.5 mg/m3; fine
Ti02:22.3 mg/m ; Si02
1.3 mg/m3
6 hr/day, 5 days/wk for 3 mo
6- or 12-mo recovery before
sacrifice
Lung burden: Si02:0.32 mg immediately after exposure.
Nano Ti02/fineTi02:5.33/6.62 mg, 4.15/1.2 mg,
3.14/1.66 mg immediately, 6 mo, 12 mo after exposure,
respectively.
6 mo after exposure, in the lung: Si02 caused moderate
focal interstitial fibrosis and moderately severe focal
alveolitis; nano Ti02 caused slightly less fibrosis and fine
Ti02 caused least fibrosis. Increases in stainable
collagen in all three treated groups, compared to
untreated groups.
12 mo after exposure, in the lung: Si02~treated rats
showed decreased fibrosis; nano Ti02 and fine Ti02
treated rats showed largely normal amount of interstitial
fibrosis but increases in alveolar macrophage number.
Increases in stainable collagen only in Si02.
Baggs et al.
(1997.048642)
5-36
-------
Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Rat
Female CDF
(F344)/CrlBR
Mouse
Female
B6C3F1/CMBR
Hamster
Female Syrian
golden (LaklVG
[SYR] BR)
FineTI02(DuPont), rutile;
aerosol 1.36 -1.44 urn
MM AD
Nano-TI02(P25),
photocatalytic, average
primary particle size
21 nm, 1.37 urn MMAD;
aerosols: 1.29-1.44 urn
MMAD
Subchronic inhalation
Fine Ti02:0,10, 50 or
250 mg/m3
nano-TiO,:0,0.5,2, or
10 mg/m
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
Ti02 and nano-Ti02,
respectively) wk of recovery
before sacrifice
Lung burden of fine Ti02:
Immediately after exposure: lung burden of fine Ti02:
mice > rats > hamsters at 50 and 250 mg/m3:
rats > mice > hamsters at 10 mg/m3. The lung burden
decreased with time after exposure.
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).
Pulmonary clearance kinetics of fine Ti02: 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/m .
Lung burden of nano-Ti02:
Lung burden of nano-Ti02: rats 2 mice > hamster.
Immediately after exposure, at 10 mg/m3, rats and mice
had same lung burdens for nano-Ti02. 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-Ti02: 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/m , rats, mice and hamsters
had biphasic decreases in lung burn, and rats only had
detectable nano-Ti02 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-Ti02 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
Ti02 had a consistent lower weight during the recovery
period, but rats exposed to 250 mg/m3 fine Ti02 had a
consistent heavier weight. Hamster exposed to fine Ti02
had decreases in weight gain after exposure and
recovery 6 wk postexposure. Hamsters exposed to
nano-Ti02 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 age-
related renal diseases.
FineTi02:
Bermudez et al.
(2002, 055578)
Nano-Ti02:
Bermudez et al.
(2004, 056707)
Comparison of
fine and
nano-Ti02 data
reported in
Bermudez et al.
(2002, 055578)
and
Bermudez et al.
(2004.056707):
Hextetal. (2002,
157878:2005.
090567)
5-37
-------
Animal Testing Material Treatment Conditions Summary of Major Effects Reference
Pulmonary inflammation after fine Ti02 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 Ti02 exposure: Increases in neutrophil %,
lymphocyte %, and macrophage number in BALfluid 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 Ti02 exposure: LDH levels in BAL fluid transiently
increased in mice and rats
Pulmonary inflammation after nano-Ti02 exposure: Rats
and mice had pulmonary inflammation.
Nano-Ti02 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 Ti02 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 Ti02 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-Ti02 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 Ti02 or 10 mg/m3
nano-Ti02.
Lung Ti02 burdens and tissue responses in mice, rat and
hamsters exposed for 13 wk to 10 mg/m3 nano-Ti02 or to
50 mg/m3 fine Ti02 were similar for all three species.
5-38
-------
Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Rat
Female Wistar
Mouse
Female NMRI
Nano-TI02(P25),
photocatalytic, 80%
anatase/20% rutile,
primarily particle size
15-40 nm, 0.8 urn MMAD
Chronic inhalation
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)
18 or 19 hr/day, 5 days/wk in
whole body chamber
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
Rats:
Increases in lung weight, and retention of inhaled
nano-Ti02 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-Ti02 exposure to
3.3 mg/lung at 4 mo postexposure).
Increased half-time of pulmonary clearance of tracer
particles
For inhaled 0.35 um labeled tracer particles
After 3-, 12-, 18-mo nano-Ti02 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 um labeled tracer particles
After 3-, 12-, 18-mo nano-Ti02 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 tracheo-
bronchial region of the long and apparently higher
clearance rates.
Rats did not have increases in DNAadducts in the lung:
No increases in DNAadduct 2 (nuclease P1-sensitive
adduct) in the lung.
Decreases in DNAadduct 1 (age-related, putative
l-compound) in peripheral lung DNA compared to filtered
air-exposed rats, probably due to adduct dilution through
cell proliferation induced by particle exposure.
Rats:
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 Ti02 (0 out of 18 in control) had
lung tumors. 24-mo exposure: 4/9 rats in Ti02 (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.
Creutzenberg et
al. (1990,
157963)
Gallagher et al.
(1994.045102)
Heinrich et al.
(1995.076637)
5-39
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Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Mouse
Male C57BL/6
Nano-Ti02
(Nanostructured and
Amorphous Materials),
anatase, measured
average primary particle
size 3.5 ±1.0nm, BET
surface area 219 ± 3 m /g,
surface functionalization:
0,0-H,H20
(manufacturer reported
primary particle 5 nm,
surface area 210 m2/g)
Aerosol size geometric
mean 120-128 ±1.6-
1.7 nm for acute (two
concentrations) and
subacute (one
concentration) exposures
Acute inhalation
Doses - 0,0.77, or 7.22 mg/m3
Single exposure of 4 hr in
whole-body chamber
No recovery time
No adverse effect/Minimal pulmonary inflammation.
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.
Grassian et al.
(2007, 090606)
Subacute inhalation
Doses - 0 or 8.88 mg/m3
4 hr/day for 10 days in whole-
body chamber
0,1, 2, or 3 wk of recovery
before sacrifice
Moderate but significant pulmonary inflammatory
response that lasted for at least 2 wk but resolved by
wk 3 after exposure.
No changes in most parameters measured to gauge
inflammatory response [total protein, LDH activity, and
cytokine (IFN-v, IL-6, or IL-1p) concentrations in BAL
fluid were not changed, and no effects on lung
histopathology.
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 Ti02
particles and less so at wk 3 postexposure.
Rat Nano-Ti02 (Baker &
Male Wistar 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 nf/g. Zeta
potential was 16.5 ±2.2
mV in 1 mM KCI.
Aerosols: 0.7-1.1 urn
MMAD (geometrical
standard deviations
2.3-3.4). Small and large
agglomerates in the
atmospheres, ranging
from below! 00 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.
Short-term inhalation
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
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.
Absolute lung weight was increased at 50 mg/m3 Ma-Hock et al.
immediately after exposure, but not after 16-day (2009,193534)
recovery.
