NIST Special Publication 1200-4 

Preparation of Nanoscale TiO2 

Dispersions in Biological Test 

Media for 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.1200-4 
 

 

 

NIST Special Publication 1200-4 

NanoEHS Protocols 

Preparation	of	Nanoscale TiO2

Dispersions	in	Biological	Test	Media

for 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.1200-4 

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 1200-4 

Natl. Inst. Stand. Technol. Spec. Publ. 1200-4, 13 pages (June 2012) 

CODEN: NSPUE 
 

 

 

Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for 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(http://ceint.duke.edu) and it, along with any other previous version, is superseded by this 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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Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for Toxicological 

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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 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 

In this protocol, TiO2 nanoparticle dispersions in relevant biological media are produced by 

following a series of steps applied to a stock TiO2 aqueous nanoparticle dispersion. To prepare 

the stock TiO2 aqueous dispersion, refer to the previous protocol in this series [12]. 

This protocol focuses on two representative media broadly used for in vitro assays: phosphatebuffered saline solution (PBS) and Dulbecco's Modified Eagle Medium containing 10 % fetal 

bovine serum (DMEM-FBS). In both cases, bovine serum albumin (BSA) is utilized as a 

stabilizing agent; series specific serum albumin has been demonstrated to function as a nonspecific biocompatible stabilizer [9, 13, 14]. This protocol is proposed for the preparation of 

dispersions for generic acute bioassay applications; its use for chronic toxicity evaluations is 

beyond the scope of this work and should be validated by use of proper controls. 

The method described herein, if applied correctly, yields 100 g/mL monomodal nanoscale TiO2 

dispersions in PBS or DMEM-FBS, characterized by mean particle diameters of ≈ 75 nm (in 

PBS) and ≈ 83 nm (in DMEM-FBS) and pH values from 7.2 to 7.4 (in PBS) and ≈ 7.8 (in 

DMEM-FBS). The dispersions retain their particle size distribution for 48 h under relevant 

incubation conditions (in DMEM-FBS) and at room temperature (in PBS). Dispersions prepared 

following this protocol should be stored so as to minimize exposure to light (e.g., in amber vials). 

a

 Information regarding SRM 1898 can be accessed at http://www.nist.gov/srm/. 

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Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for Toxicological 

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3. Terminology 

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

International 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 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. 10 mg/mL stock TiO2 aqueous nanoparticle dispersion. 

Note—Refer to [12] for materials and guidelines for the preparation of the 10 mg/mL TiO2 aqueous dispersion. 

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

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Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for Toxicological 

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

necessary. 

4.1.3. 0.1 mol/L HCl (aq) and 0.1 mol/L NaOH (aq) reagent grade solutions 

4.1.4. Bovine serum albumin (BSA), 99+ % high purity reagent grade (lipid and IgG free) 

powder (e.g., SeraCare Life Sciences, Code No. AP-4510-80) 

4.1.5. For dispersion in PBS: 

PBS (10x) solution with 0.067 mol/L (PO4), calcium and magnesium free, passed through 

0.1 μm sterile filter (e.g., HyClone Laboratories, Cat. No. SH30258.02) 

4.1.6. For dispersion in DMEM with 10 % FBS: 

DMEM, 4.5 g/L glucose and sodium pyruvate without L-glutamine & phenol red, Sterile 

(e.g., Mediatech, Inc., Cat. No. 17-205-CV), with: Penicillin-Streptomycin (5000 U/mL 

Penicillin and 5000 ug/mL streptomycin), (e.g. Invitrogen Cat. No. 15070-063), Fungizone 

Antimycotic (250 ug/ml amphotericin B and 205 ug/mL Sodium deoxycholate), (e.g. Invitrogen 

Cat No. 15290-018), and Glutimax Supplement (L-Alanyl- L-Glutamine, 200 mM), (e.g. 

Invitrogen, Cat. No. 35050-161) 

The above preparation is referred to herein as DMEM 

FBS (e.g., Gemini Bio-Products, Cat. No. 100-106) 

4.2. Materials 

4.2.1. 10 mL and 30 mL sterilized amber glass vials 

4.2.2. Aluminum or polystyrene weighing dishes 

4.3.2. Calibrated pipettes and sterile 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 

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Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for Toxicological 

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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, as well as the 

optimization of the BSA-TiO2 mixing sequence prescribed in this protocol, refer to [11]. 

