Unclassified ENV/JM/MONO(2012)40

Organisation de Coopération et de Développement Économiques 

Organisation for Economic Co-operation and Development 18-Dec-2012 

___________________________________________________________________________________________

English - Or. English 

ENVIRONMENT DIRECTORATE 

JOINT MEETING OF THE CHEMICALS COMMITTEE AND 

THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY 

GUIDANCE ON SAMPLE PREPARATION AND DOSIMETRY FOR THE SAFETY TESTING OF 

MANUFACTURED NANOMATERIALS 

Series on the Safety of Manufactured Nanomaterials 

No. 36 

JT03332780 

Complete document available on OLIS in its original format 

This document and any map included herein are without prejudice to the status of or sovereignty over any territory, to the delimitation of 

international frontiers and boundaries and to the name of any territory, city or area. 

Unclassified ENV/JM/MONO(2012)40 English - Or. English
ENV/JM/MONO(2012)40 

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OECD Environment, Health and Safety Publications 

Series on the Safety of Manufactured Nanomaterials 

No. 36 

Guidance on Sample Preparation and Dosimetry for the Safety Testing of 

Manufactured Nanomaterials

Environment Directorate 

ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT 

Paris, 2012 
ENV/JM/MONO(2012)40 

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Also published in the Series of Safety of Manufactured Nanomaterials: 

 No. 1, Report of the OECD Workshop on the Safety of Manufactured Nanomaterials: Building 

Co-operation, Co-ordination and Communication (2006)

 No. 2, Current Developments/ Activities on the Safety of Manufactured Nanomaterials: Tour 

de table at the 1st Meeting of the Working Party on Manufactured Nanomaterials (2006) 

 No. 3, Current Developments/ Activities on the Safety of Manufactured Nanomaterials: Tour 

de table at the 2nd Meeting of the Working Party on Manufactured Nanomaterials 

(2007) 

No. 4, Manufactured Nanomaterials: Programme of Work 2006-2008 (2008) 

 No. 5, Current Developments/ Activities on the Safety of Manufactured Nanomaterials: Tour 

de table at the 3rd Meeting of the Working Party on Manufactured Nanomaterials 

(2008) 

 No. 6, List of Manufactured Nanomaterials and List of Endpoints for Phase One of the OECD 

Testing Programme (2008) 

 No. 7, Current Developments/ Activities on the Safety of Manufactured Nanomaterials: Tour 

de table at the 4th Meeting of the Working Party on Manufactured Nanomaterials 

(2008) 

 No. 8, Preliminary Analysis of Exposure Measurement and Exposure Mitigation in 

Occupational Settings: Manufactured Nanomaterials (2009) 

 No. 9, EHS Research Strategies on Manufactured Nanomaterials: Compilation of Outputs 

(2009) 

 No. 10, Identification, Compilation and Analysis of Guidance Information for Exposure 

Measurement and Exposure Mitigation: Manufactured Nanomaterials (2009) 

 No. 11, Emission Assessment for the Identification of Sources and Release of Airborne 

Manufactured Nanomaterials in the Workplace: Compilation of Existing Guidance 

(2009) 

 No. 12, Comparison of Guidance on Selection of Skin Protective Equipment and Respirators for 

Use in the Workplace: Manufactured Nanomaterials (2009) 

 No. 13, Report of an OECD Workshop on Exposure Assessment and Exposure Mitigation: 

Manufactured Nanomaterials (2009) 

 No. 14, Guidance Manual for the Testing of Manufactured Nanomaterials: OECD Sponsorship 

Programme (2009) 

 No. 15, Preliminary Review of OECD Test Guidelines for their Applicability to Manufactured 

Nanomaterials (2009) 

 No. 16, Manufactured Nanomaterials: Work Programme 2009-2012 (2009) 
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 No. 17, Current Development/ Activities on the Safety of Manufactured Nanomaterials: Tour de 

table at the 5th Meeting of the Working Party on Manufactured Nanomaterials (2009) 

 No. 18, Manufactured Nanomaterials: Roadmap for Activities during 2009 and 2010 (2009) 

 No. 19, Analysis of Information Gathering Initiative on Manufactured Nanomaterials (2009) 

 No. 20, Current Development/ Activities on the Safety of Manufactured Nanomaterials: Tour de 

table at the 6th Meeting of the Working Party on Manufactured Nanomaterials (2010) 

 No. 21, Report of the Workshop on Risk Assessment of Manufactured Nanomaterials in a 

Regulatory Context (2010) 

 No. 22, OECD Programme on the Safety of Manufactured Nanomaterials 2009-2012: 

Operational Plans of the Projects (2010) 

 No. 23, Report of the Questionnaire on Regulatory Regimes for Manufactured Nanomaterials 

(2010) 

 No. 24, Preliminary Guidance Notes on Sample Preparation and Dosimetry for the Safety 

Testing of Manufactured Nanomaterials (2010) 

 No. 25, Guidance Manual for the Testing of Manufactured Nanomaterials: OECD Sponsorship 

Programme: First Revision (2010)

 No. 26, Current Development/ Activities on the Safety of Manufactured Nanomaterials: Tour de 

table at the 7th Meeting of the Working Party on Manufactured Nanomaterials (2010) 

 No. 27, List of Manufactured Nanomaterials and List of Endpoints for Phase One of the 

Sponsorship Programme for the Testing Manufactured Nanomaterials: Revised (2010) 

 No. 28, Compilation and Comparison of Guidelines Related to Exposure to Nanomaterials in 

Laboratories (2010) 

 No. 29, Current Development/ Activities on the Safety of Manufactured Nanomaterials: Tour de 

table at the 8th Meeting of the Working Party on Manufactured Nanomaterials (2011) 

 No. 30, Regulated Nanomaterials: 2006-2009(2011) 

 No. 31, Information Gathering Schemes on Nanomaterials: Lessons Learned and Reported 

Information (2011) 

 No. 32, National Activities on Life Cycle Assessment of Nanomaterials (2011) 

 
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No. 33, Important Issues on Risk Assessment of Manufactured Nanomaterials (2012) 

 No. 34, Current Development/ Activities on the Safety of Manufactured Nanomaterials: Tour de 

table at the 9th Meeting of the Working Party on Manufactured Nanomaterials (2012) 

No. 35, Inhalation Toxicity Testing: Expert Meeting on Potential Revisions to OECD Test 

Guidelines and Guidance Document (2012)

© OECD 2012 

Applications for permission to reproduce or translate all or part of this material 

should be made to: Head of Publications Service, RIGHTS@oecd.org, OECD, 2 

rue André-Pascal, 75775 Paris Cedex 16, France 
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ABOUT THE OECD 

The Organisation for Economic Co-operation and Development (OECD) is an intergovernmental 

organisation in which representatives of 34 industrialised countries in North and South America, Europe 

and the Asia and Pacific region, as well as the European Commission, meet to co-ordinate and harmonise 

policies, discuss issues of mutual concern, and work together to respond to international problems. Most of 

the OECD’s work is carried out by more than 200 specialised committees and working groups composed 

of member country delegates. Observers from several countries with special status at the OECD, and from 

interested international organisations, attend many of the OECD’s workshops and other meetings. 

Committees and working groups are served by the OECD Secretariat, located in Paris, France, which is 

organised into directorates and divisions. 

The Environment, Health and Safety Division publishes free-of-charge documents in eleven different 

series: Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides; 

Biocides; Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety of 

Novel Foods and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; Emission 

Scenario Documents; and Safety of Manufactured Nanomaterials. More information about the 

Environment, Health and Safety Programme and EHS publications is available on the OECD’s World 

Wide Web site (www.oecd.org/ehs/). 

This publication was developed in the IOMC context. The contents do not necessarily reflect the 

views or stated policies of individual IOMC Participating Organizations. 

The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) was 

established in 1995 following recommendations made by the 1992 UN Conference on 

Environment and Development to strengthen co-operation and increase international coordination in the field of chemical safety. The Participating Organisations are FAO, ILO, UNDP, 

UNEP, UNIDO, UNITAR, WHO, World Bank and OECD. The purpose of the IOMC is to 

promote co-ordination of the policies and activities pursued by the Participating Organisations, 

jointly or separately, to achieve the sound management of chemicals in relation to human health 

and the environment. 
ENV/JM/MONO(2012)40 

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This publication is available electronically, at no charge. 

For this and many other Environment, 

Health and Safety publications, consult the OECD’s 

World Wide Web site (www.oecd.org/ehs/) 

or contact: 

OECD Environment Directorate, 

Environment, Health and Safety Division

2 rue André-Pascal 

75775 Paris Cedex 16 

France 

Fax: (33-1) 44 30 61 80 

E-mail: ehscont@oecd.org 
 ENV/JM/MONO(2012)40 

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EXECUTIVE SUMMARY 

The unique properties of manufactured nanomaterials have raised the question as to whether the 

current OECD Test Guidelines are adequate to appropriately address their characterisation and the 

assessment of their toxicological properties. Since it was recognised that there is a need to develop a 

guidance document on sample preparation and dosimetry, to which special attention should be paid in 

using test guidelines when considering the chemical and physical characteristics of nanomaterials, the first 

version of the guidance document, Preliminary Guidance Notes on Sample Preparation and Dosimetry for 

the Safety Testing of Manufactured Nanomaterials [ENV/JM/MONO(2010)25] was published in May 

2010. 

As this guidance document has been recognised as a living document, it is subject to amendment and 

refinement as researchers gain greater understanding of how to handle nanomaterials in test situations. It 

was proposed, at the 8th meeting of the WPMN held in March 2011, to set up a specific task group to 

review the first version of the Guidance Notes on Sample Preparation and Dosimetry (GNSPD) with 

experience gained through both the sponsorship programme analyses of data from the sponsored 

nanomaterials and other efforts. 

This document is the revision which was initially prepared by the task group and was reviewed by 

Steering Group four of OECD Working Party on Manufactured Nanomaterials (WPMN) taking into 

account the latest experience on testing of manufactured nanomaterials as well as the scientific knowledge 

from relevant experts. Also, it is worth noting that the results/recommendations from the OECD-WPMN 

horizontal expert meeting with regard to inhalation toxicity testing for nanomaterials 

[ENV/JM/MONO(2012)14], held in October 2011, has contributed to this update. Section V-A (PhysicalChemical Properties) was updated taking into consideration the progress achieved by ISO. Several 

endpoints (such as dustiness, crystallite size) have been newly introduced. 

This revised document “Guidance on Sample Preparation and Dosimetry” has also been circulated to 

the experts of Working Group of National Coordinators of the Test Guidelines Programme (WNT), for 

their comments.
ENV/JM/MONO(2012)40 

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TABLE OF CONTENTS 

SECTION I: GENERAL INTRODUCTION ............................................................................................... 12

SECTION II: TERMINOLOGY ................................................................................................................... 13

Dispersion versus solubility ...................................................................................................................... 13

SECTION III: CONSIDERATIONS ON APPROPRIATE DOSE-METRICS ........................................... 15

SECTION IV: COMMON ISSUES REGARDING SAMPLE PREPARATION AND DOSIMETRY ...... 16

1. Storage and stability of test material ..................................................................................................... 16

2. The chemical composition of the test media ......................................................................................... 17

3. Characterisation of stock dispersions .................................................................................................... 18

4. Characterisation of samples (prepared from stock dispersions) prior to administration/testing ........... 20

5. References ............................................................................................................................................. 21

SECTION V: SPECIFIC CONSIDERATIONS ........................................................................................... 22

A. PHYSICAL-CHEMICAL PROPERTIES .............................................................................................. 22

A.1 Tentative Guidance Relevant to Sample Preparation and Dosimetry for Physical-Chemical 

Characterisation ......................................................................................................................................... 24

A.1.1 Particle size, shape, and size distribution ..................................................................................... 24

A1.2 Particle Size Distribution .............................................................................................................. 26

A.1.3 Aggregation and Agglomeration .................................................................................................. 26

A.1.4 Chemical description (composition and identification) ............................................................... 26

A.1.5 Specific surface area .................................................................................................................... 27

A.1.6 Surface chemistry ........................................................................................................................ 27

A.1.7 Surface charge, zeta potential and Hamaker constant .................................................................. 29

A.1.8 Influence of water chemistry on nanomaterial properties and dispersion behaviour ................... 30

A.1.9 Preparation of liquid dispersions; octanol/water partition coefficients (Kows) ............................ 31

A.1.10 Crystal structure ......................................................................................................................... 33

A.1.11 Interfacial tension ...................................................................................................................... 33

A.1.12 Dustiness .................................................................................................................................... 34

A.1.13 Crystallite Size ........................................................................................................................... 34

A.1.14 Electron Microscopy .................................................................................................................. 35

A.1.15 Photocatalytic Activity............................................................................................................... 35

A.1.16 Pour Density .............................................................................................................................. 36

A.1.17 Porosity ...................................................................................................................................... 36

A.1.18 Octanol-Water Partition Coefficient ......................................................................................... 36

A.1.19 Radical Formation Potential ...................................................................................................... 37

A.2 References .......................................................................................................................................... 37

B. GUIDANCE ON PREPARING SAMPLES OF NANOMATERIAL IN EXPOSURE MEDIA FOR 

ECOTOXICITY STUDIES .......................................................................................................................... 47

B.1 Introduction ........................................................................................................................................ 47
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B.2 Aquatic Media Preparation ................................................................................................................. 48

B.2.1 Methods of suspension ................................................................................................................. 48

B.2.2 Media quality ............................................................................................................................... 49

B.2.3 Physical-Chemical Characterisations ........................................................................................... 49

B.2.4 Reporting results for media preparation approaches .................................................................... 49

B.3 Non-Aqueous Media Preparation ....................................................................................................... 50

B.3.1 Method of nanomaterial introduction ........................................................................................... 50

B.3.2 Media quality ............................................................................................................................... 50

B.3.3 Physical-Chemical Characterisations ........................................................................................... 50

B.3.4 Reporting results for media preparation approaches .................................................................... 50

B.4 References .......................................................................................................................................... 50

C. GUIDANCE ON PREPARING NANOMATERIAL SAMPLES FOR DEGRADATION, 

TRANSFORMATION AND ACCUMULATION STUDIES ..................................................................... 54

C. 1 Introduction ....................................................................................................................................... 54

C. 1.1 Environmental behaviour ........................................................................................................... 54

C. 1.2 Degradation and transformation .................................................................................................. 55

C. 1.3 Bioaccumulation ......................................................................................................................... 56

C. 2 Test Method Applicability and Dosimetry ........................................................................................ 57

C. 2.1. Environmental behaviour ........................................................................................................... 57

C. 2.2 Degradation and transformation .................................................................................................. 58

C. 2.3 Bioaccumulation ......................................................................................................................... 59

C. 3 References ......................................................................................................................................... 64

D. HEALTH EFFECTS, PREPARATION OF A TEST SUBSTANCE AND DOSIMETRY .................... 69

D.1 Knowledge transfer from environmental chemistry and stock dispersion preparation ...................... 70

D.2 Recommended control measurements on test dispersions and test vehicles during experiments ...... 72

D.3 Special considerations for vehicles e.g. physiological media used in cell-free, cell-based, and 

mammalian studies .................................................................................................................................... 74

D.4 Routes of delivery and the behaviour of nanomaterials dispersions in mammalian studies .............. 75

D.4.1 Respiratory tract exposures .......................................................................................................... 76

D.4.2 Oral Exposure .............................................................................................................................. 81

D.4.3 Dermal Exposure ......................................................................................................................... 82

D.4.4 Injection routes ............................................................................................................................ 84

D.4.5 Cell cultures and dispersion of NPs in culture media .................................................................. 84

D.5 References .......................................................................................................................................... 85
ENV/JM/MONO(2012)40 

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SECTION I: GENERAL INTRODUCTION 

1. In its review of the OECD harmonised test guidelines, Steering Group 4 (SG4) of the Working 

Party on Manufactured Nanomaterials (WPMN) recommended the development of guidance on sample 

preparation and dosimetry for tests using manufactured nanomaterials. Such guidance would be a separate 

document from the OECD’s existing guidance on difficult substances Guidance Document on Aquatic 

Toxicity Testing of Difficult Substances and Mixtures [ENV/JM/MONO(2000)6] and would be written 

primarily to inform the WPMN’s exploratory testing programme (OECD Sponsorship Programme for the 

Testing of Manufactured Nanomaterials) as well as other users involved in the testing of manufactured 

nanomaterials. A drafting group consisting of volunteer experts from the WPMN and the OECD member 

countries' delegations developed the guidance during 2008-2009. The first version of the guidance, 

Preliminary Guidance Notes on Sample Preparation and Dosimetry for the Safety Testing of Manufactured 

Nanomaterials [ENV/JM/MONO(2010)25] was published on 31 May 2010. 