Lung burden: 118.4, 544.9 and 1,635 ug/lung
immediately after inhalation of 2,10 and 50 mg/m3
nano-Ti02, respectively. 16 days of recovery later, the
lung burdens were 93.4, 400.4 and 1,340 ug/lung,
respectively. Calculated clearance half-times were 47,
36 and 56 days for 2-, 10- and 50-mg/m3 groups,
respectively.
In the mediastinal lymph nodes, Ti02 was only detected
in the 50-mg/m3 group, and the nano-Ti02
concentrations were higher at 16 days after the last
exposure (mean 11.01 ug in collected lymph nodes) than
immediately after exposure (mean 2.34 ug). No Ti02 was
detected in the liver, kidney, spleen or basal brain with
olfactory bulb (detection limit 0.5 ug per organ).
BAL fluid: increases in total cell count at 50mg/m3 and
polymorphonuclear neutrophils at 10 mg/m3 and
50 mg/m , 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/m groups, but not in 50 mg/m 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
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Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Rat
Male F344
Nano-Ti02,20 nm,
anatase (Degussa); in
aerosols: agglomerates
0.71 ±1.9|jmMMAD
Fine Ti02, 250 nm,
anatase (Fisher
Scientific); in aerosols:
agglomerates
0.78±1.7|jmMMAD
Subchronic inhalation
Nano-Ti02:23.5 ± 2.9 mg/m3;
fine Ti02:22.3 ± 4.2 mg/m3
6hr/day, 5days/wk,for 12 wk
Recovery for 4, 8,12, 29 or
64 wk before sacrifice
Nano-Ti02 caused more severe and prolonged (~1 yr) Oberdb'rster et
pulmonary inflammatory response (i.e., increase in al. (1994,
alveolar macrophages, polymorphonuclear neutrophils, 046203)
and lavagable protein) than fine Ti02.
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-Ti02 and fine Ti02 shared the
same dose response curve.
More severe and prolonged impairment of alveolar
macrophage-mediated particle clearance in rats exposed
to nano-Ti02 than rats exposed to fine Ti02. Seven mo
after Ti02 exposure, fine Ti02 exposed (but not
nano-Ti02 exposed) rats showed normal clearance
rates.
Pathological changes in the lung: nano-Ti02 caused
greater epithelial effects (Type II cell proliferation,
occlusion of pores of Kohn) and more interstitial fibrotic
foci than fine Ti02.
Dosimetry:
Nano-Ti02 and fine Ti02 had a similar mass deposition in
the lower respiratory tract and same retention in the
alveolar space up to 1 yr after exposure.
Nano-Ti02 showed longer total pulmonary retention
(retention halftime: -500 days for nano-Ti02, -170 days
for fine Ti02), 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-Ti02 depends mainly
on mucocillary clearance, while fine-Ti02 depends on
clearance to the gastrointestinal tract) than fine Ti02.
5-41
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Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Rat
Male Wistar,
Strain Crl:
WI(Han)
Nano-Ti02, 20-30 nm
(measured by TEM), 70%
anatase, 30% rutile, BET
surface area 48.6 m /g,
uncoated isoelectric point
(IEP)waspH7in10mM
KCI, MMAD 1 .0 urn in
aerosol
FineTi02, median size
200 nm in ethanol
(measured by DLS), rutile,
BET surface area 6 m2/g,
IEP
-------
Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Rats
Female
Sprague-
Dawley,
Hla:(SD)CVF
Fine Ti02, primary particle
<5 |jm, 99% rutile
(reported vendor), BET
surface area 2.34 m /g
(reported inSageretal.,
2008,157499) (Sigma-
Aldrich, product#224227);
MMAD of the aerosols
402nmwithaGSDof2.4,
CMD of the aerosols
710 nm
Nano-Ti02(P25), primary
particle 21 nm, 80%
anatase, 20% rutile
(reported by vendor), BET
surface area 48.08 m2/g
(reported inSageretal.,
2008,157499); MMAD of
the aerosols 138 nm with
a GSDof2.2.CMD of the
aerosols 100nm
Short-term inhalation
Whole body chamber exposure
Exposures selected which do
not alter BAL markers of
pulmonary inflammation or lung
damage
Exposure to fine Ti02: aerosol
concentration x exposure time
(actual deposition in lung)
15mg/m3 x480 min (90 ug)
16mg/m3 x300 min (67 ug)
12mg/m3 x240 min (36 ug)
6 mg/m3 x 240 min (20 ug)
3 mg/m3 x 240 min (8 ug)
Exposure of nano-Ti02: aerosol
concentration x exposure time
(calculated/actual deposition in
lung)
10 mg/m3 x 720 min that took
place over 3 days (38 ug)
12 mg/m3 x240 min (19ug)
6 mg/m3 x 240 min (10 ug)
3 mg/m3 x 480 min (10ug)
12 mg/m3 xi20 min (10ug)
3 mg/m3 x 240 min (6 ug)
1.5 mg/m3 x 240 min (4 ug)
Sham exposure (control):
0 mg/m3 x 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
Histology of the lung:
No significant inflammation.
Particle accumulation in alveolar macrophage. Anuclear
alveolar macrophages were seen in both nano-Ti02 and
fine Ti02 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 Ti02 and nano-Ti02 exposures impaired
arteriolar dilation in a dose-dependent manner, and
nano-Ti02 exposure produced greater impairment than
fine Ti02 at similar pulmonary load doses. No-effect dose
of fine Ti02 was 8 ug (as in lung deposition), and for
nano-Ti02was 4 ug.
On a mass base, nano-Ti02 was approximately one
order of magnitude more potent than fine Ti02; on total
particle surface area base calculated by BET surface
area, fine Ti02 would be more potent than nano-Ti02
(the authors suspected overestimation of the total
nano-Ti02 surface area delivered, since no
agglomeration was considered).
Additional nano-Ti02 exposure conditions (12 mg/m3 x
2 hr; 4 mg/m3 x 6 hr; 8 mg/m3 x 3 hr) yielded the same
level of impairment of systemic arteriolar dilation,
suggesting the response is dependent on the exposure
concentration (of product) xtime.
Nurkiewicz et al.
(2008,156816)
Same exposure conditions as
above (Nurkiewicz et al., 2008,
156816) for endogenous
microvascular NO production
tests, but only three groups in
all other tests: aerosol
concentration x exposure time
(actual deposition in lung)
Sham exposure (control):
0 mg/m3 x 240 min
Fine Ti02:16mg/m3 x 300 min
(67 ug)
Nano-Ti02:6 mg/m3 x 240 min
(10ug)
24 hr postexposure, sample
collection, including and
exteriorizing spinotrapezius
muscle as described in
Nurkiewicz et al. (2008,
156816) and excising
spinotrapezius muscles from
separate groups of rats for
measurement of NO,
microvascular oxidative stress,
and nitrotyrosine staining
Same impairment of arteriolar dilation at 67 ug fine Ti02 Nurkiewicz et al.
and 10 ug nano-Ti02more than 50% decrease compared (2009,191961)
to sham treated controls after Ca2+ ionophore A23187
injection at 20 and 40 psi ejection pressures.