5.1. Dispersion in PBS 

5.1.1. Prepare a 10 mg/mL stock TiO2 aqueous nanoparticle dispersion, per [12]. 

5.1.2.	 Prepare 50 mL of PBS (1x) by doing a 1:10 dilution of the PBS (10x) with DI water, i.e., 

add 5 mL of PBS (10x) to 45 mL of DI water. Measure the pH of the prepared PBS (1x). 

5.1.3.	 Adjust the pH of the prepared PBS (1x) solution to a value in the 7.2 to 7.4 range by 

addition of 0.1 mol/L HCl (aq) and/or NaOH (aq) as needed. Mix thoroughly after each acid 

or base addition step to allow for proper homogenization and attainment of equilibrium pH. 

Note—The pH range was selected as acceptable for typical biologically relevant assays. If other values are 

desired, the stability of the dispersions must be tested accordingly. 

5.1.4.	 After addition of HCl (aq) and/or NaOH (aq) to achieve the desired pH, add an adequate 

amount of PBS (10x) to compensate for the dilution of the PBS (1x) solution by the water 

added with the acid or base solutions; e.g., if 2 mL of 0.1 mol/L HCl (aq) were added to the 

PBS (1x) to obtain the desired pH, then add 0.22 mL (2 x 0.11) PBS (10x). After pH 

adjustment and equilibration, verify that the pH of the obtained PBS (1x) is in the range from 

7.2 to 7.4. 

5.1.5.	 Weigh 0.8 g of BSA powder and transfer to a 10 mL amber glass vial. Add 10 mL of DI 

water to the vial with the BSA, seal and gently shake to allow for complete dissolution of the 

BSA while minimizing foam formation. Do not use the BSA solution until visible material is 

completely absent (allow approximately 1 h). The final product is a transparent 80 mg/mL 

BSA solution in DI water. 

5.1.6.	 Add 300 L of the 80 mg/mL aqueous BSA solution (5.1.5) into a clean 10 mL amber 

glass vial. 

5.1.7.	 Add 150 L of the 10 mg/mL stock TiO2 aqueous dispersion (5.1.1) into the vial with 

300 L of BSA solution (5.1.6). 

5.1.8.	 In a 30 mL amber glass vial, add 14.5 mL of the PBS (1x) prepared in 5.1.4 and 50 L of 

PBS (10x). 

5.1.9. Using a calibrated pipette, transfer 450 L of the TiO2/BSA/water mixture obtained in 

(5.1.7) into the 30 mL vial with 14.55 mL of PBS solution (5.1.8), to yield a dispersion 

containing 100 g/mL TiO2 and 1.6 mg/mL BSA in PBS (1x). The pH of the resulting 

dispersion should be comparable to that of the original buffer medium (5.1.4). 

5.1.10. For toxicological assays, the user is advised to conduct a control for BSA in the test 

medium (1.6 mg/mL) without TiO2. 

5.1.11. The resulting dispersion, stored in the 30 mL amber vial, retains its PSD for at least 48 h 

at room temperature. 

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Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for Toxicological 

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5.2. Dispersion in DMEM with 10 % FBS 

5.2.1. Prepare a 10 mg/mL TiO2 aqueous dispersion, per [12]. 

5.2.2.	 Prepare 50 mL of DMEM-FBS by mixing 5 mL of FBS with 45 mL of DMEM (1x). The 

resulting pH should be ≈ 7.8. 

5.2.3.	 Weigh 0.8 g of BSA powder and transfer to a 10 mL amber glass vial. Add 10 mL of DI 

water to the vial with the BSA, seal and gently shake to allow for complete dissolution of the 

BSA. Do not use the BSA solution until visible material is completely absent (allow 

approximately 1 h). The final product is a transparent 80 mg/mL BSA solution in DI water. 

5.2.4.	 Add 18.75 μL of the 80 mg/mL aqueous BSA solution (5.2.3) into a clean 10 mL amber 

glass vial. 

5.2.5.	 Add 150 μL of the 10 mg/mL TiO2 aqueous dispersion (5.2.1) into the vial with 18.75 μL 

of BSA solution (5.2.4). 

5.2.6.	 Using a calibrated pipette, add 14.83 mL of the DMEM-FBS prepared in 5.2.2 to a 30 mL 

amber glass vial. 