2. From the beginning, it has been recognised that this guidance should be a living document, 

subject to amendments and refinements as researchers gain greater understanding of how to handle 

nanomaterials in test situations. Actually, as best methods for sample preparation, dosimetry, and safety 

testing do not yet have full consensus within the field, detailed methods cannot be prescribed. Thus, 

regular updates of this guidance can be anticipated. A significant outcome of the WPMN exploratory 

program is the knowledge gained in preparing test samples and administering doses across a wide range of 

testing scenarios and material types. Accordingly it was proposed, at the 8th meeting of the WPMN held in 

March 2011, to set up a task group to update/revise the Guidance Notes on Sample Preparation and 

Dosimetry. And in due course, the first version has been reviewed and revised during 2011-2012. 

3. This document is the 2nd version which was developed between the 8th and the 10th WPMN 

(March 2011 to June 2012). It is worth noting that the results/recommendation from some of the WPMN 

horizontal expert meetings with regard to testing of nanomaterials have contributed this update. 

4. The original purpose of this guidance was primarily to assist sponsors as they conduct testing in 

support of the WPMN exploratory program. It therefore focuses on the kinds of tests that address the 

endpoints and the types of nanomaterials being tested under the sponsorship programme. Nevertheless, the 

WPMN hopes that this guidance will also be of use to the scientific community at large, in particular to 

those investigators conducting tests to advance understanding of the environmental, health, and safety 

implications of manufactured nanomaterials. It is recognised, however, that due to the wide variety of 

nanomaterials, it is difficult to develop advice applicable to all nanomaterials; accordingly, the performer 

of a study will have to exert some judgment on a case-by-case basis on the applicability of the 

recommendations given in this guidance to their particular material. 

5. Last but not least, this guidance refers and applies to water insoluble manufactured nanomaterials 

as the WPMN considered that soluble nanomaterials are unlikely to need different sample preparation 

techniques than other chemicals, apart from precautions dictated by the specific reactivity of each material. 

However their size will still affect where they are being deposited e.g. in the lung. It should be noted that 

nanomaterials that can release soluble species, e.g. silver, are also considered in this guidance. Because 
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few, if any, standard testing approaches have been developed for nanomaterials, this guidance is not a 

“book of recipes” for preparing samples and administering doses, but rather outlines – often in a general or 

descriptive manner – considerations based on early results with nanomaterials or other experience with 

chemicals and particulates. It is a guide in the most basic sense, designed to point researchers in directions 

that at present seem most promising for yielding meaningful and reproducible test results. 

SECTION II: TERMINOLOGY 

Dispersion versus solubility 

6. Most dosing techniques require the test material to be in a liquid phase (generally aqueous) for 

delivery and (eco) toxicologists sometimes use the terms “in solution” or “solubility” to infer this. 

However, in particle chemistry these terms are inappropriate. The introduction of an insoluble or very 

sparingly soluble nanomaterial to a liquid or other aqueous medium with the intention of making a stock 

“solution” will involve dispersion. A stable dispersion of a nanomaterial in a liquid is referred to as a 

colloidal dispersion. The term “colloid” applies to particles or other dispersed material in the 1 nm - 1μm 

size range (IUPAC 1997). Colloids are dispersed rather than dissolved in a medium, and the term 

“dispersion” rather than “solution” is used in this document to mean the addition of solid nanomaterial to a 

liquid phase, where the solid and liquid phases co-exist. Some metal nanoparticles may release ions from 

the surface into the surrounding water (corrosion/degradation) and it is therefore possible that these 

nanomaterials will eventually degrade completely (Liu and Hurt 2010). Because of the particle size of 

many nanomaterials, it can be difficult to distinguish between when a nanomaterial is dispersed and when 

it is dissolved. The term “dissolved” is used in other OECD documents and historically in toxicology to 

mean the component of a liquid sample that has passed through a 0.45 μm (or similar) filter. However, as 

(colloidal) dispersions of nanoparticles might also pass through such filters, it is recommended that where 

both liquid and particulates are present the term “dispersed” should be used. The terminology used in this 

document comes directly from colloid science and may need revision for more complex (second generation 

and beyond) nanomaterials. 

7. In addition, nanoparticles may interact with the liquid phase components, partially or totally 

yielding degradation or transformation products that may influence the overall toxicity and fate processes. 

This possibility needs to be taken into account when selecting the media and procedures as well as in the 

assessment of the results of any experiment. 

8. The formation of other colloidal systems, such as emulsions (dispersed liquid drops in another 

immiscible liquid) have not yet been considered in this guidance, although they will become more relevant 

as manufactured nanomaterials are further modified and functionalised. 

Consideration of stability in sample preparation 

9. Many nanoparticles are prepared in the form of aqueous dispersion (some may exist in the form 

of organic or oil-based dispersions). The particles may be maintained in dispersion by stabilizers (for 

example surfactants and polymers), surface modifications/coatings, or by charge repulsion. Generally, 

three different forces are encountered in normal dispersions of particles: electrostatic and steric hindrance, 

and Van der Waals forces, and, for magnetic particles, an additional magnetic attraction force (the same 

forces would apply to a dispersion of nanoparticles in an aerosol for inhalation tests). Nanoparticles in such 
ENV/JM/MONO(2012)40 

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dispersions can exist as primary particles, agglomerates, or aggregates. The stability of the dispersion 

depends on the net effect of all these forces, which is determined mainly by the properties of the particle 

and the dispersing medium and particle surface properties, i.e. surface chemistry. For example, particles in 

an aqueous dispersion sterically stabilised by non-ionic surfactants would be less sensitive to pH and 

electrolyte conditions than those that are only electrostatically stabilised. For particles stabilised by (extra) 

surfactants, dilution during sample preparation may lead to desorption of the surfactant from the particle 

surface and hence agglomeration of particles may occur. For particles stabilised by surface charges, pH and 

ionic strength in the medium used for sample preparation may cause agglomeration of the particles. The 

test results for agglomerated particles may differ from what would be the case in the stable dispersion. In 

such cases, consideration of suspension chemistry is important to create kinetically stable suspensions. It is 

also important to consider the fundamental relevance of surface treatment or modifications of 

nanomaterials. In absence of these, many nanomaterials will tend to form large agglomerates and the 

primary particles will not disperse in water. Sonication or stirring can break up agglomerates into smaller 

sizes and can result in their temporary suspension in the medium. However, once sonication or stirring is 

stopped, in the absence of stabilizers the smaller agglomerates will tend to re-agglomerate into larger ones 

and precipitate. 

10. The zeta potential (at a specified pH and ionic strength) and/or the isoelectric point of the 

particles (in case the particles are stabilised by surface charges) should be determined and provided so it 

can be used for the fate assessment of particles in a dispersion. For sterically stabilised particles, the zetapotential may not be a suitable parameter to estimate the fate of the particles a priori. 

Agglomerate (Working definition: from ISO TS27687 2008)1

11. Agglomerate: collection of weakly bound particles, 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 

• NOTE 1. The forces holding an agglomerate together are weak forces, for example van der 

Waals forces, or simple physical entanglement. 

• NOTE 2. Agglomerates are also termed secondary particles and the original source particles are 

termed primary particles. 

Aggregate (Working definition from ISO TS28687 2008)1

12. Aggregate: 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 

• NOTE 1. The forces holding an aggregate together are strong forces, for example covalent bonds, 

or those resulting from sintering or complex physical entanglement. 

• NOTE 2. Aggregates are also termed secondary particles and the original source particles are 

termed primary particles. 

 

1

 These definitions differ from the ones described in the British Standards Institution Standard (BS 2955: 1993) and 

Nichols, Gary, et al. A Review of the Terms Agglomerate and Aggregate with a Recommendation for 

Nomenclature Used in Powder and Particle Characterization, Journal of Pharmaceutical Science, Vol. 91 

2103-2109, 2002. The more up-to-date and currently more widely accepted definitions from ISO are 

preferred in this document. 
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SECTION III: CONSIDERATIONS ON APPROPRIATE DOSE-METRICS 

13. Dosimetry refers to estimating or measuring the amount (in terms of mass, number, surface area, 

volume, etc.) of a particle at a specific biological target site at a particular point in time2

. Mass is currently 

the most commonly used dose-metric in ecotoxicity and mammalian toxicity studies. Accordingly, it is 

usually expected that results of such studies should always report mass concentration (e.g. mg/l). This 

dose-metric is the basis of the current risk assessment process and the linkage to past work in both 

exposure and (eco) toxicology. 

14. However, the mass metric appears not always to be the most appropriate or relevant one. Indeed 

for some nanomaterials the results may be better expressed as a function of surface area or particle number 

because particle size and specific area may play a main role in determining the toxicity of nanomaterials. 

This seems to be the case for many nanomaterials and there is a trend in toxicology to relate potential toxic 

effects of nanomaterials with these properties (e.g. inhalation) individually or in combination with each 

other. In addition, it appears that mass concentration is not sufficient for comparison of nanomaterials of 

the same chemical composition. 

15. Conversion between the metrics of mass, number and surface area remains challenging. 

Accordingly, measurements that enable expression of data in terms of surface area and particle number 

should be made where and whenever possible (e.g. particle number counts in test media and surface area 

measurements on the dry nanomaterial). 

16. For the environment, it seems too early to tell whether, for instance, dose by mass should and/or 

could be substituted with size or surface area. Too few studies have actually investigated alternative dose 

metrics at this point in time and correlated these with the observed effects. 

17. Adequate characterisation of the material and consideration of the scope of applicability of the 

test is required, along with consideration of the design of the test (e.g. selected doses, sample preparation to 

minimise uncertainty/bias) and the selection of the most appropriate instrumentation/method. There are 

currently no definitive conclusions on the best metrics. However, there is growing consensus that when 

tests on nanomaterials are performed, there should be a sufficient characterisation allowing the doseresponse to be expressed in the different metrics discussed: number, surface area and mass. It is also 

critical to report each of these metrics as well as the methods that were used to derive the measurements. 

18. Particle size distributions are a function of particle mass concentration, and so any size 

distribution measurements or surface area measurements would need to be done at each dose. It is also 

recognised that surface area measurements are made in dry state and assuming that no aggregation but only 

agglomeration occurs. Agglomeration in the suspensions may be a slow process especially at low mass 

concentrations. Hence, it is recommended that the determination of the particle size be repeated at regular 

intervals to ensure that dynamic changes to the dose are detected and recorded. 

19. Since specific surface area of particles in liquids cannot be measured directly, the derivation of 

surface area from size measurements has to be done with great care. Most sizing techniques will report a 

fraction of the outer diameter of existing or forming agglomerates (depending on which technique is used). 

The back-calculation from this diameter to a surface area is likely highly erroneous. 

 

2

 Adapted from Miller, F.J. 2000. Dosimetry of particles in laboratory animals and humans in relationship to issues 

surrounding lung overload and human health risk assessment: a critical review. Inhalation Toxicology, 

V12:19-58. 
ENV/JM/MONO(2012)40 

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SECTION IV: COMMON ISSUES REGARDING SAMPLE PREPARATION AND DOSIMETRY 

20. There are some common features of sample preparation and dosimetry that apply to both 

toxicology and ecotoxicology testing, as well as different routes of exposure/delivery. These common 

aspects are outlined below, with deviations from these described in the relevant section of this document. 

One aspect that always deserves particular attention is the fact that small impurities can have a strong 

impact on the physical-chemical properties of nanomaterials. Therefore, it is important to pay attention to 

the presence of impurities. Substances with different impurities may behave very differently, even if the 

main constituent is the same. 

21. Further, there is considerable interest in producing nanomaterials with specific surface 

functionalities/modifications. Such modifications can significantly affect the chemical reactivity of a 

nanomaterial, and thereby its potential effects on (or interactions with) living organisms and the 

environment. In addition, certain modifications (e.g. DNA or protein attachment) have been shown to have 

an effect on the uptake of nanoparticles into cells in vitro. Therefore, the surface functionality of a 

nanomaterial is likely to have a strong impact on its (eco) toxicological behaviour. Surface 

functionalisation is often employed to minimise nanomaterial agglomeration and/or to introduce functional 

groups or specific chemistry to the surface of the particle. But, the size of the material dispersed (as 

primary particles, agglomerates, or aggregates) may differ substantially for a given nanomaterial 

depending on whether or not it has been surface functionalised. 

22. The issues described here apply to tests from all sections of the OECD Test Guidelines. It is 

critical to document in detail the steps used in sample preparation in order to understand the methodologies 

used, and allow for replication of test samples as needed. Keeping in mind that preparation protocols may 

be strongly dependent on the nature of the sample to be dispersed, it is suggested that media preparation 

methods be consistent among similar media types, wherever possible. For example, when a testing 

programme includes both tests for environmental behaviour and for ecological effects in aquatic media, an 

effort should be made to prepare suspensions using a common method (where this is feasible). This 

approach will help to minimize variation in material properties and foster strong linkage between results of 

fate and exposure testing and effects on organisms. 

23. Other common features include: 

1. Storage and stability of test material 

24. Nanomaterials should be stored according to the manufacturer’s recommendations, but some 

general issues are highlighted when manufacturer’s information is limited. The usual considerations for 

storing chemicals will apply, including avoiding extremes of temperature, sunlight, and moisture. 

Nanomaterials that are supplied as dry powders or dispersions should be stored so that they remain dry or 

under liquid respectively. For any other variations on storage and re-dispersion, manufacturer’s 

recommendations should be followed. Clearly, experimenters will need to make stock dispersions from 

the original material supplied by the manufacturer. These dispersions should be stored taking into account 

the usual considerations above for any chemical, but also considering the reactivity of the material. For 

example, photoreactive materials should be kept in the dark and precautions should be taken to avoid 
 ENV/JM/MONO(2012)40 

 17

adsorption of other chemicals from the laboratory atmosphere when handling the dry material (e.g. storage 

and handling under an inert gas atmosphere). Once stock dispersions are prepared, and a full 

characterisation of the freshly prepared stock dispersion has been made, additional checks should be done 

to assess the shelf life of the material or confirm the information given by the manufacturer. Two key 

aspects need to be investigated: (i) whether or not the nanomaterial gradually dissolves or 

transforms/degrades such that the solid material disappears (e.g. for metal particles that form free metal 

ions in the external medium) (ii) any temporal changes in the particle size distribution and surface charge 

in the stock dispersion. If changes occur, then protocols should be developed to restore the particle size 

distribution (e.g. re-sonicating the dispersion just before dosing in the case of agglomeration). If the stock 

dispersion cannot be restored, it should be made fresh from the same batch number of the test material and 

re-characterised. If a different batch number of test material is used, then additional physical-chemical 

characterisation will be required. 

2. The chemical composition of the test media 

25. The chemical composition of the test media will affect particle aggregation/agglomeration. The 

following parameters should therefore be measured for media used in ecotoxicology, mammalian, and in

vitro studies (in addition to other routine measurements):

• Ionic strength- it is likely that many types of nanomaterials will agglomerate in very dilute 

brackish water, or any saline conditions, where studies have shown that even 2 % salt in seawater 

will do this. Thus, for any marine or estuarine studies the salinity and NaCl concentration in the 

water should be recorded. In natural fresh water or seawater, NaCl is likely to be the bulk 

electrolyte. Similarly, for salines or dispersion solutions used in cell-free, cell-based or 

mammalian studies, the composition should be given so that the ionic strength can be calculated. 

It is also highly likely that the typical salt concentrations in physiological salines (e.g. 0.9 % 

NaCl) and culture mediums would cause agglomeration of some nanomaterials. 