No change in arteriolar dilation in response to sodium
nitroprusside (NO donor) in either 67 ug fine Ti02 or 10
ug nano-Ti02 exposed rats, indicating no change in
vascular smooth muscle sensitivity to NO.
Increased ROS amount in the microvascular wall in both
67 ug fine Ti02 and 10 ug nano-Ti02 groups at the same
level as measured by ethidium bromide fluorescence.
Increased nitrotyrosine expression in 10 ug nano-Ti02
treated rats (not measured in fine Ti02 group) in lung
(3 folds) and spinotrapezius microcirculation (4 folds), as
compared to sham exposure, suggesting nitrosative
injury in lung and systemic microcirculation.
Decreased Ca2+ ionophore A23187-stimulated
endogenous microvascular NO production in fine Ti02
and nano-Ti02 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.
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 ug fine Ti02 and 10 ug
nano-Ti02 groups.
5-43
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Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Instillations
Mouse Nano-Ti02(Degussa),
Male ICR rutile, highly dispersed
and hydrophilic fumed
nano-Ti02, diameter
19-21 nm (average
primary particle size
21 nm), surface area of
50 ±15 m/g, purity
> 99.5%
To avoid aggregation, the
nano-Ti02 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.
Single intratracheal instillation
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
Gross morphology and histology of the lung: Chen et al.
Emphysema-like lung injuries were seen at 0.1 and (2006, 090139)
0.5 mg/mouse (more severe at 0.5 mg) at 3 days, 1 wk,
and 2 wk after the instillation.
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
(cxcH, cxcIS, and cc!3), 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.
Rat
Female Wistar
(HsdCpbWU)
Nano-Ti02 (P25), Repeated weekly intratracheal
photocatalytic, hydrophilic, instillation
80% anatase/20% rutile,
primarily particle size
25 nm, BET specific
surface area 52 m2/g
Increased primary benign tumors and malignant cancers
in lung in all tested doses.
Instilled doses:
5 instillations x 3 mg
5 instillations x 6 mg
10 instillations x6 mg
Mohretal.
(2006, 097493)
Pott and Roller
(2005,157790)3
Nano-Ti02 (Degussa T805 Repeated weekly intratracheal
/P805) ,a crystal form not instillation
specified, coated with an |nstN|ed doses:
organic silicon compound;
21 nm;32.5m2/g 15 instillations x 0.5 mg
30 instillations x 0.5 mg
High initial acute mortality, lowered dose to 0.5 mg.
No conclusion on carcinogenicity.
Fine Ti02, hydrophilic,
anatase, primary particle
200 nm, BET specific
surface area 9.9 m /g
Repeated weekly intratracheal
instillation
Instilled doses:
10 instillations x6 mg
20 instillations x 6 mg
Increased primary benign tumors and malignant cancers
in lung in all tested doses.
Rat
Male Wistar
Nano-Ti02, rutile, primary
particle diameter 4-6 nm,
rod shape (synthesized in
the lab by a soft chemistry
technique); BET surface
for instilled nano-Ti02 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 urn.
Single intratracheal instillation
(acute effects)
1 or 5 mg/kg nano-Ti02or
vehicle only (150 uL)
Single intratracheal instillation
nano-Ti02was suspended in
saline containing 0.01 % Tween
80 (a surfactant and emulsifier)
Blood collection and necropsy
at 24 hr after instillation
Pulmonary inflammation: increases in macrophage and Nemmar et al.
neutrophil numbers in BAL fluid at 5 mg/kg. most (2008,157514)
nano-Ti02 aggregates in BAL fluid were inside
macrophages.
Pulmonary and cardiac edema: increases in the wet
weight-to-dry weight ratios of lung and of heart at 1 and
5 mg/kg.
Systemic inflammation: increases in monocyte and
granulocyte (but not lymphocyte) numbers in blood at
5 mg/kg.
Platelet aggregation: decreases platelet number in blood
of rats exposed to 5 mg/kg nano-Ti02, suggesting
platelet aggregation [in vitro supporting evidence: adding
2 or 10 ug/mL (but not 0.4 ug/mL) nano-Ti02 directly into
untreated rat whole blood caused platelet aggregation].
5-44
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Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Rats
Male F344
Nano-Ti02
anatase
Fine Ti02,
anatase
, -20 nm,
-250 nm,
Single intratracheal instillation
(acute effects)
500 ug of either anatase
nano-Ti02 or anatase fine Ti02
Anatase nano-Ti02 induced more inflammatory response
and higher interstitial access in the lung than anatase
fine Ti02 of the same mass dose.
Oberdb'rster et
al. (1992,
045110)
A single intratracheal instillation,
followed by 24-hr recovery
Nano-Ti02, -20 nm,
anatase (free anatase
nano-Ti02 )
Alveolar macrophage
collected 24 hr after
donor-rat received 200 ug
anatase nano-Ti02 via
intratracheal instillation
(containing phagocytized
anatase nano-Ti02)
Alveolar macrophage
collected from untreated
rat lung
PMNs from peripheral
blood of untreated rats
Single intratracheal instillation
(acute effects)
Free anatase nano-Ti02,
104ug
Phagocytized anatase
nano-Ti02 104 ug + 9.5 x 106
alveolar macrophages + 3.9 x
106 polymorphonuclear
neutrophils
Alveolar macrophages 6.8 x
106
Polymorphonuclear neutrophils
2.2 x 106
Serum-exposed anatase
Free anatase nano-Ti02 and serum-exposed anatase
nano-Ti02 caused pulmonary inflammatory reaction
(same level) and interstitial distribution.
Phagocytized anatase nano-Ti02 alone did not
contribute significantly to inflammatory reaction, because
the reaction can be explained by the alveolar
macrophages and polymorphonuclear neutrophils.
Phagocytized anatase nano-Ti02 showed less interstitial
distribution than free anatase nano-Ti02.
Serum-exposed anatase
nano-Ti02 (incubated in
rat serum for 1 hrand
then washed twice)
nano-Ti02100ug
A single intratracheal instillation
followed by 24-hr recovery
Fine Ti02, -250 nm,
anatase
Nano-Ti02, -20 nm,
anatase
Fine Ti02, -220 nm, rutile
(from Dr. Siegal at
Argonne National
Laboratory, Argonne, IL)
Nano-Ti02,-12 nm, rutile
Carbon black, -30 nm
(Cabot, 660R)
A single intratracheal instillation
of 500 ug each; anatase fine
Ti02 was also tested at 1,000
ug; anatase nano-Ti02 was also
tested at 65,107,200, and
1,000ug
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-Ti02 at high doses.
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.
Higher fractions of nano-Ti02 (anatase and rutile
nano-Ti02) were interstitialized (translocated into
interstitium or epithelium cells) than other particles.
5-45
-------
Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Rat
[strain/stock not
specified]
Nano-Ti02, -20 nm,
surface area is estimated
to be 10 times of surface
area of -250 nm Ti02
Fine Ti02, -250 nm
Single Intratracheal instillation
(acute effects)
Nano-Ti02:30,~150,500ug
Fine Ti02: -150, 500, 2,000 ug
Pulmonary inflammation (neutrophil % in lung lavage)
was seen at 24 hr postexposure. At the same mass
dose, nano-Ti02 induced more inflammation than fine
Ti02. When doses are expressed as surface area, fine
Ti02 and nano-Ti02 shared the same dose-response
curve.