5.2.7.	 Transfer 168.75 L of the TiO2/BSA/water mixture obtained in (5.2.5) into the 30 mL 

vial with 14.83 mL of DMEM-FBS (5.2.6), to yield a 100 g/mL (TiO2), 100 g/mL (BSA) 

dispersion in DMEM-FBS. The pH of the resulting dispersion should still be comparable to 

that of the original medium (5.2.2). This procedure results in a 1.1 % dilution of the DMEMFBS by the water added with the TiO2 and BSA stocks. 

5.2.8.	 If used for toxicological assessments, the user is advised to conduct separate control tests 

for the BSA (100 g/mL) in the medium and for the water-diluted medium (1.1 %), in the 

absence of TiO2. 

5.2.9.	 The resulting dispersion, stored in the 30 mL amber vial, retains its PSD and pH (± 0.1 

units) for at least 48 h under relevant incubation conditions (37 oC, 90 % humidity, 5 % CO2 

atmosphere). 

6. Expected outcome 

Note—The particle size distribution (PSD) of the resulting dispersions was monitored for 48 h, corresponding 

to the typical acute toxicity assay timeframe. Although beyond the scope of this protocol, dispersions may 

remain stable for longer periods of time. This behavior has been validated only in pure media, without the 

presence of cells or other added components. 

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

6.1. Dispersion in PBS 

6.1.1.	 The obtained dispersions should have a white but translucent appearance if prepared 

using SRM 1898 or commercial 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. 

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Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for Toxicological 

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

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

If measured using LDS: 

Dm ≈ (71 to 79) nm 

D10 ≈ (60 to 64) nm 

D90 ≈ (80 to 96) nm 

If measured using DLS: 

Dm ≈ (122 to 149) nm 

D10 ≈ (69 to 91) nm 

D90 ≈ (160 to 200) nm 

The expected size range 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. 

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 [18] for details on PSD characterization 

and validation criteria, as well as representative PSD profiles. 

6.1.3	 The pH of dispersions should be comparable (± 0.1 pH units) to the pH of the originating 

medium. 

6.2. Dispersion in DMEM with 10 % FBS 

6.2.1.	 The obtained dispersions should have a white but translucent appearance if prepared using 

SRM 1898 or commercial 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. 

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

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

If measured using LDS:
 

Dm ≈ (81 to 85) nm
 

D10 ≈ (61 to 69) nm 

D90 ≈ (94 to 115) nm
 

If measured using DLS:
 

Dm ≈ (138 to 170) nm
 

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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Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for Toxicological 

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D10 ≈ (86 to 113) nm 

D90 ≈ (162 to 337) 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. 

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 [18] for details and discussions on PSD 

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

6.2.3.	 The pH of dispersions should be comparable (± 0.1 pH units) to the pH of the originating 

medium. 

7. Abbreviations 

BSA bovine serum albumin 

DI de-ionized 

DLS dynamic light scattering 

DMEM-FBS Dulbecco's Modified Eagle Medium containing 10 % FBS 

ENM engineered nanomaterial 

FBS fetal bovine serum 

HEPA high-efficiency particulate air 

ISO International Organization for Standardization 

IUPAC International Union of Pure and Applied Chemistry 

LDS laser diffraction spectrometry 

PBS phosphate buffered saline 

PSD particle size distribution 

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

Standards and Technology) 

8. Acknowledgements 

We thank John Elliott, NIST Biochemical Science Division, for providing access to an incubator 

and for providing the DMEM-FBS test media used in the development of this protocol. 

9. References 

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

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

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Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for Toxicological 

Assessment, Version 1.1 

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, 

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, Preparation of a nanoscale TiO2 

aqueous dispersion for toxicological or environmental testing. NIST SP 1200-3, National 

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

13.	 MacCuspie, R.I., A.J. Allen, and V.A. Hackley, Dispersion stabilization of silver 

nanoparticles in synthetic lung fluid studied under in-situ conditions. Nanotoxicology, 

2010. Early Online: p. 1-17. 

14.	 Tsai, D.H., et al., Adsorption and conformation of serum albumin protein on gold 

nanoparticles investigated using dimensional measurements and in situ spectroscopic 

methods. Langmuir, 2011. 27(6): p. 2464-2477. 

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. 

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Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for 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). 

13