• Calcium concentration and hardness- divalent metal ions can also have a significant effect on 

agglomeration, especially at low salinity (freshwater). Therefore, in all freshwater ecotoxicology 

studies the calcium (Ca) concentration of the water should be measured. In addition, magnesium 

(Mg) concentration, and total hardness would be useful. In mammalian studies, if drinking water 

is used to deliver nanomaterials then Ca, Mg and total hardness of the water should similarly be 

measured. These procedures are well known. In mammalian studies special attention should be 

given to reporting the Ca and Mg concentrations in salines, including the anion (e.g. whether 

MgSO4 or MgCl2 was used in their preparation). 

• pH- this should be routinely determined in any experiment. pH affects agglomeration of charged 

nanomaterials. Physiological salines usually contain pH buffering, and the buffers should be 

reported precisely (e.g. the specific type of Tris buffer with the full chemical name, or exact 

details of phosphate buffers). Where commercially available buffer tablets or ready-made 

solutions are used, the full composition of the buffer should be reported. 

• Dissolved organic matter- it is evident that the precise type of organic matter, and the ligands it 

presents, will have potentially large effects on agglomeration and dispersion of nanomaterials. It 

would therefore be prudent to have some general information about the organic matter in any 

water. This could be something simple like a measurement of total organic matter, or dissolved 

organic carbon. This would at least give an overview of the general type of water. For cell-free, 

cell-based, or mammalian studies, the addition of bovine serum albumin (BSA) and antibiotic 

preparations to salines represent a source of organic matter. It would be prudent to use high 

purity reagents in these cases (e.g. fatty acid-free BSA or similar) rather than cheaper reagents. 
ENV/JM/MONO(2012)40 

 18

Since any charge-carrying organic substance which may be adsorbed on the surface of the 

nanoparticles will change the charge properties of the surface, and hence the dispersion behaviour, 

all the organic substances (proteins, antibiotics) added should be stated. It is important to also 

consider that different surface treatments on the same parent form (e.g.TiO2) may interact quite 

differently with the dissolved organic matter. 

• Alkalinity- this may affect agglomeration and similar arguments to pH apply. This is a routine 

measurement in ecotoxicology, but not in in vitro or mammalian toxicology. This will be 

especially important where bicarbonate buffers are used as the main method to control pH in 

salines. 

• Dispersing agents- in case an added surfactant is used to stabilise the dispersion, it would 

normally be of high concentration, considering the high specific surface area presented by many 

nanomaterials. Distribution of the dispersing agents between the aqueous phase and the particle 

surfaces would occur. Therefore, information regarding structural formula and concentration of 

the agent should be provided. The use of strong dispersing or stabilising agents may modify the 

bioavailability of the nanomaterial and, in addition, if an added agent has been used to stabilise 

the stock dispersion, this may not be appropriate for studies that investigate the fate and 

behaviour of nanomaterials in natural conditions. Accordingly, care should be taken in the 

conduction of tests and the interpretation of the test results when the use of such agents is 

unavoidable. It should also be noticed that in most cases, in absence of dispersing agent(s) (e.g. 

pigments in paint application), particles will be present in form of aggregates. 

3. Characterisation of stock dispersions 

26. In addition to routine water quality measurements in ecotoxicity testing, or reporting saline 

quality in mammalian studies, or optimized solution characteristics for in vitro methodologies, there is 

some essential information required about the nanomaterials (discussed in Handy et al., 2008; Crane and 

Handy, 2007; Crane et al., 2008, Taurozzi et al., 2012, Ma, et al., 2012). The following would apply to 

stock dispersions and arguably, this list could be common to human health and ecotoxicology studies: 

Any information from the manufacturer on the test material. 

Measured mean primary particle size (for example by electron microscopy (EM)). The method of particle 

size determination should be described and the character of the mean (number, volume, z- or intensity) 

must be given. If a certain given mean/average value is calculated from a primary data (e.g. volume 

average derived from dynamic light scattering z-average) the calculation procedure should be described. In 

some cases, it may not be possible to measure particle size in the stock dispersion (e.g. the concentration 

may be too high, EM cannot measure directly in dispersion, etc...). In this case, it should be measured in 

other relevant media such as the diluted preparations used in the actual OECD test, further dilution may be 

necessary. 

Particle size distribution and indications of mono or polydispersity (e.g. by dynamic light scattering or 

similar optical method)3

, or other attempt to describe aggregates, agglomerates or ranges of particle sizes in 

the stock dispersion, including distribution of primary particles. The methodology to derive this size 

distribution either must be standardised or must be described together with the applied procedures. If a 

buffer or saline is used to make the dispersion, then the exact composition of the medium, measured pH, 

 

3

 Taking into account possible limitations of the applied technique, e.g. Calzolai L, Gilliland D, Garcìa CP, Rossi F. 

Separation and characterization of gold nanoparticle mixtures by flow-field-flow fractionation. J 

Chromatogr A. 2011 Jul 8;1218(27): 4234-9. 
 ENV/JM/MONO(2012)40 

 19

temperature and any aeration or gassing of the dispersion should also be reported as this may affect particle 

size distributions. The method of dispersion (stirring, sonication ) should be fully described (duration, stir 

speed, sonication power (preferably in terms of energy input in J/L (joules/litre)), time, type of sonicator, 

probe and immersion depth if used, volume of sample, etc.4

). Observations regarding e.g. concentration or 

limitations of EM as in ii) above also apply for size distribution. 

Mass concentration (measured) in the stock dispersion (e.g. mg/l).Mass concentrations as such will not be 

sufficient for metal-based nanomaterials that dissolve. In such cases measuring the release of ions appears 

essential, e.g. by centrifuging suspensions and determine metal concentrations in the supernatant, in 

addition to determining the total mass of metal in suspension. In some cases ion specific electrodes may be 

used, or dialysis, etc. 

Surface area measurements of the primary particles will allow results to be calculated on a surface area 

basis. When the nanoparticles are not highly aggregated, it is understood that the surface area of dry 

nanomaterial is a good estimate of free primary particles in the dispersion, however extrapolation from dry 

materials to the aqueous dispersions should be done cautiously. 

For some charged particles, surface charge may be critical to the agglomeration process and so the surface 

charge may be indirectly assessed via measurements of zeta potential. Since the value of the zeta potential 

is a function of the ionic strength and composition of the dispersing medium, the conditions during 

determination should be standardised or reported. It would also be important to measure or fix other abiotic 

factors that might alter this, such as solution pH and ionic strength. 

Any other measurement that is particularly relevant for a specific particle type, for example, aspect ratio 

for fibres, length of nanotubes, surface functionality. 

27. A detailed analysis of the composition of the stock dispersion should be undertaken with special 

attention to the possible impurities in it. Contaminants can be incorporated into the nanomaterial at any 

point during production, handling and dispersing. Examples include iron contamination of carbon 

nanotubes during fabrication (Jurkschat et al., 2006), tetrahydrofurane (THF) (and its breakdown products) 

contamination of fullerene during solvent-exchange dispersion preparation (Markovic et al., 2007), and 

endotoxin contamination during manufacturing and handling (Vallhov et al., 2006). In some cases these 

“contaminants” are intrinsic components of the nanomaterial likely to be encountered during real world 

exposure, such as polyaromatic hydrocarbons on diesel exhaust particles (Xia et al., 2004), in which case 

their quantities should be measured to compare their impact across studies. In other cases, such 

contaminants may not be intrinsic to the original nanomaterial (such as contaminants in dispersing agents) 

and may be accounted for in controls. Ultrasonication processes sometimes produce contaminating 

particles by ablation of the probe tip and vessel. Alternatively, a purification step may need to be added to 

stock dispersion preparation. 

28. While a test of the material for metal impurities is relatively easy to perform, a thorough test for 

unknown organic impurities may sometimes not be easy or feasible. Here the information from the 

manufacturers about additives and possible by-products is indispensable but encompasses both 

technological and policy implications. 

29. The shape, volume and material type of the container using during the dispersion preparation 

may also have some influence on the size distribution and purity of the stock dispersion and should be 

considered and reported (Taurozzi, et al., 2012). 

 

4

 See also next section 
ENV/JM/MONO(2012)40 

 20

4. Characterisation of samples (prepared from stock dispersions) prior to administration/testing 

30. The general recommendations about the characterisation of stock dispersions (above) should be 

followed. 

31. The key point is to know the exact composition of the prepared sample, and to report how it was 

made. As particle size and concentration may vary with depth after stock dispersions are left to settle, a 

consistent sampling point for very heterogeneous samples over time could provide better precision (Ma 

and Bouchard 2009), also stirring or vortexing periodically and/or just before sampling can be helpful. The 

following information is required: 

i) Volumes prepared, type of water or solvents used; 

ii) pH and use of any buffers; 

iii) Exact details of any sonication times (or preferably energy input in J/L) given in terms of 

durations/intensity/instrument used/frequency of ultrasound; 

iv) Exact details of how long after sonication or mixing/stirring before the test dispersion was added 

to test vessels. Any extra (precautionary) period of mixing or sonication (e.g. 30 min or 

preferably energy input in J/L) immediately prior to dosing to the experimental model may be 

helpful and should be recorded. Re-characterisation of subsamples from stock suspensions after 

pH modification, sonication or other treatments should be considered; 

v) Exact details of volumes added to tanks or test vessels, and how they were mixed in the tanks/test 

vessels. For example, passively by diffusion, stirred in, allowed to mix with air bubbling around 

the system. Details about the depth of the liquid under treatment in tanks or vessels or details on 

the depth of the probe that is inserted under the liquid surface should be recorded and kept 

constant in all related tests; and 

vi) pH, ionic strength, dissolved organic matter. 
 ENV/JM/MONO(2012)40 

 21

5. References 

Crane, M., Handy, R. D., Garrod J., and Owen, R. (2008), “Ecotoxicity test methods and environmental 

hazard assessment for engineered nanoparticles”, Ecotoxicology 17, pp.421–437. 

Crane, M. and Handy, R. D. (2007), “An assessment of regulatory testing strategies and methods for 

characterising the ecotoxicological hazards of nanomaterials”, Report for Defra, London, UK. 

http://randd.defra.gov.uk/Document.aspx?DocumentID=2270/ (Accessed in September, 2012) 

Handy, R. D., Kammer, F. v. d., Lead, J. R., Hassellöv, M., Owen, R., and Crane, M. (2008), “The 

ecotoxicology and chemistry of manufactured nanoparticles”, Ecotoxicology 17, pp. 287-314. 

IUPAC (1997), IUPAC Compendium of Chemical Terminology, 2nd Edition. IUPAC. 

Jurkschat K., Ji X., Crossley A., Compton R.G., and Banks C.E. (2006), “Super-washing does not leave 

single walled carbon nanotubes iron-free”, Analyst 132, pp. 21-3. 

Ma H, Brennan A, Diamond SA. (2012), “Phototoxicity of TiO2 nanoparticles under solar radiation to two 

aquatic species: Daphnia magna and Japanese medaka”, Environmental Toxicology and Chemistry

31, pp.1621 

Ma, X., and Bouchard, D.(2009), “Formation of Aqueous Suspensions of Fullerenes”, Environmental 

Science and Technology 43 (2), pp. 330-336 

Markovic Z., Todorovic-Markovic B., Kleut D., Nikolic N., Vranjes-Djuric S., Misirkic M., Vucicevic L., 

Janjetovic K., Isakovic A., Harhaji L., Babic-Stojic B., Dramicanin M., and Trajkovic V. (2007), 

“The mechanism of cell-damaging reactive oxygen generation by colloidal fullerenes”, Biomaterials

28, pp.5437-48. 

Taurozzi J.S., Hackley V.A. and Wiesner M.R. (2012), “Reporting guidelines for the Preparation of 

Aqueous nanoparticle dispersions from dry materials”, Ver.2.1. CEINT/NIST Protocol. 

Vallhov H., Qin J., Johansson S.M., Ahlborg N., Muhammed M.A., and Scheynius A., and Gabrielsson S. 

(2006), “The importance of an endotoxin-free environment during the production of nanoparticles 

used in medical applications”, Nano Letters 6, pp.1682-6. 

Xia T., Korge P., Weiss J.N., Li N., Venkatesen M.I., Sioutas C., and Nel A. (2004), “Quinones and 

aromatic chemical compounds in particulate matter induce mitochondrial dysfunction: implications 

for ultrafine particle toxicity”, Environmental Health Perspectives112, pp.1347-58. 
ENV/JM/MONO(2012)40 

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SECTION V: SPECIFIC CONSIDERATIONS 

A. PHYSICAL-CHEMICAL PROPERTIES 5

32. Observations from the groups that have addressed the problem of sample preparation for 

physical-chemical characterisation often do not distinguish between characterisation appropriate in order to 

assess human health and environmental effects. Therefore, the term (eco) toxicology in this section is 

generally used unless a clear distinction is necessary. 

33. As for any other materials or chemicals, the inherent ability of a nanomaterial to cause an effect 

(being it desirable or adverse) relates to its chemical and physical properties, including its impurities. 

Physical-chemical characterisation of a nanomaterial is fundamental both for determining its identity and 

to properly execute and interpret the results of (eco) toxicological tests, including comparisons with other 

experiments with the same or similar materials. Indeed, characterization of the nanomaterial's physicalchemical properties before (eco) toxicity testing ensures that the results are related to the nanomaterial 

intended for the testing. Generally, characterisation of the nanomaterial should be conducted at least ‘as 

received’ (that is, as sampled directly from the received package) and ‘as administered’ (that is, after 

preparing the material for introduction into the test system)6

 and ‘after administration or in situ’ (that is, 

once the material has been introduced into the (eco) toxicological test system, as appropriate). 

34. In recognition of the unique properties of manufactured nanomaterials, several regional, national 

and international organisations formed Physical-Chemical expert panels for the purpose of issuing 

recommendations concerning the applicability of existing standardised test procedures (e.g. U.S. EPA 

Series 830 [U.S. EPA, 2007b] and the OECD Series 100 [OECD, 2007] test guidelines) to these materials. 

These workgroups identified a number of standardised test guidelines that are unlikely to be directly 

applicable to insoluble manufactured nanomaterials (for example, test guidelines for aqueous solubility and 

octanol/water partition coefficients); these same issues have ramifications for standardised test procedures 

in the areas of ecotoxicology, human health effects and in assessing the environmental fate (transport, 

degradation and accumulation) of these materials, The sample preparation step is crucial, so precise SOPs 

are required for every measurement technique. 

35. In addition, development of some new physical-chemical characterisation procedures may be 

required, in order to allow nanomaterials to be adequately characterised and to assist in assessing the risks 

associated with intentional or unintentional exposure of humans or the environment to these materials. 

Characteristics requiring determination might include (but are not limited to): particle size, size distribution, 

aggregation, agglomeration state, shape, chemical composition, surface area, surface chemistry, 

dissociation constant, crystal structure, surface charge, zeta potential, Hamaker constant, interfacial tension, 

 

5

 Note: It is recommended to characterising both stock dispersion and the diluted/prepared dispersions for dosing, as 

appropriate. 

6

 The subject of this section A. 
 ENV/JM/MONO(2012)40 

 23

and porosity (Oberdörster et al., 1994; Hunt et al., 1996; Oberdörster et al., 2002; Kreyling et al., 2004; 

Oberdörster et al., 2005; Nel et al., 2006; Champion and Mitragotri, 2006; Elder and Oberdörster, 2006; 

Zhu et al., 2006; Warheit et al., 2007; Limbach et al., 2007; Ji et al., 2007; Teeguarden et al., 2007; 

Murdock et al., 2008; ISO, 2008; Utterback et al., 2008; OECD, 2008; Loux and Savage, 2008). A review 

highlights the difficulties associated with the characterisation of more complex nanomaterials that cannot 

be considered as simple colloids (Richman et al., 2009). The relevance of these characteristics will depend 

on the specific nanomaterial(s) considered. Moreover, the recommendation for a definition of 

nanomaterials, adopted by the European Commission (EC) on 18 October 2011, to be used for regulatory 

purposes7

poses quite important questions. The answers to the questions certainly require additional work 

on the metrological aspects, to establish the methods and procedures to assess whether the substance under 

consideration corresponds to this definition and/or the presence of nanomaterials in consumer products. 