Oberdb'rster
(2000, 036303)
Nano-Ti02
Polytetrafluoroethylene
(PTFE) (Teflon) fume,
count median diameter
-18 nm
Repeated inhalation of PTFE
fume (5 x 105 particles/cm3 =
-50 ug/cm3, 5 min/day for
3 days) followed by a single
intratracheal instillation of
100 ug nano-Ti02
Cross tolerance: nano-Ti02 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.
Nano-Ti02, -20 nm
Fine Ti02,-250 nm
Inhalation of LPS followed by a
single intratracheal instillation of
nano-Ti02 and fine Ti02 (acute
Lipopolysaccharide (LPS), effects)
an endotoxin found in . _„ .„ . ,„
gram negative bacteria LpS-~12 mm exposure -70
a a endotoxin units (estimated
alveolar dose)
Nano-Ti02 and fine Ti02:50 ug
Within 30 min of inhalation of
LPS or saline, intratracheal
instillation of nano-or fine Ti02
24 hr of recovery
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).
50 ug nano-Ti02, but not fine Ti02, further increased
inflammatory response in compromised hosts with mild
pulmonary inflammation.
Neutrophil % in rats exposed to (LPS and then
nano-T|02) > (LPS and then fine Ti02), LPS alone,
nano-Ti02 alone > fine Ti02 alone, negative control.
It is unclear whether fine Ti02 at a dose that increases
inflammatory response would further increase
inflammatory response in compromised hosts.
Rat
Wistar
Nano-Ti02(P25),
photocatalytic, 80%
anatase/20% rutile,
untreated, hydrophilic
surface, primarily particle
size -20 nm
Nano-Ti02 (Aeroxide®
T805), photostable, 80%
anatase/20% rutile,
silanized,
trimethoxyoctylsilane-
treated hydrophobic
surface, primarily particle
size -20 nm
Single intratracheal instillation
(subchronic effects)
Doses: 0, 0.15, 0.3, 0.6, or
1 .2 mg nano-Ti02 (positive
control: 0.6 mg quartz DQ12) in
0.2 mL saline supplemented
with 0.25% lecithin
3, 21 , or 90 days of recovery
Transient pulmonary inflammatory responses to both
types of nano-Ti02 (mostly only at 1 .2 mg dose, some at
0.6 mg groups) (most responses returned to normal by
day 90).
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 .
Neither P25 norTSOS increased oxidative DNAadduct
(as 8-oxoguanine) in the lung on day 90.
Quartz induced persistent inflammatory response and
increased 8-oxoguanine on day 90.
Rehn et al.
(2003, 090613)
Crystalline silica and
quartz particles (DQ-12)
as positive reference
Rat
Male Wistar
Nano-Ti02 (Degussa),
mean diameter 29 nm,
BET surface area
49.78 m2/g
Fine Ti02(Tioxide Ltd),
mean diameter 250 nm
BET surface area 6.6 m2/g
Carbon black, mean
diameter 260.2 nm, BET
surface are 7.9 m2/g
Ultrafine carbon black,
mean diameter 14.3 nm,
BET surface 253.9 m/g
Single intratracheal instillation
(acute effects)
0,125, and 500 ug particles in
saline
24 hr of recovery before
sacrifice
Nano-Ti02 at 500 ug (but not nano-Ti02 at 125 ug or fine Renwick et al.
Ti02 at either 125 or 500 ug) increased neutrophil (2004, 056067)
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).
Both nano- and fine Ti02 (at 500 ug, but not at 125 ug)
decreased phagocytic function of macrophage.
Carbon black caused same changes as fine Ti02, with
the exception of increases in LDH activity at 500 ug.
Ultrafine carbon black caused same changes as
nano-Ti02, but increases in inflammation and LDH and
GGT activities were significant at 125 ug (nano-Ti02
caused significant changes at 500 ug only).
5-46
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Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Rat
Male
Crl:CD(SD)IGS
BR
Rat
Crl:CD®(SD)IG
SBR
FineTi02(DuPont):
primary particle -300 nm,
anatase, -99 wt %
Ti02/~1 wt % alumina,
BET surface area -6 m2/g
/D -inn\
(K-1UU)
Nano-Ti02 rods
(synthesized
hydrothermally): primary
particle length 92-233 nm
x width 20-35 nm,
anatase, BET surface
area 26.5 m2/g
Nano-Ti02 dots
(synthesized
hydrothermally): primary
particle diameter
5.8-6.1 nm, sphere,
anatase, BET surface
area 169.4 m2/g
Quartz (Min-U-Sil quartz):
median primary particle
-1.5 um (range! to
3 um), crystalline silica,
BET surface area 4 rrng
Nano-Ti02(DuPont),
photostable, rutile, coated
with alumina, (-98% Ti02,
-2% alumina), average
particle size of 136nmin
water and average BET
surface area of 18.2 m /g
(uf-1)
Nano-Ti02 (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
Nano-Ti02(DuPont),
photostable, rutile, coated
with silica and alumina
surface coating (-88 wt %
Ti02, -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)
FineTi02(DuPont),
photostable, rutile, coated
with alumina (-99% Ti02
and -1% alumina), an
average particle size
382 nm in water, average
BET surface area 5.8 m /g
Quartz
Single intratracheal instillation
(subchronic effects)
0, 1 or 5 mg/kg of each testing
material in PBS with polytron
dispersant
BAL fluid analysis at 24 hr,
1 wk 1 mo and 3 mo
postexposure (5 rats per group
per dose per time point)
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)
Single intratracheal instillation
(subchronic effects)
0, 1 , or 5 mg/kg
90-day recovery period
Like fine Ti02, nano-Ti02 rods and nano-Ti02 dots
caused only transient pulmonary inflammation, and not
significant lung toxicity.
All 5 mg/kg Ti02 (fine, nano rods, and nano dots), but not
1 mg/kg Ti02, caused transient, short-lived inflammation
(increases in neutrophil % in BAL fluid at 24 hr
postexposure only; increases in LDH by 5 mg/kg
nano-Ti02 rods at 24 hr postexposure only).
No changes in lung weight, tracheobronchial cell
proliferation (measured in high dose groups only) or lung
morphology (pathological changes).
Ti02 in macrophages was seen in all three types of Ti02.
Transient lung parenchymal cell proliferation in low and
high fine Ti02 at 1 wk postexposure (different from
previous studies in similar conditions).
Quartz caused sustained pulmonary inflammation and
early sign of pulmonary fibres is.
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).
Prelude offibrosis (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-Ti02 (both types of coated rutile).
Pulmonary inflammation and cytotoxic effects at highest
exposure of photocatalytic nano-Ti02 increased
bronchoalveolar lavage fluid LDH and BAL fluid
microprotein concentrations.
Increased tracheobronchial and lung parenchymal cell
proliferation rates at highest exposure of photocatalytic
nano-Ti02.
