NIST Special Publication 1200-3 

Preparation of a Nanoscale TiO2 

Aqueous Dispersion for 

Toxicological or 

Environmental Testing 

Version 1.2 

J. S. Taurozzi

V. A. Hackley

National Institute of Standards and Technology 

 Material Measurement Laboratory 

Gaithersburg, MD 20899-8520 

M. R. Wiesner

Duke University 

Department of Civil and Environmental Engineering 

Durham, NC 27708

http://dx.doi.org/10.6028/NIST.SP.1200-3
NIST Special Publication 1200-3 

NanoEHS Protocols 

Preparation	of	a	Nanoscale	TiO2

Aqueous	Dispersion	for	Toxicological		

or	Environmental	Testing	

Version 1.2 

J. S. Taurozzi

V. A. Hackley

National Institute of Standards and Technology 

 Material Measurement Laboratory 

Gaithersburg, MD 20899-8520 

M. R. Wiesner

Duke University 

Department of Civil and Environmental Engineering 

Durham, NC 27708

http://dx.doi.org/10.6028/NIST.SP.1200-3 

June 2012 

U.S. Department of Commerce 

John E. Bryson, Secretary 

National Institute of Standards and Technology 

Patrick D. Gallagher, Under Secretary of Commerce for Standards and Technology and Director 
Certain commercial entities, equipment or materials may be identified in this 

 document in order to describe an experimental procedure or concept adequately. 

Such identification is not intended to imply recommendation or endorsement by the 

National Institute of Standards and Technology, nor is it intended to imply that the 

entities, materials or equipment are necessarily the best available for the purpose. 

National Institute of Standards and Technology Special Publication 1200-3

Natl. Inst. Stand. Technol. Spec. Publ. 1200-3, 11 pages (June 2012) 

CODEN: NSPUE 
Preparation of a Nanoscale TiO2 Dispersion for Toxicological or Environmental Testing, Version 

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FOREWORD 

This special publication is one in a series of protocols resulting from a collaborative research 

agreement between the National Institute of Standards and Technology (NIST) and Duke 

University’s Center for the Environmental Implications of Nanotechnology (CEINT). The 

original version of this protocol (Ver. 1.0) was first posted on the CEINT web site 

(http://ceint.duke.edu) and it, along with any other previous version, is superseded by this 

updated special publication version. Updates to this protocol may be released in the future. Visit 

http://nist.gov/mml/np-measurement-protocols.cfm to check for revisions of this protocol or new 

protocols in the series. 

NIST and CEINT are interested in soliciting feedback on this method. We value user comments 

and suggestions to improve or further validate this protocol. Please send your name, email 

address and comments/suggestions to nanoprotocols@nist.gov. We also encourage users to 

report citations to published work in which this protocol has been applied. 
Preparation of a Nanoscale TiO2 Dispersion for Toxicological or Environmental Testing, Version 

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1. Introduction 

Toxicity and fate assessment are key elements in the evaluation of the environmental, health and 

safety risks of engineered nanomaterials (ENMs). While significant effort and resources have 

been devoted to the toxicological evaluation of many ENMs, including nanoscale TiO2 [1-4], 

obtaining conclusive and reproducible results continues to be a challenge [5]. This can be traced 

in part to the lack of standardized dispersion protocols and the inconsistent application of 

dispersion procedures in relevant biological and environmental matrices [6, 7]. In order to 

address these issues, the National Institute of Standards and Technology (NIST), jointly with the 

Center for the Environmental Implications of Nanotechnology (CEINT), have developed a series 

of standardized and validated protocols for the dispersion of ENMs from a powdered material 

source for both human health and environmental testing applications. This protocol has been 

developed and validated using NIST Standard Reference Material (SRM) 1898a

. SRM 1898 

consists of a widely studied and industrially relevant TiO2 nanomaterial with broad commercial 

penetration and a production history dating back several decades [3, 8-10]. 

While the procedures detailed in this protocol focus on the dispersion of SRM 1898 in aqueous 

media, it is believed that the adopted characterization, optimization and validation approaches 

can be more generally applied to the preparation of ENM dispersions in any relevant matrix. For 

this reason, and to allow for broader applicability, experimental details and discussions regarding 

the characterization, process optimization and validation steps adopted for the development of 

the dispersion method are detailed in a separate publication [11]. 