36. Relevant findings are in three main areas: 

Sample Preparation: Sample preparation is often unique to the characterization method to be employed 

(See also Section IV). When a procedure for generating nanomaterial preparations (such as dispersions) 

intended for (eco)toxicological studies is employed, a great attention should be paid to minimise any 

alteration of the physical, chemical or (eco)toxicological properties of the substrate (Crane et al., 2008). 

For example, grinding agglomerates may lead to the fracturing of individual particles which in turn can 

expose new sites of enhanced reactivity. Preparing aqueous nanomaterial dispersions may require the use 

of surfactants, solvents or sonication, which in turn can alter the degree of agglomeration, fracture 

individual particles, change existing surface modifications, or alter the bioavailability and toxicity of the 

parent compound. Interaction with organic material or other constituents contained within the supporting 

medium also should be taken into account. In order to practically and meaningfully extrapolate laboratory 

findings to environmental and physiological systems, the difference between sample preparation 

techniques compatible with the test protocol and the anticipated environmental/physiological processes 

should be considered. It has to be recognised that the interaction of a nanomaterial with testing media will 

always influence, if not alter, its properties, as is the case for any other chemical. Another important point 

for the preparation of aqueous dispersions of nanomaterials is the disequilibrium after mixing and slow 

reaction (mainly surface chemistry) towards equilibrium. It could be helpful to allow some time for 

dispersions to equilibrate before they are dosed in an experiment. For example, distilled and/or deionised 

water is in disequilibrium with atmospheric CO2, but CO2 dissolution into the dispersion and adsorption to 

the surface of freshly dispersed nanomaterials is an important process altering the surface charge of many 

nanomaterials and the pH of the dispersion. The same holds true for all surface reactions of the 

nanomaterials with any substance in the used medium (e.g. Bovine Serum Albumin (BSA)). There are 

protocols for dispersing nanomaterials, which are specific to both the nanomaterial and the method by 

which it will be characterized (PROSPEcT, 2010a; PROSPEcT, 2010b; ISO/TR 13097; Wang et al., 2010; 

Keller et al., 2010; Ji et al., 2010; Chowdhury et al., 2010; Taurozzi et al., 2012a, b, c) and others intended 

to be more generally applicable (Jensen et al., 2011). The preparation of pyroforic, not passivated, dry 

nanoparticles must be performed in argon atmosphere in fume hood due to the risk of ignition. 

 

7 http://ec.europa.eu/environment/chemicals/nanotech/index.htm: Nanomaterial is a natural, incidental or 

manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate 

and where, for 50% or more of the particles in the number size distribution, one or more external 

dimensions is in the size range 1 nm – 100 nm.

In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number 

size distribution threshold of 50 % may be replaced by a threshold between 1 and 50 %. By derogation 

from the above, fullerenes, graphene flakes and single wall carbon nanotubes with one or more external 

dimensions below 1 nm should be considered as nanomaterials.
ENV/JM/MONO(2012)40 

 24

Dosing: (Eco) toxicological studies typically employ dosage procedures intended to be both reproducible 

and quantitative. However, aqueous nanomaterial dispersions may be very sensitive to the techniques 

employed in their preparation and they may not necessarily follow the principles of equilibrium 

partitioning. In particular, significantly more empirical data may be required in order to develop methods 

designed to ensure reproducible and quantitative dosimetry (especially with aqueous dispersions). Another 

aspect to be taken into account in dosing is the reduced (due e.g.to agglomeration or precipitation) or 

excessive delivery of nanomaterials into test systems, such as cells during the in vitro studies and to 

animals during in vivo studies. In fact high levels of exposure can give rise to overload effects that can be 

misinterpreted as evidence of cytotoxicity (Wittmaack, 2011a) and vice versa. In all cases selected doses of 

administered nanomaterials should ensure adequate exposure, need to be scientifically justified and must 

reflect possible exposure scenarios in eco- or human toxicology. On the other hand, as nanomaterials may 

change form not only after sample preparation but also during and after release to the test system (see 

previous paragraph and next section and specific chapters on environmental and human health sample 

preparation), dosing and exposure methodologies (including monitoring) may need to be adapted if 

material modifications are reasonably anticipated. 

Physical-chemical characterisation for (eco)toxicology studies: Human and environmental toxicologists 

seek to develop rigorous mechanistic understandings of their findings for the purposes of elucidating: a) 

the toxicological response, b) possible antagonisms and synergisms with other toxicants, and c) predictive 

methodologies useful for assessing the risks posed by new products, for which there is limited knowledge 

available on certain characteristics. This last aspect is perhaps the least understood of these three main 

areas. 

A.1 Tentative Guidance Relevant to Sample Preparation and Dosimetry for Physical-Chemical 

Characterisation 

A.1.1 Particle size, shape, and size distribution 

37. The Nanotechnology Committee of the International Organisation for Standardisation (ISO /TC 

229) has developed a technical report entitled ‘ISO/PDTR 13014: Nanotechnologies — Guidance on 

physicochemical characterization for manufactured nano-objects submitted for toxicological testing’

whose purpose is to assist health scientists and experts from other disciplines to understand, plan, identify, 

and address relevant physical-chemical characterization of such materials before conducting toxicological 

tests on them. Before the publication of the overview in ISO/PDTR 13014, a number of individual ISO and 

other organisations' standard documents were available, containing relevant information on PhysicalChemical characterization of nanomaterials.8

 The reader is also advised to consult the report issued by the 

European Commission's Joint Research Centre: Requirements on measurements for the implementation of 

the European Commission definition of the term "nanomaterial" (European Commission 2012). It 

describes the requirements for particle size measurements of nanomaterials based on the European 

Commission's Recommendation on the definition of nanomaterial (European Commission 2011b), it 

discusses the related generic measurement issues and reviews the capabilities of the measurement methods 

currently available. Moreover, it illustrates with practical examples the measurement issues that remain to 

be solved. 

 

8

 There are several standards documents to be mentioned here: 

• NIOSH/DUNE interlaboratory study: evaluation of a sample preparation technique for determination of TEMbased size distribution using NIST Reference Materials 8011, 8012, and 8013: gold nanoparticles 

•
 ENV/JM/MONO(2012)40 

 25

38. An increasing number of publications on nanomaterials demonstrate that the particle size of a 

nanomaterial can strongly influence its toxicological properties (Carlson et al., 2008; Coi et al., 2008; Pan 

et al., 2007), as well as its dosimetric fate in the entire organism, including the organ of uptake, circulation 

and secondary organs of accumulation. The size characterization of nanomaterials, however, is not a trivial 

task, often nanomaterials show a distribution of sizes and the measurement of the particle size distribution 

(PSD) will be challenging, especially in dispersion (Carlson et al., 2008; Choi et al., 2008; Pan et al., 2007; 

Goodman et al., 2004; Chithrani et al., 2006; Walkey et al., 2011; Suresh et al., 2012). Particularly 

challenging is the discrimination between primary particles and their aggregates/agglomerates. 

Measurement of PSD also calls into question the metrics used for its reporting. PSD in principle can be 

expressed in mass, volume, or number of particles. Each one of these metrics presents its own issues and 

challenges. The European Commission has just recently published its definition of nanomaterials that 

require the measurement of the number PSD in the 1-100 nm size range (European Commission 

(2011/696/EU). At the moment, there is not a single technique able to satisfactorily measure the number 

particle size distribution of objects in the 1-100 nm size range. Techniques that can partially address this 

can be categorized as electron microscopy-based (Dudkiewicz et al., 2011, Buhr et al., 2009, Klein et al., 

2011); laser diffraction-based (Brar et al., 2011); centrifugation-based (Camey et al., 2011) and separationbased (Baalousha et al., 2011). In any case more than just one technique (multi-method approach) is 

required for PSD measurements. 

39. There exists a suite of standardised procedures (for example by EPA, OECD and other 

organisations) for physically characterising particles. However, many of them have minimum size cut off 

thresholds that exceed the 1 nm to 100 nm size range, although, agglomerates/aggregates may form from 

the primary particles to make secondary particles larger than 100 nm. Among all of the standardised 

characterisation procedures, these are perhaps the most easily modifiable through incorporation of more 

recent technological advances, which recognise the physical and analytical limits of those methods, and 

develop new methods, including also combinations of methods. These advances include areas such as 

transmission electron microscopy (TEM), scanning electron microscopy (SEM), confocal microscopy, 

Dynamic Light Scattering (DLS), atomic force microscopy (AFM), etc. Before quantitative measurements 

of nanomaterial characteristics are conducted, a qualitative TEM analysis is instrumental to judge the 

relevance/suitability of the below-mentioned quantitative analyses and to avoid/evaluate possible 

measurement artifacts or bias. To our knowledge no formal guidelines for the unambiguous and detailed 

description of a nanomaterial are available. Procedures can be based on methods described in the literature 

(Krumbein et al., 1963; ISO 9276-6, 2008; NIST 960-1, 2001). Such qualitative description includes at 

least: (i) representative and calibrated micrographs; (ii) the agglomeration- and aggregation status; (iii) the 

general morphology9

; (iv) the surface topology; (v) the structure (crystalline, amorphous, ...); (vi) and the 

presence of contaminants and aberrant particles. 

40. Researchers also should recognise that dimensional values obtained from different measurement 

methods will differ with regard to the measurand, and for example from hydrodynamic estimates. Suitable 

methods for the measurement of particle shapes need to take into consideration the ‘dimensionality’ of a 

nanomaterial. The EC definition explicitly states ‘in one dimension’. Techniques based on scattering, like 

DLS, and on the measurements of the hydrodynamic radius, like centrifugal sedimentation, reduce 3D 

information to 1D (e.g. radius of hypothetical sphere’) which in unequiaxal nanomaterial, like fibers, might 

lead to erroneous conclusions. Taking into account that the aim is a 1-nm-resolution, choices are limited to 

a few techniques including SEM, TEM and, in specific cases, AFM; Yet, extreme care needs to be taken 

for the sample preparation for these microscopical techniques to avoid alterations like retrograde 

nanoparticle agglomeration and particle masking by drying debris contained in the solvent. 

 

9

 Scientific Basis for the Definition of the Term “Nanomaterial” 

[http://ec.europa.eu/health/scientific_committees/emerging/docs/scenihr_o_030.pdf] 
ENV/JM/MONO(2012)40 

 26

41. Thus, values for particle size, shape, size distribution and degree of agglomeration will depend 

both on the methodology used as well as on the properties of the medium supporting the sample under 

consideration. Although EM techniques are considered the standard in the peer reviewed literature, care 

should be exercised in extrapolating properties observed under high vacuum conditions (as with SEM and 

TEM measurements) to aqueous and physiological dispersions. Lastly, nanomaterial physical properties 

that may influence any of these properties, such as magnetisation, also should be characterised. The 

assessment of particle size, shape, size distribution and agglomeration is of a paramount importance in 

defining the dose metric applicable in in vitro investigation. In fact, a recent study on silica nanoparticles 

have demonstrated that in order to avoid overload of cells, it is fundamental to determine the relative 

magnitude of transport by diffusion and gravitational settling of nanoparticles (Wittmaack, 2011b). 

A1.2 Particle Size Distribution 

42. Number-based distributions of nanomaterial size, shape and surface characteristics can be 

obtained by quantitative (semi-automatic) analysis of aggregated/agglomerated nanomaterial based on 

TEM micrographs. Essential basic general principles are (i) the traceability of information, imaging and 

results, (ii) analysis and representation of results on the per-particle level, (iii) (for practicality) automating 

of repetitive tasks. To our knowledge, no suitable and validated procedure is available. The outline of in 

house procedure can follow NIST guide-lines (NIST 960-1, 2001). The different parts of such methods 

support on different guidelines. Subsampling and suspension of samples can be done according to ISO14887(ISO 14887, 2000; ISO 14488, 2007; ISO 13322-1, 2004). Imaging and image analysis guidelines 

are given in ISO publications (ISO 13322-1, 2004; ISO 9276-2, 2001; Krumbein et al., 1963; ISO 9276-6, 

2008). Data analysis and representation can be done in combination with the methods described in several 

ISO publications (ISO 13322-1, 2004; ISO 9276-1, 1998; ISO 9276-2, 2001; ISO 9276-6, 2008). It is 

important to note that particle size may change in the test media and liquid test media in particular, where 

smaller particles may aggregate/agglomerate or larger particles may deaggregate/deagglomerate. 

A.1.3 Aggregation and Agglomeration 

43. Primary particle size tends to be a relatively robust parameter as compared to 

aggregate/agglomerate size, less influenced by environmental conditions (pH, solvent, sonication, presence 

of proteins etc). In combination with other information on the nanomaterial, it may be correlated with 

Volume Specific Surface Area (VSSA) and nano-specific properties. Currently, no generally applicable 

guidelines for measurement of primary particle size and morphology are available. Primary particle size for 

metal based nanomaterials can be inferred from the unit crystal size as determined by powder x-ray 

diffraction. Otherwise, primary particle size is usually measured manually, more frequently by TEM. In 

specific cases, like suspensions of isolated nanoparticles, a semi-automatic measurement is feasible. 

Measurands are to be selected according to the method of measurement. In some research projects, 

including the EC’s Nanogenotox Joint Action, TEM-based methodology is developed and explored for 

primary particle measurement focusing among others on the selection of the measurands and on the 

randomized selection of particles to be measured. For aggregated nanomaterial with a fractal-like 

morphology, fractal dimensions can be calculated by combining results as proposed by Brasil et al. (1999). 

Artifacts can be examined and interpreted using advanced TEM techniques including cryo-EM (Adrian et 

al., 1984; Adrian et al., 1998), and electron tomography (Van Doren et al., 2011).. 

A.1.4 Chemical description (composition and identification) 

44. A thorough chemical description of the nanomaterials comprising both purity and coating and/or 

surface modification(s) is essential. This issue encompasses both technological and policy implications. It 

is likely that nanomaterial preparations will contain impurities and the nanomaterials might receive surface 

treatments or coatings designed to generate desirable interfacial properties (Alexandre and Dubois, 2000). 
 ENV/JM/MONO(2012)40 

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From an (eco) toxicological point of view, impurities may be relevant if they are present in sufficient 

quantity to elicit (eco) toxicological effects of a significant magnitude. If technically feasible, relevant 

impurities should be chemically identified in detail to determine the exact composition of the sample used 

for toxicology testing. E.g. nanomaterials may contain residues of catalysts or other materials used in their 

production. Although there are a number of standardised test guidelines addressing the purity issue, it may 

be necessary to adjust them to focus more on the issue of surface coatings. Additional guidance also may 

be needed to specifically address surface coated nanomaterials. The composition and expected reactivity of 

coatings should be taken into account when preparing the samples for nanomaterials composition and 

purity analysis. 

A.1.5 Specific surface area 

45. Standardised procedures are available for measuring particle specific surface areas (the accessible 

surface of a sample when exposed to either gaseous or liquid adsorbate, generally normalised to the sample 

mass) that are likely to be applicable to manufactured nanomaterials (e.g. BET 10 procedures, dye 

adsorption, negative ion adsorption, particle morphology etc.). However, in many cases, specific surface 

areas are the derived quantities depending on parameters, such as the nature of the probe adsorbate 

molecule (Klobes et al., 2006). In addition, investigators may wish to evaluate whether the particle size 

distributions (and surface areas) of sparingly soluble manufactured nanomaterials are altered through 

ripening and/or phase alteration phenomena (Ohman et al., 2006; Lefevre et al., 2006). Lastly, according to 

Klobes et al. (2006), the measurement of the specific surface area might most efficiently be conducted 

concurrently with measurements of pore size, pore size distribution, porosity and perhaps even particle 

density as these properties will most probably have an important influence on the (eco) toxicological 

properties of the material. As a complement and altenative, the determination of the volumetric specific 

surface area (VSSA) is a versatile characteristic that may allow to reduce the variability caused by different 

specific densities of different nanomaterials (Kreyling et al., 2010). 