Lung inflammation/cytotoxicity/cell proliferation and
histopathological responses: quartz > nano-Ti02 P25
(anatase and rutile) > fine Ti02 (rutile) = nano-Ti02 uf-1
(rutile) = nano-Ti02 uf-2 (rutile).
Warheit et al.
(2006, 088436)
Warheit et al.
(2007, 090594)
5-47
-------
Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Mouse Nano-Ti02 (Hangzhou
Female Dayang Nanotechnology
CD10CR) Co. Ltd.), rutile, 80 nm,
measured average size
71.4 ±23.5 nm, purity
>99%
FineTi02 (Zhonglian
Chemical Medicine Co.),
anatase, 155 nm,
measured average size
155.0 ± 33.0 nm, purity
>99%
Repeated intranasal instillation
~500 ug Ti02 in pure water per
mouse every other day for 2,
10, 20, or 30 days (1,5,10 or
15 instillations, respectively)
Necropsy 1 day after last
instillation
Fortranslocation of Ti02 into
brain: 6 mice per group for each
time point.
For effects in brain: 10 mice per
group
Ti02 distribution (measured after 15 instillations): first
into olfactory bulb, and then to hippocampus. Ti
concentrations: hippocampus, olfactory bulb
> cerebellum, cerebral cortex > thalamus.
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-a 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-a and IL-1p, but not IL-6
(155 nm after 15 instillations).
Wang et al.
(2008,157474)
Wang et al.
(2008,157473)
5-48
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Animal
Testing Material
Treatment Conditions Summary of Major Effects
Reference
Mouse Nano-Ti02 (Hangzhou
CD-1 (ICR) Dayang Nanotechnology
Co. Ltd.), rutile, 25 nm,
purity >99%
Nano-Ti02 (Hangzhou
Dayang Nanotechnology
Co. Ltd.), rutile, 80 nm,
purity >99%
Fine Ti02 (Zhonglian
Chemical Medicine Co.),
anatase, 155 nm, purity
>99%
Repeated intranasal instillation
(subacute effects)
10 uL of 50 mg/kg Ti02 or water
every 2 days
Blood and brain were collected
from anesthetized mice after 2,
10, 20, or 30 days
No changes in water and food consumption or BW. Wang et al.
Brain Ti02 content (measured in all brain samples): f2007' 157616>
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 Ti02-treated mice
compared to control, but not in 25 nm Ti02-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 nmTi02-treated mice;
5-hydroxytryptamine was significantly increased in
155 nmTi02-treated mice; homovanillic and
5-hydroxyindole acetic acid were decreased in 80 and
155 nm Ti02-treated mice; dopamine was decreased in
80 nmTiOrtreated mice.
'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-Ti02 (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
et al., 2009,193534) or liver toxicity
ALT - Alanine transaminase
AST-Aspartate aminotransferase
BAL- Bronchoalveolar lavage
BET- Brunauer, Emmett, Teller method of calculating surface area
CMD - Count median diameter
DLS - Dynamic light scattering
ELISA- Enzyme-linked immunosorbent assay
F344 - Fischer 344 rat strain
GFAP - Glial fibrillary acidic protein
GGT-gamma-Y-glutamyltransferase, a marker for damage to Clara and type
II epithelial cells (Ma-Hock et al., 2009,193534)
GSD - Geometric standard deviation
GSH - Reduced glutathione
GSH-Px-Glutathione peroxidase
GST- Glutathione-S-transferase
lEP-lsoelectric point
IL-1p-lnterleukin-1 beta
IL-6 - lnterleukin-6
IFN-y - interferon-gamma
LDH - Lactate dehydrogenase, a general marker of cell injury (Ma-Hock et
al.. 2009.193534)
LPS - Lipopolysaccharide
MMAD - Mass median aerodynamic diameter
MTP - Microsomal triglyceride
NADPH - Nicotinamide adenine dinucleotide phosphate
P25-Aeroxide®P25
PBS - Phosphate buffered saline
PIGF - Placenta growth factor
PMN - Polymorphonuclear neutrophils
PTFE - Polytetrafluoroethylene
ROS - Reactive oxygen species
RT-PCR - Real-time polymerase chain reaction
SOD - Superoxide dismutase
TEM -Transmission electron microscopy
TNF-a -Tumor necrosis factor-alpha
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
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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-Ti02 particles in mammalian animal models: other
(injection, ocular) route
Animal Testing Material
Treatment Conditions Summary of Major Effects
Reference
Injection
Rat Nano-Ti02, primary particle
Male Wistar 20-30 nm (measured by TEM), BET
(strain surface area 48.6 m /g, 70%
Crl:WI(Han) anatase/ 30% rutile, uncoated, IEP
waspH7in10mMKCI
FineTi02 (Kronos International),
median size 200 nm in ethanol
(measured by DLS), rutile, BET
surface area 6 m2/g, IEP spleen » lung > kidney. The
time for the Ti02 concentration to return to normal
levels were in the same sequence. Liver had the
same Ti02 levels after 14 and 28 days. Spleen
had slightly decreased Ti02 levels 14 and 28 days
after injection. Lung and kidney had no elevated
Ti0214 days after injection. No Ti02 was detected
in blood cells, plasma, brain or lymph nodes
(mediastinal, mesenteric, popliteal) at any three
time points tested (detection limit 0.3 ug Ti = 0.5
ugTi02 per tissue).
Mouse Nano-Ti02(DegussaW740X),
Balb/c dispersion of photocatalytic
uncoated nano-Ti02 (80% anatase/
20% rutile) at 40 wt%, primary
particle 4.7 nm, mean aggregate
size < 100 nm;(Evonik, 2007,
157577: Llames, personal
communication, 2008,157528:
Weaver, personal communication,
2008.157467)
i.v. injections
5.6 mg/mouse/day for
2 days (total dose
11.2mg/mouse)
1 or 3 days of recovery
before sacrifice
Lung, liver, and spleen showed white
discoloration and phagocytosis of nano-Ti02
aggregates by macrophages under light
microscope.
Miller etal. (2007,
157668)
Mouse
Slc:ICP,
pregnant
female and
male offspring
Nano-Ti02,25-70 nm anatase,
surface area 20-25 m /g, purity
99.9% (from Sigma-Aldrich)
s.c. injections of 100 uLof
1 mg/mLnano-Ti02 (i.e.,
0.1 mg nano-Ti02) into each
time-pregnant Slc:ICP
mouse once per day at 3, 7,
10 and 14 days post-mating
Male offspring were
weighed, and sacrificed at
4 days or 6 wk of age for
evaluation.
In 6-wk-old male offspring from nano-Ti02- Takeda et al. (2009,
exposed dams 193667)
Nano-Ti02 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)
Markers of apoptosis (activation of caspase-3 and
crescent-shaped cells), occlusion of small
vessels, and perivascular edema observed in the
brain
5-50
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Animal
Mouse
Sex,
strain/stock
not specified
Testing Material
Nano-Ti02 (Degussa W740X),
dispersion of photocatalytic
uncoated nano-Ti02 (80% anatase/
20% rutile) at 40 wt%, primary
particle 4.7 nm, mean aggregate
size < 100 nm; (Evonik, 2007,
157577: Llames, personal
communication, 2008, 157528:
Weaver, personal communication,
2008. 157467)
Treatment Conditions
Subcutaneous injections:
total 0 or total 5,600 mg/kg
over 2 days
Intravenous injections: total
0 or total 560 mg/kg over
2 days
1 or 5 days of recovery
Summary of Major Effects
Subcutaneously injected mice:
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-Ti02 aggregates,
suggesting interaction between T-cells and
nano-Ti02.