2. Principles and scope 

This protocol provides a validated method for the preparation of aTiO2 nanoparticle dispersion in 

high purity de-ionized water by use of ultrasound (a process referred to here as sonication), as a 

stock preparation compatible with biological and environmental matrices. Furthermore, this 

protocol can be used in combination with matrix-specific protocols for the preparation of 

dispersions in more complex biological or environmental media. For the preparation of TiO2

dispersions in biologically relevant media, refer to [12]. For guidelines on reporting relevant 

conditions and critical parameters relating to the ultrasonic dispersion of ENMs, consult 

reference [13]. For additional relevant and general considerations on the application of 

ultrasound to prepare ENP dispersions, refer to [7, 14].

The method described herein, if applied correctly, yields aqueous monomodal nanoscale TiO2

dispersions, characterized by a mean particle diameter of ≈ 70 nm and a pH between 3.7 and 4.9. 

The method is validated for the preparation of dispersions in the (0.5 to 20) mg/mL concentration 

range. Dispersions prepared following this protocol should be stored so as to minimize exposure 

to light (e.g., in amber vials), at room temperature, and used within 24 h of preparation.

 

 

a

 Information regarding SRM 1898 can be obtained at http://www.nist.gov/srm/. 
Preparation of a Nanoscale TiO2 Dispersion for Toxicological or Environmental Testing, Version 

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3. Terminology 

This protocol complies with definitions relevant to nanotechnology as set forth in the ASTM 

standard E2456 [15] and is consistent with the draft standard ISO TS 80004-1 [16]. Additional 

guidance is derived from recommendations of the International Union of Pure and Applied 

Chemistry [17]. 

nanoparticle — sub-classification of ultrafine particle that is characterized by dimensions in the 

nanoscale (i.e., between approximately 1 nm and 100 nm) in at least two dimensions; also 

referred to as “nano-object” in ISO TS 80004-1 [16]. 

primary particle — the smallest discrete identifiable entity associated with a particle system; in 

this context, larger particle structures (e.g., aggregates and agglomerates) may be composed of 

primary particles. 

aggregate — a discrete assemblage of primary particles strongly bonded together (i.e., fused, 

sintered or metallically bonded). 

Note—The adjective "primary", when used in conjunction with the term aggregate, is employed in the present 

context to indicate the smallest achievable aggregate.

agglomerate — assemblages of particles (including primary particles and/or smaller aggregates) 

held together by relatively weak forces (e.g., van der Waals, capillary or electrostatic), that may 

break apart into smaller particles upon further processing. 

Note—Although we define them as distinct entities, the terms aggregate and agglomerate have often been used 

interchangeably to denote particle assemblies. 

dispersion — used in the present context to denote a liquid (aqueous) in which particles are 

homogeneously suspended, or the process of creating a suspension in which discrete particles are 

homogeneously distributed throughout a continuous fluid phase; implies the intention to break 

down agglomerates into their principal components (i.e., primary particles and/or aggregates). 

4. Reagents, materials and equipment 

4.1. Reagents 

4.1.1. AEROXIDE TiO2 P25 (Evonik Degussa GmbH, Germany); available from distributors or 

obtained as a certified reference material from NIST (SRM 1898). 

4.1.2. ≥ 18 M·cm resistivity Type I biological grade de-ionized (DI) water; biological grade 

implies sterility and absence of endotoxin contamination. 

Note—Pyrogens (also known as endotoxins) are shed from the outer membrane of Gram-negative bacteria 

during cell division or lysis. These toxins are relatively heat-stable and are not destroyed under typical 

sterilizing conditions. As a result, pyrogens are ubiquitous and can interfere with the accuracy of toxicity assays. 

To depyrogenize glassware, bake at 250 °C for 2 h or at 200 °C overnight. 

Note—Limulus Amoebocyte Lysate (LAL) reagent grade pyrogen-free water can be obtained from commercial 

vendors. 