A.1.6 Surface chemistry 

46. The surface properties of nanoparticles are critically important with respect to the agglomeration, 

aggregation, and toxicity behaviours. The expression surface chemistry (generally speaking, the chemical 

nature and composition of the outermost layers of the nanomaterial) may need to be considered in more 

detail or perhaps in a hierarchical manner, including coatings, functional groups, capping agents potential 

surface reactions in different media (e.g. redox reactions, coordination chemistry, catalysis). Thorough 

characterisation of the surface chemistry of a nanomaterial requires analysis of spectroscopy, interfacial 

analysis (ISO, 2008), toxicology (reactive oxygen species generation; Oberdörster et al., 2005; Nel et 

al.,2006; Balbus et al., 2007), surface complexation modelling (Loux and Savage, 2008), and colloid 

chemistry (colloidal particle stability; Shaw, 1992, Richman and Hutchinson 2009). Moreover, there are 

several reports showing that nanoparticles of the same core material, but with different surface coatings 

have quite different properties with respect to cell uptake and toxicological profiles (Goodman et al., 2004; 

Chithrani et al., 2006; Walkey et al., 2011; Suresh et al., 2012). 

47. In most applications (e.g. nanomedicine, drug delivery, teragnostic, fillers, rheological agents, 

inorganic and organic pigments, photocatalysts and anti-photocatalysts, fluorescence labelling, bio-sensors, 

diagnostics) controlling and tailoring the surface chemistry of nanomaterials is not only important but also 

required (Bui et al., 2010; Debouttière et al., 2006). As an example ZnO is surface treated for cosmetic 

applications to avoid photocatalytic activity. For instance, the possibility to synthesise core-shell 

nanostructures with well controlled surface functionalities will enable their broad application in the 

 

10 BET: Brunauer, Emmet and Teller. A classical method of determining surface area by measuring adsorption of a 

monolayer of an inert gas on the surface of a solid. 
ENV/JM/MONO(2012)40 

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biomedical science. The high level of interest in these types of multilayer nanostructures means that high 

sensitivity spectroscopic methods are developed able to characterise their surface chemistry to gain better 

insight into the chemical composition and elemental distribution (Grainger et al., 2008). For instance, 

techniques such as X-ray Photoelectron Spectroscopy (XPS) and Time of Flight Secondary Ion Mass 

Spectrometry (ToF-SIMS) have been successfully applied to characterize core-shell quantum dots (Zorn et 

al., 2011). Moreover, XPS, coupled with other techniques such as TEM, HREM, Raman and SEM, has 

been also used to investigate the surface chemistry of several nanomaterials ranging from gold (Au) 

nanoparticles (Techane et al., 2011), to carbon nanotubes (Yang et al., 2005), and from polymers (Suna et 

al., 2008) to metal oxides (Baer et al., 2007). 

48. The requirements for high-vacuum conditions of the conventional surface spectroscopy 

techniques impose a major limitation in analysing interfacial physicochemical processes at ambient 

conditions. Major efforts have been undertaken to overcome this so called “pressure-gap” to observe 

processes taking place at surfaces and interfaces under elevated or ambient gas pressure or liquid interfaces. 

For instance tremendous progress has been made in HRTEM and X-ray fluorescence spectroscopy (XRF) 

by means of the open environmental cells (E-cells) (Parson, 1974; de Jonge et al., 2011). Moreover, very 

recently, the E-cell has been adapted also to be used for the characterisation of nanomaterials by XPS. In 

particular, Kolmarov et al. (2011) have successfully used graphene oxide windows to study the surface 

composition of Au nanoparticles in liquid by means of XPS. Although this study has been conducted by 

using a synchrotron source, since the E-cell is compatible with the high vacuum, it can potentially be used 

with any commercial electron spectrometer, with no need of expensive differential pumped electron optics 

and high-flux synchrotron radiation. The sample can be kept in the cell after the XPS measurements and 

analysed also with other techniques such as SEM, TEM and Raman spectroscopy. This type of experiments, 

once standardized, will open a new chapter in the investigation of the surface and interface phenomena of 

nanomaterials. Another aspect of surface chemistry that may be particularly relevant is the surface acidity 

related to dissociation constants of surface ionisable sites. Ionisable sites may influence the surface charge 

which has been considered significant in toxicological studies (Oberdörster et al., 2005; Nel et al., 2006). 

Surface ionisation also may play a major role in colloidal particle stability (Shaw, 1992) and may even 

inhibit migration into hydrophobic phases (e.g. octanol/water partition coefficients). While surface 

chemistry is a complex property influenced by many parameters, surface ionisation will be discussed in 

more detail in this section, due to its significance. 

49. Many metal oxide nanomaterials possess surface ionisable sites (e.g. >SOH groups) that exhibit 

surface complexation reactions with the hydronium ion (and other soluble ions) of the following forms 

(where >SOH designates a reactive bound site): 

 

 

Historical mass action expressions that are sometimes used to describe these reactions are: 

 

and 

 
 ENV/JM/MONO(2012)40 

 29

where the subscripted K’s represent intrinsic acidity constants, the species in brackets represent the 

concentrations of reacting species, e is the charge of the proton, Ψ is the surface potential, k is the 

Boltzmann constant and T is the absolute temperature. 

50. These reactions are analogous to the acidity behaviour of a diprotic acid in aqueous solution with 

two exceptions: 1) the use of concentrations instead of chemical activities, and 2) the exponential 

Boltzmann term which converts bulk solution hydronium ion concentrations into interfacial values. The 

interfacial potential (Ψ) can be related to the zeta potential obtained from electrokinetic studies which in 

turn can in part be used to develop predictions of colloidal particle stability. In addition, geochemical 

speciation model predictions of surfacial ionised site concentrations can be used to interpret (eco) 

toxicological findings when the surface charge is considered a significant variable. 

51. There is a multitude of incompatible surface complexation models available for simulating these 

reactions (e.g. diffuse layer models; constant capacitance models; Gouy-Chapman-Stern-Graham models; 

triple layer models etc.), so although they display a vast potential for interpreting (eco)toxicological 

findings an agreed (set of) model(s) is yet to be developed. In addition, there is no general consensus 

within the environmental research community as to which model is demonstrably superior. The minimum 

dataset needed to utilise models of this nature then includes intrinsic acidity constants, site densities 

(requiring values for total concentrations of reactive sites and specific surface areas), solution composition 

and a multitude of binding constants useful for describing reactions with those dissolved ions commonly 

found in aqueous media. Some of these models (e.g. constant capacitance and triple layer models) may also 

require one or more capacitance terms. In summary, the area of surface complexation modelling would still 

need significant additional research and in the short term, it may be preferable to develop empirical 

datasets of the driving variables obtained from inquiries in this area: solution-composition-dependent 

surface charge densities and zeta potentials. 

A.1.7 Surface charge, zeta potential and Hamaker constant 

52. The toxicological role of surface charge is discussed in Oberdörster et al. (2005) and Nel et al.

(2006). The surface charge of manufactured nanomaterials in aqueous suspension will likely result from 

two phenomena: 1) isomorphic substitution of an ion with one valence by an ion of a different valence in 

the crystalline structure and 2) surface site reactions with the proton and other ions in aqueous solution 

(Loux and Savage, 2008; Hemraj-Benny et al., 2008). In particular, the surface charge of a given particle 

may be dependant both on pH and solution composition. Clearly, (eco) toxicologists conducting research 

in this area will need to ensure that this is measured and that the exact measurement conditions are given 

within the bounds of the fluid properties likely to occur in the medium of interest. 

53. Associated with a surface charge is a surface potential (in volts). The surface potential plays a 

major role in such phenomena as: 1) surface complexation with other ions in solution (Loux and Savage, 

2008), 2) interfacial pH and oxidation/reduction potentials (Loux and Anderson, 2001), and 3) the stability 

of colloidal particle dispersions in water (Shaw, 1992; Loux and Savage, 2008). Although it is difficult to 

measure the surface potential on nonconductive surfaces, it can be related to a zeta potential obtained from 

widely applicable electrokinetic procedures (Hunter, 1981; Delgado et al., 2007). It should be noted, 

however, that such procedures are based on models, which simplify the system to a certain degree, thus 

relating the surface potential to the zeta potential implies some model assumptions. If one can obtain 

dispersion-composition-dependent zeta potentials for particles in aqueous dispersions, one can 

subsequently employ Poisson-Boltzmann charge/potential relationships to obtain estimates of the charge 

density at the beginning of the diffuse layer, within certain model assumptions. In conjunction with a 

specific surface area measurement, one can then estimate a total charge on the surface. 
ENV/JM/MONO(2012)40 

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54. Along with the zeta potential, the Hamaker constant (which may be obtained from a variety of 

procedures; Visser, 1972; Bergstrom, 1997; Ackler et al., 1996) can be used to predict whether 

manufactured nanomaterials are likely to agglomerate in natural waters (Nowack and Bucheli, 2007; Loux 

and Savage, 2008). Predictions of this type of agglomeration will be limited to homogeneous 

agglomeration of the particles since the data needed to predict the deposition with a heterogeneous set of 

natural surfaces is often not available (e.g. Hamaker constants and zeta potentials); in general the rate of 

homo-aggregation is lower than that of hetero-aggregation. The shape and possibly size of resulting 

aggregate structures may vary too. In this case homo-aggregate studies are not easily used for real world 

hetero-aggregations processes. Please note that concentration of salts and other chemicals could affect this 

process. Agglomeration is considered to play a role in (eco) toxicological phenomena; this property may 

also be useful for toxicological interpretations since agglomeration induces the passage from the nano size 

level to a micrometric size which does not allow, for instance, the entrance into the cells. 

A.1.8 Influence of water chemistry on nanomaterial properties and dispersion behaviour 

55. Although not rigorously tested yet, Derjaguin-Landau-Verwey-Overbeek (DLVO) based theories 

exist for predicting the agglomeration behaviour of nanomaterial dispersions in water. For example, Shaw 

(1992), Ross and Morrison (1988) and Overbeek (1952) derived expressions for predicting the minimum 

ionic strength in water (or the Critical Coagulation Concentration [CCC]) needed to lead to the onset of 

room temperature colloidal particle agglomeration (Loux and Savage, 2008): 

 Shaw (1992): 

 

 

 

 

 

 Ross and Morrison (1988): 

 

 

 Overbeek (1952): 

 

 

 CCC – Critical coagulation conc. A – Hamaker constant (Joules) 

 k – Boltzmann constant z – counter ion valence 

Ψ – zeta potential T – absolute temperature 

 e – proton charge γ – (EXP(zeΨ/2kT)-1)/(EXP(zeΨ/2kT)+1) 
 ENV/JM/MONO(2012)40 

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56. Given values for both the zeta potential and Hamaker constant of a colloidal (or nano-) particle in 

aqueous dispersion, these calculated CCC values can be compared with the solution ionic strength (I; 

where I = 1/2Σ[cizi

2

]; ci is the concentration and zi is the valence of dissolved ion i) and predictions can be 

made as to whether these colloidal particles are likely to form agglomerates. By definition, this approach 

requires knowledge of the concentration of the major ions in the aqueous medium (i.e., the Water 

chemistry). This is usually defined in most test guidelines. 

57. Figure 1 compares estimated critical coagulation concentration (CCC) values obtained using the 

above three equations for particles with an absolute zeta potential of 0.025 V in a room temperature aquatic 

medium. Based on these simulations, particles with a Hamaker constant of 10-19 J (e.g. iron and aluminium 

oxides; Loux and Savage, 2008) are predicted to remain in a stable dispersion only in low ionic strength 

freshwaters and rainwater. In contrast, particles with a Hamaker constant of 10-21 J (e.g. some natural 

organic matter or organic matter coated particles; Loux and Savage, 2008) are predicted to remain in a 

stable dispersion even in hyper saline waters. Other possible influences on colloidal particle stability such 

as pH and organic matter are further discussed in parts C and D as well as in the introduction of this 

guidance document. It should be noted, however, that nanomaterials, which agglomerate – in agreement 

with DLVO-theory – can, under certain conditions, disagglomerate if the environment changes (Peters et 

al., 2012): silica nanoparticles aggregated at the simulated acid conditions of the human stomach, whereas 

disagglomeration occurred at simulated intestinal conditions at near neutral pH. 

Figure 1. Estimated critical coagulation concentration (CCC) values for a room temperature system with 

particles possessing zeta potential of ca. 0.025 V 

A.1.9 Preparation of liquid dispersions; octanol/water partition coefficients (Kows) 

58. As will be discussed later, particles suspended in fluids may well be governed by kinetic 

limitations rather than thermodynamic considerations and therefore, under these circumstances, the 

application of a standardised concept such as an octanol/water partition coefficient may be inappropriate in 

this type of system. Nevertheless, Kows play a key role in assessing the hydrophobicity of truly dissolved 

chemicals and it may be necessary to develop surrogate procedures to acquire information of this nature 

with liquid dispersions. 
ENV/JM/MONO(2012)40 

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59. It should be noted, however, that the partition coefficient has limitations for some materials, as 

has been shown for non-nanoscale materials, such as compounds that bind to proteins rather than to lipids. 

It should furthermore not be assumed that hydrophobicity is the only driving force behind accumulation 

and/or persistence; other mechanisms might dominate the effects on the nanometre scale. 

60. A point raised within workgroup deliberations concerned the need to definitively characterize 

solutions/dispersions used in (eco) toxicological studies. Specifically, there is no evidence to support (or 

refute) a contention that solutions (derived from parent nanomaterials), insoluble nanomaterial dispersions 

or dispersions of insoluble nanomaterial agglomerates and/or aggregates are likely to engender the same 

toxicological response. Hence, the lack of information of this nature may compromise the interpretation of 

any subsequently obtained (eco) toxicological results. 

A publication (Jafvert and Kulkarni, 2008) demonstrated great promise for extending the traditional 

concepts of equilibrium solubility/partitioning theory to buckminsterfullerene colloidal dispersions. It is 

not at all clear that similar successes will be achieved with larger, higher molecular weight manufactured 

nanomaterials. For example, Figure 2 illustrates the type of expected solubility behaviour for a compound 

immersed in a liquid as a function of time. Generally speaking, an undersaturated solution will dissolve 

more of the solute with time until it reaches a maximum value. In contrast, an oversaturated system (e.g. 

possibly obtained through the evaporation of solvent) will precipitate the solute onto particles with time 

until it reaches a minimum value. Therefore, with a compound exhibiting true thermodynamic solubility, 

one anticipates that one will observe a common equilibrium value in both under- and oversaturated systems 

given sufficient time. However, the required time to reach equilibrium may have implications for the 

environmental fate of these products because the kinetics of solubility phenomena is sensitive to the 

diffusive properties of the solute. In addition, it is known that very high molecular weight products can 

take extended time periods to reach equilibrium. Hence, even if a given liquid dispersion is being governed 

by thermodynamic equilibrium processes, the length of the equilibration period may be such that the local 

equilibrium assumption may be inappropriate in these systems, taking also into account that particles tend 

to accumulate at interfaces. On the other side, the surface treatment is a very important parameter when 

considering partitioning. There is literature on phase transfer of particles from aqueous to organic phases 

by changing the nature of the surface layer (Biondi et al., 2012). The particular case of Janus nanoparticles 

whose unique surface allows two different types of chemistry to occur on the same particle may pose 

additional challenges as well. 

Figure 2. Expected concentrations as a function of time for a system exhibiting true thermodynamic solubility 

behaviour. 
 ENV/JM/MONO(2012)40 

 33

61. There are standardised methods for estimating particle size distributions via the preparation of 

time-dependent aquatic suspensions in the field of soil science (e.g. Amezketa, 1999; Fristensky and 

Grismer, 2008) and the adaptation of these existing methodologies may prove to be an efficient means of 

achieving this goal. 