No changes in Con-A stimulated T-cell
proliferation.
Reference
Weaver etal.
(2007.193713)
Ocular exposure
Rabbit
Male New
Zealand
White
Nano-Ti02 (identified as uf-C , a
pre-commercial version of DuPont
Light Stabilizer 210), 79%
anatase/21% rutile, approximately
90 wt% Ti02, 7% alumina, and 1 %
amorphous silica, average particle
size 1 40.0 ± 44 nm in water,
average BET surface area 38.5
m2/g
Acute ocular irritation
Doses -0 or 57 mgtoone
eye of each animal
Single exposure (the eye
remained unwashed
following treatment)
Observation at 1 , 24, 48,
and 72 hr following
administration of the
nano-Ti02
Reversible conjunctival redness in the treated eye
(normal by 24 or 48 hr after administration of
nano-Ti02).
No corneal injury evident, no clinical signs
observed, and no BW loss occurred.
Warheit et al.
(2007.091075)
BET - Brunauer, Emmett, Teller method of calculating surface area
BW-Body weight
CDS - Cluster of differentiation 8
CDS + T cell - Cytotoxic T cell with CDS surface protein
DLS - Dynamic light scattering
lEP-lsoelectric point
P25-Aeroxide®P25
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 (I ARC) (Baan, 2007, 157717:1 ARC, 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).
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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 urn) 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 um)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 TiO2has 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 population-
based 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 case-
control 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 urn, with some collection of particles up to 10 urn.
5-52
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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 vehicle-
treated 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
(Oberdorster 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
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Table 5-8. Treatments and pulmonary tumor incidences in rats exposed to fine and nano-Ti02
through intratracheal instillation in Pott and Roller (2005) study
Treatment
Nano-Ti02,
hydrophilic
(P25)
Nano-Ti02,
hydrophobic
(Degussa
P805)c (Sigma
AL900032)C
Fine Ti02,
hydrophilic
(Sigma AL
232033)
No treatment
Crystal form;
primary
particle size;
specific
surface area
(BET)
Majority anatase;
25 nm"(21 nm
and 30 nm were
also reported);
52 m /g
(Data of Degussa
T805)c Crystal
form not
specified, coated
with an organic
silicon
compound;
21 nm;32.5m2/g
Anatase; 200 nm;
9.9 m2/g
--
in,,mh*rnf 15',*=',* Lungs with Lungs with Lungs Lungs with
Phot°- i(nc*m^nl 2SS/a Survival PrimarV PrimarV with metastases
stability filiations start/at 50o/o (wk) benign malignant tumors, of other
instillation) tumors (%) tumors (%) total (%) tumors (%)
Photo- 5 x 3.0 48/42
catalytic
5 x 6.0 48/46
10x6.0 48/46
(Currently 15x0.5 24/11
available
Degussa
T805 is a
pnotostabie 30x05 48/15
UV filter) JU*u.5 4B/ib
(Untreated 10x6.0 48/44
anatase is
photo- 20 x 6.0 48/44
catalytic)
48/46
114 21.4 31.0 52.4 14.3
114 17.4 50.0 67.4 15.2
104 23.9 45.7 69.6 15.2
86 0.0 0.0 0.0 9.1
114 6.7 0.0 6.7 6.7
108 15.9 13.6 29.5 11.4
113 38.6 25.0 63.6 2.3
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
"Rats at risk were "sufficiently examined rats which survived at least 26 wk after first instillation" according to Pott and Roller (2005,1577901.
'Regarding particle characteristics, Pott and Roller (2005,1577901 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).
'According to Pott and Roller (2005,1577901: "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 (2005,1577901 study. Currently available T805 is
photostable nano-Ti02 (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.
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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 (im, 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% bronchiole-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).
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Table 5-9. Incidence of tumor in the abdominal cavity of rats intraperitoneally injected with
photocatalytic nano-TiCh.
Animal, age at the
beginning of the
experiment
Nano-Ti02 treatment
Rats with sarcoma, mesothelioma, or
carcinoma, other than uterine tumors, in the
abdominal cavity (percentage)
Rats sacrificed when in bad health or 2.5 yr after treatment
Wistar rat,
9-wk old
Sprague-Dawley rats,
8-wk old
Wistar rats,
4-wk old
Wistar rats,
5-wk old
i.p. injection of 18 mg/rat, once per wk for 5 wk (total dose
90 mg/rat)
i.p. injection of 5 mg/rat
i.p. injection of 5 mg/rat
i.p. injections of 2, 4, and then 4 mg/rat (total dose 10 mg/rat)
6 of 113 rats examined (5.3%)
2 of 52 rats examined (3.8%)
0 of 47 rats examined (0%)
0 of 32 rats examined (0%)
Preliminary results at 28 mo after i.p. injection
Wistar rats,
8-wk old
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%)
i.p. - intraperitoneal
Source: Data from Pott et al. (1987, 0298231.
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 x 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 dose-
response 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.
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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-cell-
induced 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.
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TiO9 or nano-TiO9 in air
Deposition in respiratory tract
Decreased clearance (overload)
Inflammation diminishing over
time post-exposure
Rats
Persistent inflammation
Cellular injury, persistent cell
proliferation, fibrosis
Mutation
Lung tumors
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
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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 (im), 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-Ti02 carcinogenicity studies in animals
Exposure route Species
Result
Lowest effective dose
(highest ineffective dose)
References
Photocatalytic nano-Ti02
Intratracheal
instillation
Inhalation
Intraperitoneal
injection
Wistar rats, female
Wistar rats, female
NMRI mice, female
Wistar and
Sprague-Dawley
rats
Increased lung tumors (benign
and malignant)
Increased lung tumors
No increases in lung tumors
No increase in abdominal tumors
5 instillations at 6.0 nig/instillation
5 instillations at 3.0 nig/instillation
Approximately 12 mg/m3 for 24 moa
(Approximately 10 mg/m3for 13.5 mo)b
(5 intraperitoneal injections at 18 mg/rat per
injection)
Bormetal.(2000,
041486)
Pott and Roller (2005,
157790)
Heinrichetal. (1995,
076637)
Heinrichetal. (1995,
076637)
Pott etal. (1987,
029823)
Nano-TiO; with unspecified photoreactivityc
Intratracheal
instillation
Intramuscular
implantation
Wistar rats, female
Sprague-Dawley
rats, male
No conclusion11
No increases in tumor at
implantation sites
(30 instillations at 0.5 nig/instillation)
(not specified)
Pott and Roller (2005,
157790)
Hansen et al. (2006,
090611)
NMRI = Naval Medical Research Institute
a7.2 mg/m3 for 4 mo, followed by 14.8 mg/m3 for 4 mo and then 9.4 mg/m3 for 16 mo
b7.2 mg/m3 for 4 mo, followed by 14.8 mg/m3 for 4 mo and then 9.4 mg/m3 for 5.5 mo
°Nano-Ti02 particles not specified or have questionable identification
'Unexpected high acute toxicity; problem with ascertaining the identity of testing material
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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.
