NIST Special Publication 1200-5 

Preparation of Nanoscale TiO2 

Dispersions in an Environmental 

Matrix for Eco-Toxicological 

Assessment 

Version 1.1 

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.1200-5 
		

 

 

 

NIST Special Publication 1200-5 

NanoEHS Protocols 

Preparation	of	Nanoscale TiO2

Dispersions	in	an	Environmental	

Matrix	for	Eco‐Toxicological	

Assessment

Version 1.1 

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.1200-5 

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 1200-5 

Natl. Inst. Stand. Technol. Spec. Publ. 1200-5, 12 pages (June 2012) 

CODEN: NSPUE 
 

 

 

 

Dispersion of Nanoscale TiO2 for Eco-Toxicological Assessment, Version 1.1 

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(http://ceint.duke.edu) and it, along with any other previous version, is superseded by this 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

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Dispersion of Nanoscale TiO2 for Eco-Toxicological Assessment, Version 1.1 

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 series focus on the dispersion of SRM 1898 in specific 

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 is proposed for the preparation of dispersions for generic acute eco-toxicity 

applications; its use for chronic toxicity or other environmental studies, while potentially 

efficacious, is beyond the scope of the present work and should be validated by use of proper 

controls. 

In this protocol, a TiO2 nanoparticle dispersion in a relevant environmental matrix is produced 

by following a series of steps applied to a TiO2 aqueous nanoparticle stock. Following the 

Organization for Economic Cooperation and Development (OECD) Guideline 202 for testing of 

chemicals (12), the environmental matrix used in this work is reconstituted from four stock 

solutions defined in ISO 6341 (13), yielding a calculated hardness of ≈ 170 mg/kg (ppm) as 

CaCO3. 

In this protocol, humic acid (HA) is utilized as a stabilizing agent to disperse SRM 1898 in the 

selected test matrix. Natural organic matter – and humic acid in particular – has been 

demonstrated to function as a non-specific stabilizer in environmental matrices (14-16). 

The method described herein, if applied correctly, yields 15 mL of a 100 g/mL monomodal 

nanoscale TiO2 dispersion in the selected test matrix, characterized by a mean particle diameter 

of ≈ 75 nm and pH values in the (7.0 to 7.7) range, without the need for pH adjustment steps. 

The tested TiO2 concentration was adopted from the limit test concentration recommended in 

OECD Guideline 202, while the pH of the dispersion was validated to fall within the 

a

 SRM 1898 was in production as of the publication of this protocol, with a target date for release in early 2012. The 

NIST SRM inventory can be accessed at http://www.nist.gov/srm/. 

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Dispersion of Nanoscale TiO2 for Eco-Toxicological Assessment, Version 1.1 

Guideline’s recommended pH range. The dispersion retains its particle size distribution and pH 

at room temperature for up to 96 h, which is the maximum duration for acute toxicity assays 

(17). Dispersions prepared following this protocol should be stored in darkness or in amber glass 

vials, as TiO2 is photoactive. 

3. Terminology 

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

International standard E2456 (18) and is consistent with the draft standard ISO TS 80004-1 (19). 

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

Applied Chemistry (20). 

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 (19). 

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 dispersed particle entity. 

agglomerate—assemblage 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. 200 g/mL stock TiO2 aqueous nanoparticle dispersion. 

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

implies sterile and pyrogen-free water. 

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. 

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Dispersion of Nanoscale TiO2 for Eco-Toxicological Assessment, Version 1.1 

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. 

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

necessary. 

4.1.3. Humic acid (HA) powder (e.g., Suwanee River Humic Acid Standard II, International 

Humic Substances Society, MN, USA) 

4.1.4. ISO Test Water 6341 aqueous stocks (in order of increasing ionic strength): 

Stock A: 3 mmol potassium chloride: 0.23 g of potassium chloride, KCl, in 1 L of DI 

water 

Stock B: 31 mmol sodium bicarbonate: 2.59 g of sodium bicarbonate, NaHCO3, in 1 L of 

DI water 

Stock C: 20 mmol magnesium sulfate heptahydrate: 4.93 g of magnesium sulfate 

heptahydrate, MgSO4 • 7H2O, in 1 L of DI water 

Stock D: 80 mmol calcium chloride dihydrate: 11.76 g of calcium chloride dihydrate, 

CaCl2 • 2H2O, in 1 L of DI water 

4.2. Materials 

4.2.1. 20 mL and 30 mL sterilized and pyrogen-free glass vials 

4.2.2. aluminum or polystyrene weighing dishes 

4.3.2. calibrated pipettes and sterile and pyrogen-free disposable tips covering a (0.020 to 5.000) 

mL range; adjustable volume pipettes are most convenient 

4.3. Equipment 

4.3.1. analytical balance with readability to 0.1 mg 

For verification of expected outcome: 

4.3.3. pH meter 

4.3.4. Laser Diffraction Spectrometer (LDS), or 

4.3.5. Dynamic Light Scattering (DLS) instrument 

5. Preparation of TiO2 nanoparticle dispersions 

Note—To avoid contamination, all glassware in contact with the media or suspensions 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 to 170 °C) for 2 h to 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. 