Note—Sterility and absence of pyrogen contamination should be verified for all materials in contact with the 

dispersion. If using the LAL test for pyrogens, avoid using cellulose-based filters, as they can be a source of 

beta-glucan, which interferes with the LAL assay.
Preparation of a Nanoscale TiO2 Dispersion for Toxicological or Environmental Testing, Version 

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Note—If the dispersion is not intended for toxicological assessment, pyrogen-free conditions may not be 

necessary.

4.2. Materials 

4.2.1. 100 mL, ≈ 5 cm diameter cylindrical glass beaker 

4.2.2. 125 mm wide x 65 mm deep crystallizing glass dish 

4.2.3. 50 mL borosilicate volumetric flask or pipette 

4.2.4. Aluminum or polystyrene weighing dish 

4.2.5. Clamps or other locking device for holding beaker in place 

4.2.6. Chopped ice 

4.3. Equipment 

For the preparation of the aqueous dispersion: 

4.3.1. Analytical balance 

4.3.2. Probe-type sonicator with a standard 1⁄2 inch (1.3 cm) diameter titanium horn fitted with a 

removable flat tip (or similar ultrasonic device) 

For verification of expected outcome: 

4.3.3. pH meter and electrode 

4.3.4. Laser diffraction spectrometer (LDS), or

4.3.5. Dynamic light scattering (DLS) instrument, or

4.3.6. X-ray disc centrifuge (XDC) particle size analyzer 

5. Preparation of aqueous TiO2 nanoparticle dispersion 

Note—To avoid contamination, all glassware should be meticulously cleaned, rinsed with ethanol and dried 

prior to use. Glassware can be sterilized using an autoclave, by exposure to hot, dry air (130 °C-170 °C) for 2-4 

h in an oven or by prolonged contact with alcohol. Avoid detergents if possible; if detergents are used, rinse with 

copious amounts of DI water prior to rinsing with ethanol and drying. Store and work in high-efficiency 

particulate air (HEPA) filtered clean bench if available; if not, containers should be capped or sealed with 

thermoplastic (e.g., Parafilm). 

Note—Use clean, sterile pipette tips and sterile procedures. 

Note—For details on the validation of the particle size distribution (PSD) of dispersions, the optimization of the 

TiO2 sonication sequence, as well as stability, validation and applicability considerations, refer to [18]. 

5.1. Using an analytical balance and an aluminum or polystyrene weighing dish, weigh an 

adequate mass of dry TiO2 powder to achieve the desired concentration in a 50 mL water 

volume. For guidance on mass-concentration relationships, refer to the table below.

 
Preparation of a Nanoscale TiO2 Dispersion for Toxicological or Environmental Testing, Version 

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Mass 

(g) 

Concentration in 50 

mL volume (mg/mL) 

0.025 0.5 

0.05 1 

0.5 10 

1 20 

Note—For greater accuracy and reproducibility with respect to mass concentration, the TiO2 powder should be 

dried overnight at an elevated temperature between (105 and 150) oC and then allowed to cool to room 

temperature in a desiccator prior to use.

5.2. Add the weighed mass of powder to a 100 mL, ≈ 5 cm diameter cylindrical glass beaker. 

Add 50 mL of DI water to the beaker with the powder. 

5.3. Place the beaker inside the 125 x 65 mm glass dish and secure the beaker in the center of the 

dish by use of clamps or other locking device to ensure that the beaker remains in place 

during sonication. 

5.4. Fill the 125 x 65 mm glass dish with enough water and chopped ice to allow for the ice 

water bath level to encase the beaker to approximately the level of the water contained in the 

beaker. 

5.5. Immerse the sonicator horn into the liquid in the beaker down to about 2.5 cm below the 

liquid level in the beaker. Center the horn in the beaker; the horn should not touch the sides 

or the bottom of the beaker. 

5.6. Select a sonicator setting that yields a delivered power of approximately 50 W. 

Note—Refer to [14] for details on the recommended calibration procedure for the determination of the 

sonicator’s delivered power. 

5.7. Operate the sonicator at this delivered power level for 15 min, using an 80 % pulsed 

operation mode (e.g., 80 % on / 20 % off during each second of operation time), or similar 

on/off time sequence. 