A.1.10 Crystal structure 

62. Standardised powder X-ray diffraction (XRD) procedures exist for determining the crystal 

structures of colloidal particles (at least larger ones). Analysis of physical-chemical properties also may 

provide valuable information in this area. As well as basic crystalline structure, XRD may be used to 

determine average crystallite size of crystalline materials (which for mono-crystalline primary particles 

also means average primary particle size), and may also give indications on particle morphology. XRD is 

useful for distinguishing different crystal phases of materials of the same chemical composition. In turn, 

this can provide insight into whether historical data can be used to further characterize a given material. A 

major concern however is whether a given manufactured nanomaterial has been derivatised; in particular, 

an amorphous surface coating will not be revealed by the use of x-ray diffraction (unless a significantly 

high volume of the amorphous material can be detected). In that case TEM will enable the amorphous 

layer to be seen. 

A.1.11 Interfacial tension 

63. Thus far, the discussion has focused largely on water insoluble nanomaterials. Some 

manufactured nanomaterials will likely exhibit sparingly soluble behaviour. If the solubility or the 

transformation in the aqueous media of a sparingly soluble nanomaterial leads to aqueous toxicant 

concentrations in excess of (eco)toxic levels, then these products may also be of concern e.g. ions released 

into solution. 

64. Due to their extremely small size, manufactured nanomaterials possess an extremely high 

specific surface area and, relatively speaking, also possess an extremely high fraction of atomic/molecular 

constituents on the surface (compared to the number of constituents contained internally). As these surface 

species have fewer bonds with adjacent species than do internal constituents, it takes less energy to remove 
ENV/JM/MONO(2012)40 

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surface species from the particle. A consequence of this phenomenon is that in comparison with bulk 

material of the same composition, many of these nanoparticulate species will have lower melting points 

and enhanced solubility or degradation in solvents. For nanomaterials that can degrade in solution, e.g. 

metal nanoparticles, a quantitative expression relating aqueous “solubility” to the particle specific surface 

area (SSA) of the solid phase and the solid/water interfacial tension (γ) is given below (Stumm and 

Morgan, 1981): 

log(Ksp, SSA) = log(Ksp, SSA=0) + (2/3)γ(SSA)/2.303RT 

where Ksp,SSA is the solubility product of a material with a specific surface area SSA, Ksp,SSA=0 is the 

solubility product of the bulk material, γ is the solid/water interfacial tension, R is the ideal gas constant 

and T is the absolute temperature. 

65. It should be noted that the equation given above holds only for pure water as a solvent but other 

aqueous solvents have other solubility products which are partly determined in the literature; this equation 

needs to be generalized. 

66. In most cases, finely divided materials are significantly more soluble than large, bulk products of 

the same composition or release ions into solution at a significantly faster rate. Similarly, with some 

materials, larger particles will grow larger at the expense of smaller particles in a given dispersion; the net 

result is that the aqueous solubility of a material also will decrease with time due to this phenomenon, also 

called Ostwald ripening. Within the context of the present discussion, many nanomaterials are likely to 

display enhanced aqueous solubilities when compared to the bulk material. Alternatively, given a value for 

the interfacial tension, one also can calculate the solubility of a nanomaterial provided that the temperature 

and solubility of the larger bulk material also is known. 

A.1.12 Dustiness 

67. The propensity of a material to become suspended in air is of interest when nanomaterials are 

manufactured or handled. The methods that are readily available were generally developed with an aim to 

assess the likelihood of workplace exposures to powders and were not designed with nanomaterials in 

mind (Hamelmann and Schmidt, 2003). An additional challenge is that many methods require a large mass 

of material which is often not available for nanomaterials (Ogura et al., 2009). It has been shown that some 

methods alter the starting nanomaterials and can fracture aggregated/agglomerated nanomaterials into 

smaller entities (NanoCare, 2009). The methods chosen will need to take these factors into account. The 

Vortex Shaker method has been specifically developed for nanomaterials. Other specific methods related 

to tests of agglomerate stability of nanomaterials are a method based on shear forces in a nozzle 

(Stahlmecke et al., 2009), the rotating drum and continuous drop method (e.g. Kuhlbusch et al., 2009; 

Perosh, Nanodustiness Project, 2012). Standardization of dustiness of nanomaterials is still ongoing and e.g. 

on topic of the EU-FP7 Project MARINA. 

A.1.13 Crystallite Size 

68. For crystalline materials, microscopy methods have commonly been used to assess crystallite size 

(which for mono-crystalline primary particles also means average primary particle size) including AFM, 

SEM and TEM. These methods may have limited utility for materials that are aggregated and 

agglomerated. Alternatively, diffraction methods may be used. The techniques used to prepare samples 

that are suitable for analysis can affect the measurement so the microscopy methods are most useful for 

durable nanomaterials that can withstand the preparation conditions. Also, the measured particles typically 

are laid on a surface. Most microscopic techniques do not provide a good statistical assessment and the 
 ENV/JM/MONO(2012)40 

 35

methods generally measure the properties of tens or hundreds of particles. Nevertheless, if the particles 

measured can be demonstrated to be representative of the larger sample these methods can provide useful 

information about the shape and surface structure of the particles in the sample. (e.g. 

http://www.malvern.com/labeng/products/iwtm/particle_size_analysis.htm). 

69. For nanomaterials that can be treated as spherical dynamic light scattering can be useful. DLS 

works best on particles in a medium, most frequently in a liquid medium. Thus medium effects have to be 

taken into account. The method should be used with caution for polydisperse nanomaterials (Calzolai et al., 

2011). Due to the fact that the aggregation and agglomeration processes are time-dependent and that the 

method cannot discriminate between primary particles and aggregates, the measurements of samples in 

solutions under the DLS may display time-dependent character. It is important to state what condition the 

solution of particles is expected to be in. In most cases equilibrium state is required. 

A.1.14 Electron Microscopy 

70. Applicable methods may include ISO TS 10797 for TEM and ISO TS 10798 for SEM. These 

methods have been assessed for carbon nanotubes and may have more general applicability for 

nanomaterials in general. As noted above, the techniques used to prepare a sample that are suitable for EM 

analysis can affect the measurement so the microscopy methods are most useful for durable nanomaterials 

that can withstand the preparation conditions. Most microscopic techniques do not provide a good 

statistical assessment and the methods generally measure the properties of tens or hundreds of particles. 

Nevertheless, if the particles measured can be demonstrated to be representative of the larger sample these 

methods can provide useful information. Other more advanced electron microscopy techniques such as 

Electron Tomography (ET), Atomic Force Microscopy (AFM) or Scanning Transmission Electron 

Microscopy (STEM. A. Engel, Biozentrum Basel) should also be considered. Detailed protocols on how to 

measure ‘Photocatalytic Activity’ and ‘Photocatalytic Radical Formation Potential’ are given in JRC 

Report, 2011. 

71. In some cases, SEM can be used to characterise/calibrate another technique that may be much 

more productive (DLS for example). The SEM would give a good indication of shape of particles and the 

second technique would give much more statistical account of the sample. 

72. Recent developments in scanning electron microscopy applying the transmission mode (STEM) 

have demonstrated traceable measurements of number-based nanoparticle size distributions. The analysis 

is performed on per-particle level and (semi-)automatic image analysis tools help to improve statistics 

(Buhr et al., 2009, Klein et al., 2011). 

A.1.15 Photocatalytic Activity 

73. The photocatalytic activity gives an indication of the potential for transformations in the 

environment which in turn represents an important point of concern when evaluating the full life-cycle of 

the nanomaterial. On the other side, photocatalytic activity is an extremely important technical 

specification for nanomaterials that have this characteristic. The surface treatment is critical in this field 

and information on it is essential to understand the behaviour of the nanomaterial under consideration (See 

http://uscc.dreamscapesdesigners.net/documents/Photocatalytic.pdf). The photocatalytic activity of a 

substance can be characterized qualitatively by describing relevant observations or by reporting one or 

more specific photocatalytic chemical reaction equations. Photocatalytic activity can also be expressed by 

giving a quantitative value of the moles converted per g of sample per s of irradiation. It is also possible to 

give a microscopic description of the photocatalytic activity by reporting the turn-over frequency (TOF, 
ENV/JM/MONO(2012)40 

 36

unit: 1/s), which is the maximum number of molecules converted per catalytic site per second. When 

reporting quantitative data it is necessary to specify the light source (wavelength) and the irradiation power 

in the method description or in the SOP. When comparing photocatalytic activities of different 

nanomaterials, it is advantageous to use an assay which is recognized also for other particulate substances, 

which are not necessarily nanomaterials. One example is the zero order rate of the photocatalytic oxidation 

(reactive oxygen species) of liquid propan-2-ol to propanone, under oxygenated conditions for a fixed mass 

of particulate, as has been done for particulate ZnO. See also: SCCNFP/0649/03 The Scientific Committee 

On Cosmetic Products And Non-Food Products Intended For Consumers Opinion Concerning Zinc Oxide 

[http://ec.europa.eu/health/ph_risk/committees/sccp/documents/out222_en.pdf and 

http://sourcedb.ipe.cas.cn/zw/lwlb/200908/P020090901337110041200.pdf]. 

A.1.16 Pour Density 

74. The 'pour density' is a useful parameter for estimating the weight-to-volume ratio of a material; it 

is often used to estimate technical settings in industrial processes. It is defined as the apparent density of a 

bed of material formed in a container of standard dimensions when a specified amount of the material is 

introduced without settling. 

75. Some information about pour density measurements (of carbon black) is available in the ASTM 

Standard D1513 – 05e2. 

A.1.17 Porosity 

76. Porosity may be determined using the Barrett-Joyner-Halenda (BJH) method of analysis of 

adsorption and desorption isotherms to determine pore area, specific pore volume and pore size distribution 

independent of external area due to the particle size of the sample. The t-plot method is commonly used to 

determine the external surface area, pore volume and pore surface area in microporous solids (European 

Commission, 2011). 

A.1.18 Octanol-Water Partition Coefficient 

77. The n-octanol/water partition coefficient (KOW) is a key physico-chemical parameter that is used 

in numerous estimation models and algorithms for environmental partitioning, sorption, bioavailability, 

bioconcentration/bioaccumulation and as well for human toxicity and ecotoxicity. Traditional octanolwater partition coefficient methods might only be applicable under some circumstances to some classes of 

manufactured nanomaterials (OECD 2009,). However, traditional and alternative methods to address 

insoluble or partially soluble nanoparticles should continue to be monitored for their applicability to 

nanomaterials. 

78. The octanol-water partition coefficient determination is based on a test that measures the 

distribution of a chemical between two immiscible liquid phases. Correlations are then used to predict the 

effects of a new chemical (using structure activity relationships, for example) for which a KOW could be 

measured. KOW is a key step in determining the fate of chemicals as well as assessing their (eco) 

toxicological risks (OECD, 1995, 2004, 2006). However, while these guidelines are applicable to watersoluble nanomaterials, there are caveats in applying these guidelines to insoluble nanomaterials due to 

factors such as the aggregation and accumulation of nanomaterials at phase interfaces. See also paragraph 
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 37

92 and following as well as paragraphs 106, 109 (and, in general the general and especific sections on 

sample preparation) in these Guidances. 

79. Petersen, et al. (2010) tested traditional KOW methods, with modifications for carbon nanotubes, 

and concluded that these methods are not appropriate for generating KOW values that would be predictive of 

MWCNT(Multi Wall Carbon Nano Tube) bioaccumulation in sediment species. A lack of MWCNT 

transport across interfacial boundaries between octanol and water, and other complications, were 

encountered. Hristovski et al. (2012) have suggested additional modifications of the traditional KOW test 

methods that may make them predictive of fate, bioavailability and transport of some nanomaterials in the 

environment. They addressed the tendency of some nanomaterials to distribute to the octanol-water 

interface in test systems by developing additional distribution coefficients that increased the predictive 

nature of the tests on silver, fullerene, and hematite nanoparticles. Others (Xiao and Weisner, 2012) have 

examined methods that are not based on traditional approaches, such as those used by OECD, to measure 

nanoparticle hydrophobicity. They have applied several alternative methods developed, for example for 

nanoparticle pharmaceutical testing (including organic dye adsorption experiments), to fullerene- and 

silver-based nanoparticles. 

A.1.19 Radical Formation Potential 

80. The generation of radicals such as reactive oxygen species in vitro (ROS) has been associated 

with toxicity in in vivo experiments. ROS generation is correlated with oxidative stress, particularly via 

the inhalation route, and can lead to inflammation and cytotoxicity. ROS include singlet oxygen, 

superoxide, hydrogen peroxide, and hydroxyl radicals. These species can be generated at the particle 

surface due to factors including structural defects resulting in altered electronic properties, the presence of 

transition metals on the surface, and the photoactivation of electron hole pairs. Many of these points are 

summarized in Nel et al., 2006. 

81. There are at least four acellular assays for radical formation potential that have been tested for 

their applicability to nanomaterials, but they have not yet undergone extensive standardization. A vitamin 

C yellowing assay that measures the chemical reactivity of nanomaterials toward a vitamin C derivative as 

an antioxidant has been used to assess titanium dioxide, quartz and clay (Warheit, et al., 2010). A broad 

range of nanoparticles was assessed for intrinsic radical electron inducing capacity utilizing a 

dichlorofluorescin-based dye, which is used to detect ROS and reactive nitrogen species (Rushton, et. al, 

2010). The FRAS (ferric reducing ability of serum) assay uses human blood serum as a reagent medium in 

an assay that measures changes in the total antioxidant capacity of the serum, and has been tested with both 

particulate nanoparticles and carbon nanotubes (Bello, et al., 2009). A fourth assay has been used to assess 

ROS formation in the presence of U.V. light: the thiobarbituric acid reactive substance assay was used to 

assess ROS formation of titania in the presence of U.V. light (Sanders, et. al. 2012). 

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Harmonised 

Van Doren E., De Temmerman P-J., Francisco M. and Mast J. (2011), “Determination of the volumespecific surface area by using transmission electron tomography for characterization and definition 

of nanomaterials”. Journal of Nanobiotechnology 9(1), pp.17. 

Visser, J. (1972), “On Hamaker Constants: A comparison between Hamaker constants and Lifshitz-Van 

der Waals constants”. Advances in Colloid and Interface Science 3, pp.331-363. 

Walkey, C. D., Olsen, J. B., Guo, H., Emili, A. and Chan, W. C. W. (2011), “A Combination of 

Nanoparticle Size and Surface Chemistry Determines Serum Protein Adsorption and Macrophage 

Uptake”. Journal of the American Chemical Society 134(4), pp.2139-47. 

Wang, X., Xia, T., Ntim, S. A., Ji, Z., George, S., Meng, H., Zhang, H., Castranova, V., Mitra, S., and Nel, 

A.E. (2010), “Quantitative techniques for assessing and controlling the dispersion state and 

biological effects of multi-walled carbon nanotubes in mammalian tissue culture cells”, ACS Nano, 

4(12), pp. 7241-7252. 

Warheit, D.B., Borm, P.J.A., Hennes, C. And Lademann, J. (2007), “Testing strategies to establish the 

safety of nanomaterials: Conclusions of an ECETOC workshop”, Inhalation Technology 19, pp.631-

643. 
ENV/JM/MONO(2012)40 

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Wittmaack, K. (2011a), “Eccessive delivery of nanostructured matter to submersed cells caused by rapid 

gravitational settling”, ACS Nano 5(5), pp. 3766-3778. 

Wittmaack, K. (2011b), “Novel dose metric for apparent cytotoxicity effects generated by in vitro cell 

exposure to silica nanoparticles”, Chemical Research in Toxicology 24, pp.150-58. 

Xiao, Y and Wiesner M. (2012), “Characterization of surface hydrophobicity of engineered nanoparticles”, 

Journal of Hazardous Materials 215-216, pp.146-151. 

Yang, D-Q., Rochette, J.F., and Sacher, E. (2005), “Controlled Chemical Functionalization of Multiwalled 

Carbon Nanotubes by Kiloelectronvolt Argon Ion Treatment and Air Exposure”, Langmuir 21, 

pp.8539-45. 

Zhu, S., Oberdorster, E., and Haasch, M.L. (2006), “Toxicity of engineered nanoparticle (fullerene, C60) 

in two aquatic species, Daphnia and fathead minnow”, Marine Environmental Research 62 Suppl, 

pp.S5-S9. 