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Dispersion of Nanoscale TiO2 for Eco-Toxicological Assessment, Version 1.1 

5.1.1.	 Prepare 20 mL of 100 mg/L HA (aq.) solution, by adding 0.002 g of HA and 20 mL of DI 

water in a 20 mL glass vial. After adding both components, allow the covered solution to 

equilibrate for 48 h. The solution should have a pH of 4.0 ± 0.2 after equilibration. Proceed 

to the following steps after equilibration of the HA solution (i.e., 48 h after preparation of the 

solution). 

5.1.2.	 Prepare 50 mL of 200 g/mL TiO2 aqueous nanoparticle dispersion, by adding 0.01 g of 

SRM 1898 or equivalent into 50 mL of water, and following the sonication conditions 

prescribed in (21). 

5.1.3. In a 30 mL amber glass vial, add 3 mL of the HA solution (5.1.1) 

5.1.4. Add 7.5 mL of the TiO2 stock prepared in 5.1.2. into the vial with the HA solution (5.1.3) 

5.1.5. Add 3.46 mL of water into the above mixture (5.1.4) 

5.1.6. Add 0.26 mL of ISO stock solution A into the above mixture (5.1.5) 

5.1.7. Add 0.26 mL of ISO stock solution B into the above mixture (5.1.6) 

5.1.8. Add 0.26 mL of ISO stock solution C into the above mixture (5.1.7) 

5.1.9. Add 0.26 mL of ISO stock solution D into the above mixture (5.1.8) 

5.1.10. This procedure will yield a dispersion containing 100 g/mL TiO2 and 20 mg/L HA in 

OECD compliant hard water with a hardness of ≈ 170 mg/L (ppm) as CaCO3. 

Note—The above mentioned hardness value is calculated from the amount of Ca and Mg added with the starting 

stock solutions. 

5.1.11. If intended for toxicological assessment, the user is advised to conduct separate control 

tests for HA (20 mg/L) in the test medium (in the absence of TiO2). 

6. Expected Outcome 

Note—The particle size distribution (PSD) of the resulting dispersions was monitored for 96 h, corresponding 

to recommended acute toxicity assay timeframes ranging from 24 h to 96h (17). Although beyond the scope of 

this protocol, dispersions may remain stable for longer periods of time. This behavior has been validated 

without the presence of cells or other added components in the test media. 

Note—Serial dilutions may cause agglomeration, this should be tested accordingly. 

6.1.The resulting TiO2 dispersions should have a white but translucent appearance if prepared 

using SRM 1898 or commercial P25. 

Note—If source powders other than SRM 1898 or P25are used, the appearance may vary depending on the final 

particle size, particle concentration, and other factors. 

6.2.The particle size distribution (PSD) of the P25 dispersion should be monomodal, with the 

following volume-based mean particle diameter (Dm), D10 and D90 values:b 

If measured using LDS: 

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. 

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Dispersion of Nanoscale TiO2 for Eco-Toxicological Assessment, Version 1.1 

Dm ≈ (70 to 81) nm; 

D10 ≈ (59 to 63) nm 

D90 ≈ (79 to 102) nm 

If measured using DLS: 

Dm ≈ (120 to 127) nm; 

D10 ≈ (68 to 86) nm 

D90 ≈ (146 to 163) nm 

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

obtained following the prescribed procedure. Refer to the Appendix for details on the 

calculation of the expected size parameter ranges, and illustrations of representative PSD 

profiles. Refer to (11) for details and discussions on PSD characterization and validation 

criteria. 

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. 

6.3.The pH of dispersions after preparation should be ≈ 7.0. After (24 – 96) h, dispersions may 

experience a slight increase in pH ranging from 0.3 to 0.7 units. The dispersions should 

remain well within the OECD recommended pH range of 6 to 9 (12) during the studied 

timeframe (24 to 96) h. 

7. Abbreviations 

DI de-ionized 

DLS dynamic light scattering 

ENM engineered nanomaterial 

HA humic acid 

HEPA high-efficiency particulate air 

ISO International Organization for Standardization 

IUPAC International Union of Pure and Applied Chemistry 

LAL Limulus Amoebocyte Lysate 

LDS laser diffraction spectrometry 

OECD Organization for Economic Cooperation and Development 

PSD particle size distribution 

SRM Standard Reference Material (a registered trademark of the National Institute of 

Standards & Technology) 

8. Acknowledgements 

We thank Michelle Twyla Blickley, NIST Surface and Microanalysis Science Division, who 

provided the ISO stock solutions used in the development of this protocol. 