5.8. After sonication, transfer the aqueous dispersion to an amber borosilicate glass container and 

store at ambient temperature until further use. Do not refrigerate. 

Note—If an amber glass container is not used, store the stock dispersion in a cabinet or other container such 

that exposure to light is minimized. It is not established whether or not exposure to typical laboratory light levels 

is consequential with respect to dispersion properties or biological behavior of the TiO2 nanoparticles. 

Note—The stability of dispersions prepared according to this protocol has been validated up to 48 h; however, 

since this protocol is intended as a preliminary step for the preparation of dispersions in relevant media, the 

resulting dispersions should be used as soon as possible following preparation and in accordance with 

directions prescribed in medium-specific protocols. 
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6. Expected outcome 

6.1. The obtained TiO2 aqueous dispersion should have an opaque white appearance if prepared 

using P25. 

Note—If source powders other than P25 are used, the appearance may vary depending on the final particle size, 

particle concentration and other factors. 

6.2. The particle size distribution (PSD) of P25 dispersions prepared following this protocol 

should be monomodal, with the following volume-based mean particle (Dm), D10 and D90

valuesb: 

If measured using LDS: 

Dm ≈ (67 to 75) nm 

D10 ≈ (58 to 61) nm 

D90 ≈ (79 to 89) nm 

If measured using DLS: 

Dm ≈ (108 to 116) nm 

D10 ≈ (60 to 76) nm 

D90 ≈ (148 to 155) nm 

If measured using XDC: 

Dm ≈ (70 to 84) nm 

D10 ≈ (9 to 54) nm 

D90 ≈ (97 to 142) nm 

The expected range for size parameters was calculated from three independent replicates of 

10 mg/mL dispersions obtained following the prescribed procedure. Refer to the Appendix 

for details on the calculation of the expected size parameter ranges. 

The volume-based mean particle diameter, as well as the D10 and D90 values for aqueous P25 

dispersions prepared following the protocol should be reported by the user to allow for 

comparison with the values specified herein. Refer to [11] for details and discussions on PSD 

characterization and validation criteria, as well as illustrations of representative PSD profiles. 

6.3. The pH of P25 aqueous dispersions prepared using this protocol should be between 3.7 and 

4.9, with lower pH values obtained for higher P25 concentrations. 

Note—The successful implementation of this protocol for ENMs other than P25, including other sources of TiO2

or other metal oxide ENMs, is dependent on knowing the isoelectric point (IEP) or zero point of charge of the 

ENM; in order to obtain a stable dispersion for ENMs that are electrostatically stabilized (i.e., that do not 

contain a sterically stabilizing capping agent), the final pH should be at least 2 to 3 pH units below or above the 

IEP. This may require the addition of a compatible acid or base, and should be evaluated as part of the 

optimization process. Multiple test or practice runs may be required in order to determine the required quantity 

 

b

 D10 and D90 refer to characteristic percentile size values associated with the cumulative volume or mass less than 

10 % and 90 %, respectively, of the total volume or mass within the distribution. These parameters are routinely 

reported by LDS instruments. They may or may not be obtainable directly from commercial DLS instruments, 

depending on the manufacturer. 
Preparation of a Nanoscale TiO2 Dispersion for Toxicological or Environmental Testing, Version 

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and concentration of acid or base to add; pH adjustment should, optimally, be performed after step 5.2 and prior 

to sonication. If dispersions are to be used for toxicological or environmental tests, additives for adjusting the 

pH should be selected based on their biocompatibility and relevance to the test matrix, and considering potential 

sonication-induced physicochemical changes [7]. 

7. Abbreviations 

DI de-ionized 

DLS dynamic light scattering 

ENM engineered nanomaterial 

HEPA high-efficiency particulate air 

IEP isoelectric point 

ISO International Organization for Standardization 

IUPAC International Union of Pure and Applied Chemistry 

LDS laser diffraction spectrometry 

NIST National Institute of Standards and Technology 

PSD particle size distribution 

SRM Standard Reference Material 

XDC X-ray disc centrifugation 

8. References 

1. Landsiedel, R., et al., Testing metal-oxide nanomaterials for human safety. J. Adv. 

Mater., 2010. 22(24): p. 2601-2627. 