Zorn, G., Dave, S. R., Gao, X. and Castner, D.G. (2011), “Method for determining the elemental 

composition and distribution in semiconductor core-shell quantum dots”, Analytical Chemistry 83, 

pp.866-873. 
 ENV/JM/MONO(2012)40 

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B. GUIDANCE ON PREPARING SAMPLES OF NANOMATERIAL IN EXPOSURE MEDIA FOR 

ECOTOXICITY STUDIES 

B.1 Introduction 

82. There is currently no broad consensus on the best approaches for preparing nanomaterial samples 

in media for ecotoxicity studies. However, recent publications describe evaluations of preparation methods 

for specific nanomaterials (e.g. nano-silver), suggested operating procedures, and the use of natural 

dispersants rather than pure solvents as dispersion or suspension agents (El Badawy 2010, Allen 2010, 

Kennedy et al., 2008). Review of the literature also reveals that a wide range of suspension methods 

continue to be used and include use of strong solvents (e.g. tetrahydrofuran, THF), dispersion or stabilising 

agents (e.g. TWEENTM, citrate, etc.), bath or probe ultrasonication, stirring for a broad range of time 

periods, bead milling, etc (Klaine et al., 2008, Handy et al., 2008, Hund-Rinke et al., 2010, Handy et al., 

2011). This observation is confirmed by a recent survey of international nanomaterial researchers 

completed by Environment Canada (based on preliminary summary of responses, January 2012). Adding 

to the difficulty in evaluating these various methods is the evidence that some solvents may alter 

nanomaterial properties and toxicity (Smith et al., 2007) or even be toxic themselves (Henry et al., 2007). 

Moreover, in most cases, characterisation of the tested nanomaterials has been limited to the working or 

stock dispersions, rather than in the post-dilution exposure media or directly in the test vessels. There is 

also some probability that the methods reported might not produce similar results for all nanomaterials, or 

forms of a specific nanomaterial, for example anatase dominated or rutile dominated titanium dioxide, or 

surface and non-surface treated titanium dioxide. Finally, these evaluations will, in some cases, be strongly 

dependant on the characterization methods used, e.g. DLS might give different answers compared to TEM 

or other microscopy approaches. For all of these reasons, preparation of exposure media for ecotoxicity 

testing cannot yet be highly standardized and should continue to be viewed as experimental. 

83. Variability in nanomaterial properties, most notably agglomerate or aggregate size, has been 

shown to depend consistently and significantly on media pH, ionic strength, and concentration and form of 

dissolved organic matter (El Badawy et al., 2010, Domingos et al., 2009 and French et al., 2009). Even 

within the narrow dissolved ion range of 3.4 to 13.3 mmol/L, well within the range typical of freshwaters, 

agglomerate or aggregate size can vary two to three fold (French et al., 2009, Stolpe and Hassellöv, 2007). 

Because total surface area for a given volume of material increases as a square function of decreasing 

particle size, and the interaction of nanomaterials and biotic systems occur at the particle surface, these 

ionic strength effects have clear implications for exposure in ecotoxicity testing of nanomaterials. For 

dissolved organic matter (DOM), the extensive literature describing its interactions with chemical 

contaminants (e.g. metals) suggests that nanomaterial properties, fate, and toxicity might be strongly 

affected by these substances (Chen et al., 2011). For example, DOM has been shown in several studies to 

stabilise particles in suspension, and to reduce the agglomeration or aggregation phenomena (Hyung et al., 

2007, Loux and Savage 2008). The interaction of nanomaterials and DOM might also alter bioavailability 

and rates of uptake by test organisms (Duval and Qian, 2009). It is also important to recognise that DOM 

itself is highly variable among water sources, is difficult to characterize, and can contain a broad range of 

aromatic, lipid, protein, and other constituents; all factors that are likely to produce variation in their effects 

on nanomaterials. 

84. An additional concern is the degradation of nanomaterials during testing, most importantly the 

release of soluble species (e.g. ions) from solid surfaces. This is particularly important where soluble 
ENV/JM/MONO(2012)40 

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species (e.g. ions released) are especially toxic, or in close proximity to potential biological targets, as is 

the case with silver (Ag+

) ions released from silver nanoparticles. Ionicsilver is highly toxic to aquatic 

species, with effect levels in the low μg/L range; therefore even relatively slow release of Ag+

 from silver 

nanoparticles can results in toxic levels of these species. Degradation of both nano-scale silver and ZnO2

has been well-documented (Allen et al., 2010, Kennedy et al., 2010, Liu and Hurt 2010, Franklin et al., 

2007) and is likely an issue for nano-scale copper (Heilaan et al., 2008). 

85. Control and measurement of these factors is highly desirable to assure that outcomes of research 

projects, i.e. data are as consistent and comparable as possible. Every effort should be made to quantify 

these factors at time intervals sufficient to fully describe exposure conditions, most importantly their effect 

on bulk concentration and particle size over the duration of testing. Where possible, it is also suggested that 

additional efforts be made to determine how these factors affect particle behaviour and properties. For 

example, simple beaker tests might be done over a range of pH values typical for a test system to quantify 

the effects on particle size (See also the Guidance Manual for the Testing of Manufactured 

Nanomaterials: OECD’s Sponsorship Programme: First Revision ENV/JM/MONO(2009)20/REV). In the 

absence of broadly applicable methods for producing exposure media for biotic effects assessment, the 

following advice is generally intended to minimise intra- and inter-laboratory variability within 

sponsorship groups. 

86. Finally, it is strongly suggested that where research involves both ecotoxicity testing and 

environmental behaviour, degradation, transformation, and bioaccumulation (Section C of this document) 

common methods for media preparation be used. This approach will maximize comparability and 

integration of exposure and effects studies by increasing the probability that nanomaterials have similar 

suspension properties, at least at the initiation of each type of test. There also may be circumstances where 

well-vetted methods for media preparation exist for fate-related tests, but not for ecotoxicity tests. Due to 

the variability and uncertainty in the real exposure levels, even under controlled laboratory conditions, 

measuring and reporting exposure in terms of internal doses and/or body burdens at the end of the exposure 

period should be considered as a complementary option. Section C. 2.3 Bioaccumulation offers 

recommendations for measuring the level of nanoparticles or its transformation products in biota. 

B.2 Aquatic Media Preparation 

B.2.1 Methods of suspension 

87. Dispersion of nanomaterial might include stirring, sonication, grinding, use of solvents, and 

stabilising agents. The advantages and disadvantages of these methods are outlined in Crane et al. (2008) 

and Handy et al. (2011). This suggested that some nanomaterials are significantly altered by sonication and 

grinding (e.g. carbon nanotubes can be shortened, coatings can be removed, surfaces can be hydroxylated, 

etc.) and that the interaction of solvents with some nanomaterials might result in toxic by-products. The 

method of dispersion will also depend on the specific material to be tested and whether or not it has been 

surface treated. Best scientific judgment should be used in selecting the methods, and where there is 

evidence or an indication that a dispersion method might significantly alter toxicity, those effects should be 

controlled or quantified. The general goal of dispersion efforts is to produce consistent particle sizes with 

predictable polydispersity. It is unlikely that these properties will be stable over renewal periods (or the 

duration of a test); rather, the goal should be to have a repeatable initial test medium, and to monitor 

property changes with sufficient frequency to quantify these changes. In some cases, in order to remove the 

larger particles, filtration with 0.45 μm or 0.22 μm filters might be employed (e.g. Ma and Bouchard, 

2009). This approach should be altered where information is available concerning environmentally relevant 

particle sizes. This might include manufacturer information on specific sizes produced and incorporated 

into products, and unlikely to undergo post-production processes that might alter particle size or 

distribution. It is assumed that tests will be conducted using periodic renewal approaches to avoid expense 
 ENV/JM/MONO(2012)40 

 49

and waste production. Test media quality (pH, ionic strength, DOM concentration) should be harmonised 

as much as possible between comparative studies. One source of Standard Operating Procedures is the US 

Center for the Environmental Implications Nanotechnology (CEINT). In collaboration with the US 

National Institute for Standards and Technology (NIST), SOPs for sonication and suspension of TiO2 have 

been made available (CEINTa-d, http://www.ceint.duke.edu/research/transport-and-transformations, last 

accessed 07 November, 2011). 

B.2.2 Media quality 

88. It is essential that pH, ionic strength, and DOM concentrations be quantified and made as 

uniform as possible among tests (and replicates), endpoint measurements, and laboratories. Careful 

consideration should be given to assuring that physical-chemical properties determinations are 

representative of all other test conditions, e.g. a full suite of physicochemical determination of a material in 

de-ionised water are very unlikely to accurately predict many of those values in high ionic strength media. 

Accordingly, it is recommended that physico-chemical characterisation be made in the actual test media, 

whenever possible, (Antunovic et al., 2011). Media quality determinations should be made at intervals 

sufficient to determine their variability, both in stock solutions and in test media. Typically, intervals 

described (and the methods used) in current guidelines should be sufficient, however, more determinations 

should be made where there is evidence or indication of increased variability, perhaps due to tested 

nanomaterials, e.g. interaction of DOM with fullerenes or carbon nanotubes. See also the paragraph 87. 

B.2.3 Physical-Chemical Characterisations 

89. It is likely that agglomeration will occur during most toxicity tests and will alter and likely reduce 

test organism exposure either due to reduced particles counts, surface area, or loss of bulk concentration. 

For this reason, particle and/or agglomerate size distribution and material concentration should be assessed 

at intervals sufficient to quantify exposures. Because few nanomaterial studies have measured these 

parameters across dilution series or at intervals during exposure, it is difficult to prescribe a specific 

approach. However, at a minimum and where possible, these determinations should be made immediately 

prior to, and after, media renewal. It is also desirable to measure particle/aggregate/agglomerate size 

distribution using two or more methods, e.g. dynamic light scattering and SEM, TEM (possibly cryo-TEM), 

and other microscopy techniques. Where possible, it is also suggested that the size-determination methods 

differ in the approach used, e.g. DLS measures size based on hydrodynamic diameter and electrophoretic 

mobility (and can bias size determination due to charge layering), whereas microscopy provides for direct 

observation and visual measurement of physical size, but limits the size of particle population that can be 

measured. [See section A.1.1 regarding comparability of results using different methods and annex III of 

the Guidance Manual for the Testing of Manufactured Nanomaterials: OECD’s Sponsorship Programme 

ENV/JM/MONO(2009)20 ]. These confirmatory measurements might be made in preliminary studies with 

the test media, or in a subset of tests or treatment levels to establish the comparability of measurement 

techniques. These measurements should also be taken in media in the presence of test organisms (and food 

when feeding is required) as they will likely alter particle behaviourconsiderably... Finally, it is recognised 

that many physicochemical properties cannot be determined in wet media, most notably surface area, 

which relies upon dry samples. Refer to Section A of this document for more in-depth discussion of 

characterization approaches. 

B.2.4 Reporting results for media preparation approaches 

90 bis. It is important that results of various media preparation approaches be reported in detail. 

Negative results, i.e. cases where preparation methods lead to excessive agglomeration or complete failure 

in exposure, are as important as positive results in providing the basis for future specific guidance for 

testing. 
ENV/JM/MONO(2012)40 

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B.3 Non-Aqueous Media Preparation 

B.3.1 Method of nanomaterial introduction 

90. This section covers media preparation for all non-aqueous tests, including sediment (e.g. OECD 

TG 218), soil (e.g. OECD TG 222), dung (e.g. OECD TG 228), and direct application (e.g. OECD TG 214) 

testing. Given the lack of methods for detecting or quantifying many nanomaterials in these more complex 

media, these tests will necessarily be relatively more exploratory compared with aquatic testing. Materials 

may be delivered to test media in the form of water-based dispersions or mixed as dry material. An 

advantage of using wet suspensions is that the starting point for material addition can be made uniform 

between aquatic and sediment or soil testing. If the nanomaterial is introduced and homogenised directly in 

solid form to the media, care should be taken in homogenisation so that the test material is not 

unintentionally damaged, and detailed description of the methods should include homogeniser type, speeds 

or force applied, and duration. In either wet or dry application, the problem of characterizing materials in 

complex media such as soil, sediment, or sludge, has yet to be resolved. However, one exception is 

extraction and analysis of SWCNTs using solvents and near infrared fluorescence spectroscopy (Schierz et 

al., 2011). TEM has also been used to characterize materials in solid test matrices (e.g. Kool et al., 2011). 

B.3.2 Media quality 

91. All of the media quality issues discussed for aquatic tests apply mutatis mutandi as well to 

preparation of dispersions for delivery to other media. In addition, all guidance on characterising these 

media described in appropriate OECD test guidelines should be followed. Best scientific judgment should 

be used in determining whether nanomaterial testing might require additional or more frequent 

measurement. All efforts should be made to minimise variation in these media variables between tests and 

sponsor laboratories. One approach to address this issue is to homogenise and distribute natural media 

among all researchers, or to use a single batch of laboratory-constructed media, following test guideline 

recipes and procedures. 

B.3.3 Physical-Chemical Characterisations 

92. It is recognised that methods for many physical-chemical properties, most importantly, particle 

size, have yet to be developed for complex media. However, where possible, and using best scientific 

judgment, methods for doing so should be investigated, e.g. identifying and perhaps measuring carbon 

nanotubes using microscopy techniques. Where methods exist for digesting or extracting materials for 

determination of bulk concentration, these measurements should be made. The intervals for such 

measurement should be sufficient to document accurate and consistent delivery of materials to test media. 

B.3.4 Reporting results for media preparation approaches 

93. It is important that results of various media preparation approaches be reported in detail. 

Negative results, i.e. cases where preparation methods lead to excessive agglomeration or complete failure 

in exposure, are as important as positive results in providing the basis for future specific guidance for 

testing. 

B.4 References 

Allen, H.J., Impellitteri, C.A., Macke, D.A., Heckman, J.L., Poynton, H.C., Lazorchak, J.M., 

Govindaswamy, S., Roose, D.L., and Nadagouda, M. (2011), “Effects from filtration, capping agents, 
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and presence/absence of food on the toxicity of silver nanoparticles to Daphnia magna”, 

Environmental Toxicology and Chemistry 29, pp.2742–2750. 

Andrievsky, G.V., Kosevich, M.V., Vovk, O.M., Shelkovsky, V.S., and Vashenko, L.A. (1995), “On the 

production of an aqueous colloidal solution of fullerenes”, Journal of the Chemical Society, 

Chemical Communications 12, pp.1281–1282. 

Antunović, B., Barlow, S., Chesson, A., Flynn, A., Hardy, A., Jany, K-D., Jeger, M-J., Knaap, A., Kuiper, 

H., Larsen, J-C., Lovell, D., Noerrung, B., Schlatter, J., Silano, V., Smulders, S. and Vannier, P. 

(2011), “Guidance on the risk assessment of the application of nanoscience and nanotechnologies in 

the food and feed chain”, EFSA Journal 9, pp.2140. 

Brant, J.A., Labille, J., Bottero, J-Y., and Wiesner, M.R. (2006), “Characterising the impact of preparation 

method on fullerene cluster structure and chemistry”, Langmuir 22, pp.3878–3885. 

Brant, J., Lecoanet, H., Hotze, M., and Wiesner, M. (2005), “Comparison of electrokinetic properties of 

colloidal fullerenes (n-C60) formed using two procedures”, Environmental Science and Technology 

39, pp.6343–6351. 

CEINT/ NIST (a). Protocol for Preparation of Nanoparticle Dispersions from Powdered Material Using 

Ultrasonic Disruption, Ver.1.1, www.ceint.duke.edu/sites/ceint.duke.edu/files/NIST.SP_.1200-

2.pdf .( Accessd on 07 November, 2011) 

CEINT/ NIST (b). Reporting Guidelines for the Prepartion of Aqueous Nanoparticle Dispersions from Dry 

Nanomaterials, Ver.2.1, www.ceint.duke.edu/sites/ceint.duke.edu/files/NIST.SP_.1200-

1.pdf .(Accessed in September, 2012). 