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Dispersion of Nanoscale TiO2 for Eco-Toxicological Assessment, Version 1.1 

9. References 

1.	 Landsiedel, R., L. Ma-Hock, A. Kroll, D. Hahn, J. Schnekenburger, K. Wiench, and W. 

Wohlleben, 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., T.R. Webb, K.L. Reed, S. Frerichs, and C.M. Sayes, 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., W. Liu, C.B. Xue, S.C. Zhou, F.L. Lan, L. Bi, H.B. Xu, X.L. Yang, and F.D. 

Zeng, 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., S.H. Ramirez-Garcia, V.A., M. Roesslein, F. Klaessig, V. Kestens, I. 

Lynch, C.M. Garner, A. Rawle, A. Elder, V. Colvin, W. Kreyling, H.F. Krug, Z. 

Lewicka, S. McNeil, A. Nel, A. Patri, P. Wick, M.R. Wiesner, T. Xia, G. Oberdörster, 

and K. Dawson, Interlaboratory reproducibility of size and surface charge measurements 

on nanoparticles prior to biological impact assessment. J. Nanopart. Res., 2011. 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, 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, 

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

Paris, France. 

10.	 Porter, D., K. Sriram, M. Wolfarth, A. Jefferson, D. Schwegler-Berry, M. Andrew, and 

V. Castranova, 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, Just 

Accepted, 2012. DOI:10.3109/17435390.2012.665506. 

12.	 OECD Guideline 202 for Testing of Chemicals, Daphnia sp., Acute Immobilisation Test. 

2004, Organization for Economic Cooperation and Development. 

13.	 ISO 6341: Water quality - Determination of the inhibition of the mobility of Daphnia 

magna Straus (Cladocera, Crustacea) - Acute toxicity test, Third Edition. 1996, 

International Standards Organization, Geneva, Switzerland. 

14.	 Thio, B.J.R., D.X. Zhou, and A.A. Keller, Influence of natural organic matter on the 

aggregation and deposition of titanium dioxide nanoparticles. J. Hazard. Mater., 2011. 

189(1-2): p. 556-563. 

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Dispersion of Nanoscale TiO2 for Eco-Toxicological Assessment, Version 1.1 

15.	 Keller, A.A., H.T. Wang, D.X. Zhou, H.S. Lenihan, G. Cherr, B.J. Cardinale, R. Miller, 

and Z.X. Ji, Stability and aggregation of metal oxide nanoparticles in natural aqueous 

matrices. Environ. Sci. Technol., 2010. 44(6): p. 1962-1967. 

16.	 Gao, J., S. Youn, A. Hovsepyan, V.L. Llaneza, Y. Wang, G. Bitton, and J.C.J. Bonzongo, 

Dispersion and toxicity of selected manufactured nanomaterials in natural river water 

samples: Effects of water chemical composition. Environ. Sci. Technol., 2009. 43(9): p. 

3322-3328. 

17.	 EPA-821-R-02-012: Methods for measuring the acute toxicity of effluents and receiving 

waters to freshwater and marine organisms, Fifth edition. 2002, U.S. Environmental 

Protection Agency. 

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

International 

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

International Standards Organization, Geneva, Switzerland. 

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

International Union of Pure and Applied Chemistry (IUPAC). 

21.	 Taurozzi, J.S., V.A. Hackley, and M.R. Wiesner, Preparation of a nanoscale TiO2 

aqueous dispersion for toxicological or environmental testing. Special Publication 1200­

3, National Institute of Standards and Technology, Gaithersburg, MD, 2012. 

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Dispersion of Nanoscale TiO2 for Eco-Toxicological Assessment, Version 1.1 

Appendix 

A.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). 

A.2. Representative PSD profiles 

The following figure illustrates representative LSD and DLS PSD profiles obtained for 

dispersions prepared following the procedure described in this protocol. 

0 

5 

10 

15 

20 

25 

30 

35 

40 

10 100 1000 10000 100000 

Volume (%) 

Diameter (nm) 

0 

5 

10 

15 

20 

25 

10 100 1000 10000 100000 

Volume (%) 

Diameter (nm) 

Figure 1. LDS (left) and DLS (right) volume-based PSD profiles of SRM 1898 dispersions prepared following the 

procedure described in this protocol. Measurements were performed using the dispersion medium in the 

measurement cell. The x-axis is shown on a log scale. 

12