2. Pichat, P., A brief survey of the potential health risks of TiO2 particles and TiO2-

containing photocatalytic or non-photocatalytic materials. J. Adv. Oxid. Technol., 2010. 

13(3): p. 238-246. 

3. Warheit, D.B., et al., Pulmonary toxicity study in rats with three forms of ultrafine-TiO2 

particles: Differential responses related to surface properties. Toxicology, 2007. 230(1): p. 

90-104. 

4. Wu, J.H., et al., Toxicity and penetration of TiO2 nanoparticles in hairless mice and 

porcine skin after subchronic dermal exposure. Toxicology Lett., 2009. 191(1): p. 1-8. 

5. Titanium Dioxide (TiO2) IARC Monograph 93. International Agency for Research on 

Cancer, 2006. 

6. Roebben, G., et al., Interlaboratory reproducibility of size and surface charge 

measurements on nanoparticles prior to biological impact assessment. Nature Nanotech., 

2010. 13(7): p. 2675-2687. 

7. Taurozzi, J.S., V.A. Hackley, and M.R. Wiesner, Ultrasonic dispersion of nanoparticles 

for environmental, health and safety assessment – Issues and recommendations. 

Nanotoxicology, 2011. 5(4): p. 711-729. 

8. Standard Reference Material 1898 (in production), National Institute of Standards and 

Technology, Gaithersburg, MD, http://www.nist.gov/srm/. 

9. List of Manufactured Nanomaterials and List of Endpoints for Phase One of the OECD’s 

Testing Program, Series on the safety of manufactured nanomaterials, Number 6, 2008, 
Preparation of a Nanoscale TiO2 Dispersion for Toxicological or Environmental Testing, Version 

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Environmental Diractorate, Organization for Economic Co-operation and Development. 

Paris, France. 

10. Porter, D., et al., A biocompatible medium for nanoparticle dispersion. Nanotoxicology, 

2008. 2(3): p. 144-154. 

11. Taurozzi, J.S., V.A. Hackley, and M.R. Wiesner, A standardized approach for the 

dispersion of titanium dioxide nanoparticles in biological media. Nanotoxicology, 2011. - 

submitted. 

12. Taurozzi, J.S., V.A. Hackley, and M.R. Wiesner, Protocol for the preparation of 

nanoscale TiO2 dispersions in biological test media for toxicological testing. NIST SP 

1200-4, National Institute of Standards and Technology, Gaithersburg, MD, June 2012. 

13. Taurozzi, J.S., V.A. Hackley, and M.R. Wiesner, Reporting guidelines for the preparation 

of nanoparticle dispersions from dry materials. NIST SP 1200-1, National Institute of 

Standards and Technology, Gaithersburg, MD, June 2012. 

14. Taurozzi, J.S., V.A. Hackley, and M.R. Wiesner, Preparation of nanoparticle dispersions 

from powdered material using ultrasonic disruption. NIST SP 1200-2, National Institute 

of Standards and Technology, Gaithersburg, MD, June 2012. 

15. E2456-06: Standard terminology relating to nanotechnology. 2006, ASTM International 

16. ISO TS 80004-1: Nanotechnologies - Vocabulary - Part 1: Core terms. 2007, 

International Standards Organization, Geneva, Switzerland. 

17. Version 2009-09-07, Release 2.1.5: Compendium of chemical terminology. 2009, 

International Union of Pure and Applied Chemistry (IUPAC). 

18. Taurozzi, J.S., V.A. Hackley, and M.R. Wiesner, A standardized approach for the 

dispersion of titanium dioxide nanoparticles in biological media. Nanotoxicology, 2011. 

Submitted. 

9. Appendix 

9.1. Calculation of expected particle size parameters 

The expected range for Dm, D10 and D90 values was obtained using the following equation: 

range ൌ ൬࢞ െ ࢚. ࢙

࢔√

൅ ࢞	to	

࢙ .࢚

࢔√

൰

Where x and s are the average and standard deviation, respectively, of the measured size 

parameter from three independent replicates, t is the student test parameter for a 95% confidence 

interval and two degrees of freedom (t = 4.30), and n is the number of tested samples (n = 3).