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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, liquid-
phase techniques are used following sample extraction. As presented in Table 1-4 in Section 1.6.2,
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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 product-
specific 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
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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 nano-
TiO2 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 fixed-
matrix 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 um
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 nano-
TiO2, although conventional TiO2 is likely to contain nanoscale TiO2. Production estimates of nano-
TiO2 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
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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
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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 ultrafme 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 nano-
TiO2; 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.
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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 offish. 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 um2/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-
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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 offish,
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
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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 nano-
silver 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.
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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 nano-
TiO2 intended for use in sunscreen. However, coatings used to increase the photostability of nano-
TiO2 could be removed in the environment through weathering or biotransformation, yielding nano-
TiO2 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
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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 ug/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 long-
term 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.
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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-Ti02
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 (NOT)," 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 physic-
chemical 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 nano-
TiO2, 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).
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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.
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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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Annex A. Nano-Ti02 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-Ti02
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.
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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-Ti02 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
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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-l.
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).
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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 Optisol™, 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
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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 Kobe'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
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microstructure. This effect can be counteracted by using suitable surfactants or polymers (Hewitt,
1996. 1579361
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-Ti02 or Micronized Ti02
as Active Ingredient
Table A-l 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-l is likely not exhaustive. Also, product formulations and labels could change over time.
A-6
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Table A-1. Ti02 content in various sunscreen products.
Brand/ Manufacturer
Abella
Alba Botanica
B. Kamins
BABOR
BABOR
BENEV
Bliss
California Baby
California Baby
California Baby
Cellex-C
Cellex-C
Cellex-C
Cellex-C
Colorescience
Derma logica
Dermalogica
EmerginC
Fallene/Total Block
Fallene/Total Block
Fallene/Total Block
Fallene/Total Block
Jan Marini
June Jacobs
Lancome
Lancome
Peter Thomas Roth
Pevonia Botanica
ProCyte
ProCyte
ProCyte
ProCyte
ProCyte
Total Skin Care LLC
Wilma Schumann
Product
Solar Shade, SPF 45
Chemical Free Sunscreen, SPF 18
Chemist Bio-Maple Sunbar Sunscreen, SPF 30 Fragrance-Free
High Protection Lotion, SPF 30
Moderate Protection Sun Cream, SPF 20
Pure Ti02
Oil-free Sunban Lotion for the Face, SPF 30
SPF 30 & Fragrance Free Sunscreen; also available as Sunblock Stick, SPF 30
Sunscreen SPF 30+ - Everyday Year Round; also available as Sunblock Stick
Water Resistant, Hypo-Allergenic Sunscreen, SPF 30
Sunscreen, SPF 15
Water Resistant Sunscreen, SPF 30
Sun Care Broad Spectrum UV-A, UV-B Sunblock & Moisturizer, SPF 15
Sun Care, SPF 30
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
Oil Free Matte Block, SPF 20
Ultra Sensitive FaceBlock, SPF 25
Sun 30 (and tinted version)
Total Block Clear, SPF 65
CoTZ, SPF 58
Total Block Cover-Up/Make-Up, SPF 60
Total Block Tinted, SPF 60
Bioglycolic Facial Lotion, SPF 15
Micronized Sheer, SPF 30
Soleil High Protection Face Cream - Gel, SPF 30
Soleil Soft-Touch Moisturizing Sun Lotion, SPF 15
Instant Mineral, SPF 30
Pevonia Soleil Sun Block, SPF 15
TiSilc Sheer, SPF 45
TiSilc Sheer, SPF 45 (tinted)
TiSilc Sunblock, SPF 60+
TiSilc Untinted, SPF 45
Z-Silc Plus Sunblock, SPF 30+
pH Advantage Basics Sun Blocker, SPF 15
Wilma Schumann Sunscreen, SPF 20
Percentage Ti02
N/A
7.0%
2.04%
N/A
4.5%
N/A
6%
4.5%
4.5%
N/A
2%
2%
N/A
2%
12%
4%
14%
N/A
4%
10%
10%
10%
5.5%
14.5%
4.5%
4.5%
15%
N/A
N/A
3.5%
8%
3.5%
4.0%
N/A
N/A
N/A-Not available.
Source: Used with permission from the Environmental Working Group, for their Skin Deep Database (EWG, 2008,1963431.
A-7
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REFERENCES
Barker PJ; Branch A (2008). The interaction of modern sunscreen formulations with surface coatings. Progr Org Coating,
62: 313-320. http://dx.doi.Org/10.1016/i.porgcoat.2008.01.008180141
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
EWG (2008). Sunscreen investigation: Skin deep-cosmetic safety reviews. Retrieved June 02, 2009 from
http://www.cosmeticsdatabase.com/special/sunscreens2008/. 196343
Hewitt J (2002). A moment of clarity. Soap, Perfum Cosmet, 75, 47-50. 093307
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-of-
particle-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-
313. http://dx.doi.0rg/10.1586/17469872.3.3.307 193559
Newman KA (2006, December). Sun protection report. GCI Magazine.
http://www.gcimagazine.com/marketstrends/segm ents/suncare/4829426.html?page=l. 157745
Oxonica (2005). Technical notes: Example formulation details and protocol tips to obtain optimal dispersion of Optisol
(TM) UV Absorber. Retrieved April 04, 2007 from
http://www oxomca.com/media/mediajromoliterature.php?start=6. 157793
Packaged Facts (2001). The U.S. market for suncare and lipcare products. Retrieved March 31, 2001 from
http://www.mindbranch.com/listing/product/R567-393.html. 196053
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 Colipan 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/outl 35_en.pdf. 092740
A-8
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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
http://www.fda.gov/nanotechnology/meetings/kobo_files/textmostly/index.htm. 093309
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.
http://www.koboproductsinc.com/Downloads/IFSCC2004.pdf. 157825
TGA (2006). A review of the scientific literature on the safety of nanoparticulate titanium dioxide or zinc oxide in
sunscreens. Australia: Australian Government, Department of Health and Ageing, Therapeutic Goods
Administration, http://www.tga.gov.au/npmeds/sunscreen-zotd.pdf. 089202
U.S. Pharmacopeia (2006). Titanium dioxide . In U.S. pharmacopeia official monographs (p. 3364). Rockville, MD: U.S.
Pharmacopeia. 155639
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
A-9
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Annex B. Nano-Ti02 in Sunscreen:
Manufacturing Processes
B.1. Overview of Nano-Ti02 Manufacturing Process
A generic manufacturing process for nano-TiO2 for sunscreen applications is outlined in
Figure B-l.
Nuclei Synthesis
^^^^^^^m
Filtration / Washing
Drying
Dispersion Milling
Source: Dransfield (2005,1578091
Figure B-1. Generic manufacturing process for nano-T!O2 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.
TiO2 Processes Compared
Sulfate Process
Chloride Process
llmenite etc. + H2SO
Digestion
Crystallization
i
Hydrolysis, Filtration
& Washing
I
Calcination
Milling & packing
of untreated TiO2 pigment
Drying, milling & packing
of surface treated TiO2 Pigment
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-l.
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
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 "[ajlthough 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.
Feedstock
Additives
Control of
flow rates,
temperature,
drop size etc
Additives
Additives
Solution
preparation
^
r
Spray Hydrolysis
Calcining
Milling
Hydrated product
Calcined product
• "Whiffle balls"
with nano-sized
structure
* Milled slurry
Spray Drying
Micronizing
Nano-sized
product
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 + 4HC1) and three
additional wet processes (TiOCl2 + 2NaOH -» TiO2 + 2NaCl + H2O ; Na2TiO3 + 2HC1 -» 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 900°C. 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) + 2C12 + C -> TiCl4 + CO2
TiCL, + O2 -> TiO2 + 2C12
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 (HC1) 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 HC1 (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, andAltair Processes
Milling breaks apart the hollow crystalline lattice 1 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 40-100°C 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 300°C. 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-Ti02 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 Bl (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-Ti02 particles used in
Particle name
T805 Degussa20/80 RU/AN
T817 Degussa79/12/2 RU/AN/Fe
UV-TitanM160
UV-TitanM212
UV-TitanX161
UV-Titan X200
EusolexT-2000
TT051A
TT051C
MT-100AQ
MT-100AR
MT-100T-L-1
MT-100SA
MT100TV(orMT-100TV)
MT100Z(orMT-100Z)
MT-500SA
MirasunTiW60
UV-Titan M262
Tioveil dispersions
Manufacturer
Degussa
Degussa
Kemira
Kemira
Kemira
Kemira
Merck
Merck
Merck
Mitsubishi/Tayca
Mitsubishi/Tayca
Mitsubishi/Tayca
Mitsubishi/Tayca
Mitsubishi/Tayca
Mitsubishi/Tayca
Mitsubishi/Tayca
Rhodia
Rhodia and Kemira
Uniqema
Crystal type
rutile/ anatase
rutile/ anatase
rutile
rutile
rutile
rutile
unknown
rutile
rutile
rutile
unknown
rutile
rutile
rutile
rutile
rutile
anatase
rutile
rutile
sunscreen
Average
crystal size
21 nm
21 nm
17-20nm
20 nm
15 nm
20 nm
14nm
35 nm
35 nm
15 nm
15 nm
15 nm
15 nm
15 nm
15 nm
35 nm
60 nm
20 nm
10-28nm
Coating materials and concentrations
silicone dioxide <2.5%
silicone dioxide <2.5% (also doped with di-iron trioxide 2%)
alumina 5.5-7.5%, stearic acid 10%
alumina 5-6.5%, glycerol 1%
alumina 8.5-11.5%, stearic acid 10%
none
alumina 8-11%, simethicone 1-3%
alumina 11%, silica 1-7%
alumina 11%, silica 1-7%, stearic acid 3-7%
alumina 4-8%, silica 7-11%
alumina 4-8%, silica 7-10%
alumina 3.3-7.3%, stearic acid 5-11%
alumina 4-7.5%, silica 2-4%
alumina 1-15% or 3-8%; aluminum stearate 1-13% or
1-1 5% or stearic acid 5-11%
alumina 6-10%, stearic acid 10-16%
alumina 1-2.5%, silica 4-7%
alumina 3-7%, silica 12-18%
alumina 5-6.5%, dimethicone 1-4%
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, 0927401.
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
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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).
Optisol™ 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. Optisol™ 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).
Uniqema/Croda1 manufactures several TiO2 sunscreens, including aline of Solaveil™ 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-Ti02
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.
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Table B-2. Formula SC-383-1 for "Weightless Morning Dew with Sun Protection"
Ingredients %
Part A
Water QS
Hydroxypropyl starch phosphate3 1.00
Arlatone V-150 [steareth-100 (and) steareth-2 (and) mannan (and) xanthan gum] 0.50
Arlatone LC 2.00
Pricerine™ 9088 (glycerin) 4.00
Solaveil CT-10W [water (and) Ti02 (and) isodeceth-6 (and) oleth-10 (and) aluminum stearate (and) 5.00
alumina (and) simethicone]
PartB
Solaveil CT-200 Ui02 (and) isohexadecane (and) 2.00
triethylhexanoin (and) aluminum stearate (and) alumina (and) polyhydroxystearic acid]
Ethyl methoxycinnamateb 4.00
BRIJ™ 721 (steareth-21) 2.00
Arlamol PS15E (PPG-15 stearyl ester) 5.00
PartC
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-1OW. 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 PartC. Stir to room temperature.
Note: QS means a sufficient quantity.
"Structure XL, National Starch
bEusolex 2292, Merck KGaA
'Phenonip XB, Clariant
Source: Croda (2009,1938801.
B-8
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Table B-3. Formula KSL-17 for High SPF Transparent Sunscreen
Ingredients %
Parti
Rose Talc-MS2 - Kobo Products : Talc (and) Methicone 1.00
Velvesil 125-Momentive/Kobo Products : Cyclopentasiloxane (and) C30-45Alkyl Cetearyl Dimethicone Crosspolymer 3.00
Net-WO - Barnet: Cyclopentasiloxane (and) PEG-10 Dimethicone (and) Disteardimonium Hectorite 0.20
CM3K40T4 - Kobo Products : Cyclopentasiloxane (and) Ti02 (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 40°C 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. (2009,1960451.
B-9
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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
MitchnickM; O'Lenick AJ Jr (1996). PatentNo. 5565591 (class: 556/10). United States: United States Patent Office.
http://www.freepatentsonline.com/5565591 .html. 157935
Osterwalder N; Capello C; Hungerbilhler 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 Colipan 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/outl 35_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
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Umicore (2008). Nano-sized oxide powders for UV applications. Abstract from Innovation for Sustainable Production (i-
SUP2008), 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
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Annex C. Nano-Ti02 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 HEPAH14 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 powder-
collection 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)1.
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
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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.I.I.
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 (im. 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 (im thick) was more effective against nanoparticle
C-2
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penetration than woven cotton (650 (im thick) and woven polyester (160 (im 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).
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REFERENCES
BASF (2008). Guide to safe manufacture and for activities involving nanoparticles at workplaces in BASF AG. Germany:
BASF, http://basf.eom/group/corporate/en/function/conversions:/publish/content/sustainability/dialogue/in-
dialogue-with-
politics/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
http://www.nanosafe2008.org/home/liblocal/docs/Oral%20presentations/O6-l_Golanski.pdf. 196048
Guizard B; Tenegal F (2008). Liquid Recovery of TiO2 nanoparticles synthesized by laser pyrolysis. Retrieved March 30,
2009 from http://www.nanosafe2008.org/home/liblocaVdocs/Oral%20presentations/O6-4_Guizard.pdf. 196049
Harford AJ; Edwards JW; Priestly BG; Wright PFA (2007). Current OHS Best Practices for the Australian nanotechnology
industry: A position paper by the NanoSafe Australia Network. Victoria, Australia: NanoSafe Australia Network.
http://mams.rmit.edu.au/72nuxiavskpg.pdf. 196051
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