CEINT/ NIST (c). Protocol for Preparation of Nanoscale TiO2 Aqueous Dispersion for Toxicological or 

Environmental Testing, Ver.1.2, www.ceint.duke.edu/sites/ceint.duke.edu/files/NIST.SP_.1200-

3.pdf. (Accessed in September, 2012) 

CEINT/ NIST(d). Protocol for Preparation of Nanoscale TiO2 Dispersions in Biological Test Media for 

Toxicological Assessment, Ver.1.1, www.ceint.duke.edu/sites/ceint.duke.edu/files/NIST.SP_.1200-

3.pdf. (Accessed in September, 2012) 

Crane, M., Handy, R.D., Garrod, J., and Owen, R. (2008), “Ecotoxicity test methods and environmental 

hazard assessment for engineered nanoparticles”, Ecotoxicology 17(5), pp.421-37. 

Domingos, R.F., Tufenkji, N., and Wilkinson, K.J. (2009), “Aggregation of titanium dioxide nanoparticles: 

Role of fulvic acid”, Environmental Science and Technology 43, pp.1282-1286. 

El Badawy, A.M., Luxton, T.P., Silva, R.G., Scheckel, K.G., Suidan, M.T., and Tolaymat, T.M. (2010), 

“Impact of environmental conditions (pH, ionic strength, and electrolyte type) on the surface charge 

and aggregation of silver nanoparticles suspensions”, Environmental Science and Technology 44: 

pp.1260–1266. 

EPA (2010), “State of the Science Literature Review: Everything Nanosilver and More”, Sientific, 

Technical Research, Engineering and Modeling Support Final Report, Contract No. EP-C-05-057 

Task Order No. 95, Washington, D.C. 

Franklin, N.M., Rogers, N.J., Apte, S.C., Batley, G.E., Gadd, G.E., and Casey, P.S. (2007), “Comparative 

Toxicity of Nanoparticulate ZnO, Bulk ZnO, and ZnCl2 to a Freshwater Microalga 
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(Pseudokirchneriella subcapitata): The Importance of Particle Solubility”, Environmental Science 

and Technology 41 (24), pp. 8484–8490. 

French, R.A., Jacobson, A.R., Kim, B., Isley, S.L., Penn, R.L., and Baveye, P.C. (2009), “Influence of 

ionic strength, pH, and cation valence on aggregation kinetics of titanium dioxide nanoparticles”, 

Environmental Science and Technology 43,pp.1354–1359. 

Handy, R.D., Cornelis, G., Fernandes, T., Tsyusko, O., Decho, A., Sabo-Attwood, T., Metcalfe, C., 

Steevens, J.A., Klaine, S.J., Koelmans, A.A., and Horne, N. (2012), “Ecotoxicity test methods for 

engineered nanomaterials: Practical experience and recommendations from the bench”, 

Environmental Toxicology and Chemistry 31, pp.15–31. 

Handy, R.D., Kammer, F., Lead, J.R., Hassellöv, M., Owen, R., and Crane, M. (2008), “The ecotoxicology 

and chemistry of manufactured nanoparticles”, Ecotoxicology 17, pp.287-314. 

Heinlaan, M., Ivask, A., Blinova, I., and Dubourguier, H-C. (2008), “Toxicity of nanosized and bulk ZnO, 

CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus 

platyurus”, Chemosphere 71, pp.1308–1316. 

Henry, T.B., Menn, F., Fleming, J.T., Wilgus, J., Compton, R.N., and Sayler, G.S. (2007), “Attributing 

effects of aqueous C60 nano-aggregates to tetrahydrofuran decomposition products in larval 

zebrafish by assessment of gene expression”, Environmental Health Perspectives115, pp.1059-1065 

Hund-Rinke, K., Schlich, K., and Wenzel, A. (2010), “TiO2 nanoparticles - Relationship between 

dispersion preparation method and ecotoxicity in the algal growth test”, Umweltwiss Schadst Forsch

22, pp.517 - 528. 

Hyung, H., Fortner, J.D., Hughes, J.B., and Kim, J-H. (2007), “Natural organic matter stabilises carbon 

nanotubes in the aqueous phase”, Environmental Science and Technology 41, pp.179–184. 

Kennedy, A.J.H., Hull, M.S., Steevens, J.A., Dontsova, K.M., Chappell, M.A., Gunter, J.C., and Weiss, 

C.A. Jr. (2008), “Factors influencing the partitioning and toxicity of nanotubes in the aquatic 

environment”, Environmental Toxicology and Chemistry 27, pp.1932–1941. 

Kennedy, A.J., Hull, M.S., Bednar, A.J., Goss, J.D., and Gunter, J.C. (2010), “Fractionating nanosilver: 

importance for determining toxicity to aquatic test organisms”, Environmental Science and 

Technology 44, pp. 9571-9577. 

Klaine, S.J., Avarez, P.J.J., Batley, G.E., Fernandes, T.F., Handy, R.D., Lyon, D.Y., Mahendra, 

McLaughlin, M.J., and Lead, J.R. (2008), “Nanomaterials in the environment: Behavior, fate, 

bioavilability, and effects”, Environmental Toxicology and Chemistry 27, pp.1825–1851. 

Liu, J., and Hurt, R.H. (2010), “Ion release kinetics and particle persistence in aqueous nano-silver 

colloids”. Environmental Science and Technology 44, pp. 2169-2175. 

Loux, N.T., and Savage, N. (2008), “An assessment of the fate of metal oxide nanomaterials in porous 

media”, Water, Air, and Soil Pollution 194, pp.227-241. 

Ma, X., and Bouchard, D. (2009), “Formation of aqueous suspensions of fullerenes”, Environmental 

Science and Technology 43, pp.330-336. 
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Schierz, P., Parks, A.N., and Ferguson, P.L. (2011), Abstract: Novel Approaches for the Detection of 

Single Walled Carbon Nanotubes in Environmental Matrices, Society for Environmental Toxicology 

and Checmistry, North America 32nd Annual Meeting. 

Smith, C.J., Shaw, B.J., and Handy, R.D. (2007), “Toxicity of single-walled carbon nanotubes on rainbow 

trout (Oncorhyncus mykiss): Respiratory toxicity, organ pathologies, and other physiological 

effects”, Aquatic Toxicology 82, pp.94-109 

Stolpe, B., and Hassellöv, M. (2007), “Changes in size distribution of fresh water nanoscale colloidal 

matter and associated elements on mixing with seawater”, Geochimica et Cosmochimica Acta 71, pp. 

3292–3301 
ENV/JM/MONO(2012)40 

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C. GUIDANCE ON PREPARING NANOMATERIAL SAMPLES FOR DEGRADATION, 

TRANSFORMATION AND ACCUMULATION STUDIES 

C. 1 Introduction 

94. This part presents a scan of the existing published literature on environmental behaviour and fate 

studies. 

C. 1.1 Environmental behaviour 

95. Regarding the currently tested nanomaterials it is highly likely that they will not stay dispersed 

in natural freshwaters (Klaine et al., 2008). The tendency to undergo agglomeration or aggregation is 

however dependent on so many parameters that particles need to be tested for their agglomeration or 

aggregation behaviour until methods are available which are able to assist or replace the testing by 

modelling based on first principles. However, there have also been recent indications that nanomaterial 

agglomerates or aggregates may stay dispersed in other aquatic conditions and in high concentrations of 

OM some nanomaterial can de-agglomerate. Moreover also the settled nanoparticles attached to the 

suspended particulate matter may not be deposited but will travel along with the other particulate material 

e.g. to river estuaries. Depending on their chemistry and the receiving environment, interaction between 

the nanomaterials and the natural organic matter (NOM, which may enhance agglomeration, and thus 

sedimentation, or lead to dispersion) and sediment is likely. Depending on the nanomaterial type and 

receiving environment, the interaction with NOM may lead to enhanced (e.g. Hyung et al., 2007) or 

reduced (Baalousha et al., 2008) dispersibility and hence possible bioavailability for pelagic organisms. 

96. The fate of nanomaterials in the marine environment is likely to be characterised by enhanced 

agglomeration and thus sedimentation (Klaine et al., 2008, Stolpe and Hassellöv 2007), due to the high 

ionic strength of seawater. Although bioavailability could be diminished, it is possible that biological 

systems may become clogged by e.g. agglomerated nanomaterial and other particles, and thus their activity 

impaired (e.g. Nielsen et al., 2008). This considers only nanoparticles without specific surface 

functionalization and only possible electrostatic interaction based on surface charge and the variation of the 

surface charge by interaction with e.g. NOM. Non-charge effects as steric/entropic stabilisation through 

e.g. polymers attached to the surface, will make the dispersion stability more independent from simple 

electrostatic effects, hence more independent from ionic strength or the presence of NOM. It is not yet 

clear if those NPs particle types will be prone to quick aggregation in e.g. salt water / marine conditions. 

Reactions like bridging flocculation caused by e.g. natural polysaccharides, seem to be very effective 

(more effective than simple increase in ionic strength) and still not quantitatively understood for complex 

systems such as the marine environment. High energy environmental and biological processes, e.g. 

waves and turbulent stream conditions or flow through the fish opercula-gill system may re-suspend the 

nanomaterials and affect the agglomeration status and bioavailability. 

97. Aggregation of nanomaterials and their tendency to concentrate at interfaces violate the 

assumption that the test substance has free diffusion through the phases, making log KOW difficult to 

determine in practice. Jafvert and Kulkarni (2008) have studied the octanol-water partition coefficient (log 

KOW) of fullerene (C60) and its aqueous dispersability. They obtained a value for log KOW of 6.7, and a 

value for the solubility of C60 in water-saturated octanol of 8 ng/L. Hence based on this high KOW- value, it 

is expected that C60 has high affinity for lipids and organic matter. This indicates that in the natural 
 ENV/JM/MONO(2012)40 

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environments, C60 will tend to adsorb to solid phases. C60 behaves like an intermediate: partly as 

nanoparticle, partly as a large organic molecule. Hence some of the classical techniques (like a toluene 

extraction and LC-MS) work for C60. Despite its hydrophobic characteristic, fullerene C60 has been 

dispersed in water by stirring or sonicating or via solvent exchange (e.g. Pycke et al., 2011; Ma and 

Bouchard, 2009). The fullerene agglomerates are able to form stable water suspensions (nC60 or aqu/C60; 

e.g. Isaacson et al., 2011; Scharff et al., 2004). 

98. Some modelling work has been published for titanium oxide (TiO2), silver nanoparticles and 

carbon nanotubes (Mueller and Nowack, 2008; Boxhall et al., 2007, Gottschalk et al., 2010, Arvidsson et 

al., 2011, Quick et al., 2011). However, current knowledge of the behaviour of nanoparticles in natural 

waters does not provide sufficient basis for the full assessment of environmental exposure concentrations 

or amounts of dispersed nanomaterials. Many of these early modelling studies were only based on 

estimates and calculations and not on environmental measurements of nanomaterial content. 

99. In terrestrial systems some nanomaterials may preferentially bind to NOM and thus become less 

bioavailable (e.g. Li et al., 2008), although sediment and soil ingesters (e.g. oligochaetes) may be able to 

take up these nanomaterials. In fact they may preferentially ingest nanomaterial if they are associated with 

NOM, e.g. see Roberts et al. (2007) and they may be released and bound to the tissues (strip de-associate) 

within the gastrointestinal tract (GIT). The binding to decaying organic matter, detritus and the associated 

microorganisms may facilitate the dietary exposure of detritivorous and omnivorous organisms. The 

organisms selected for toxicity tests should address both types of exposure, i.e. in liquid matrix (in 

dispersion/solution) or associated to solid matrix (particle bound). It would be preferable to know the 

distribution of nanomaterial and the relevant type of exposure in advance. 

100. Determining the agglomeration/aggregation and sorption characteristics of the nanomaterials can 

provide valuable information when developing new test guidelines, using existing test guidelines with 

modifications or interpreting the results from existing test guidelines. Comprehensive tests to assess the 

agglomeration behaviour of nanomaterial in natural waters are currently only describing the agglomeration 

of the nanomaterial with themselves, however these tests reveal some general behavioural characteristics, 

which could be proven to describe quite well the real-worlds behaviour (Kammer et al., 2010; Ottofuelling 

et al., 2011). Also the potential capacity of nanomaterials to adsorb substances and work as toxicant 

carriers should be taken into account regarding the environmental behaviour of nanomaterials. In fact for 

nanomaterial to become relevant carriers for toxicants is relatively unlikely since some critical 

prerequisites must be met. One is the mobility of the particle in surface-, ground- and seepage waters, 

another is the strong binding of the toxicant to the nanomaterial and finally the nanomaterial must be 

present in a relevant concentration, compared to other particles which compete for the binding of the 

toxicant (Hofmann and Kammer 2009). 

101. Methods for environmental analyses are now in development for various materials and 

environmental matrices (Hassellöv et al., 2008) and these methods could provide the basis for 

environmental fate testing. (See also Section V, part A of the document) 

C. 1.2 Degradation and transformation 

102. Degradation, transformation and persistence of nanomaterials in the environment depend on their 

chemical composition, of both core and surface material as well as environmental conditions including pH, 

natural organic matter, ionic concentration and composition and content of dissolved oxygen. It is likely 

that most nanomaterials which are currently available will stay in their original particulate form, though 

levels of agglomeration/aggregation can be expected to be different. Some nanomaterials might have 

biocidal effects on microorganisms and hence affect the biodegradation. There is lack of data in this area, 

although, the organic coatings could be biodegraded or transformed by environmental factors. Hartmann et 
ENV/JM/MONO(2012)40 

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al. (2011) have studied degradability of aged aquatic suspensions of C60 nanoparticles. They observed no 

biodegradation of C60 in 28 days. However, when additional organic substrate (sodium acetate) was added, 

C60 nanoparticles (20 mg/L) did not inhibit the biodegradation as this other substrate which was completely 

mineralized.For metal containing particles, the UN GHS protocol for dissolution/transformation of metals 

and sparingly soluble metal compounds can be adapted to determine the rate and extent to which metallic 

nanomaterials can produce soluble available ionic and other metal-bearing species in aqueous media under 

a set of standard laboratory conditions representative of those generally occurring in the environment. 

C. 1.3 Bioaccumulation 

103. Current work assessing uptake has focussed on exposures in media with different nanomaterial 

loads over a specific time interval, followed by total body burden assessment, especially if individuals are 

small, such as Daphnia species, copepods, Lumbriculus or Eisenia species (Roberts et al., 2007, Fernandes 

et al., 2007a, Petersen et al., 2008). For larger organisms, specific studies have focussed on detection, e.g. 

by electron microscopic methods, of loads within specific organs, such as liver, kidney, muscle, gills (e.g. 

titanium in trout, Federici et al., 2007). In terms of detection, it may not always be possible to identify the 

form of such material. This may be particularly important for materials that tend to transform and/or 

dissolve readily such as silver or for nanomaterial of chemical compositions based on elements that are 

already highly abundant in the environment or in the media used for exposure tests (e.g. Zn, Cu, etc). 

104. The first step in the uptake and possible accumulation of a substance, at least in the aquatic 

environment, is often the adsorption and attachment and possible accumulation of the material onto the 

surface of the organism (Handy and Eddy 2004; Fernandes et al., 2007b, Nielsen et al., 2008). This has 

also been shown by the agglomeration of single wall carbon nanotubes on the gill mucus of rainbow trout 

(Smith et al., 2007). 

105. As first generation nanomaterials tend to follow colloidal chemistry and colloids can eventually 

agglomerate, these nanomaterial agglomerates will end up in the sediments (Klaine et al., 2008). Thus, 

bioaccumulation studies on sediment organisms (e.g. OECD TG 317) would be especially important. 

106. Petersen et al. (2008) have indicated that carbon nanotubes (CNTs) were not readily accumulated 

by the earthworm Eisenia foetida with results indicating bioaccumulation factors 2 orders of magnitude 

smaller than those measured for pyrene. Lee et al. (2008) have detected bioaccumulation of insoluble 

copper nanoparticles in cells of emerging and growing plants when tested on agar plates. 

107. Not much work has been published on potential food chain effects of nanomaterials, although 

fish that drink water containing nanomaterials show gut pathology (Federici et al., 2007; Smith et al., 

2007). A study (Holbrook et al., 2008) on the possible transfer of quantum dots in a simplified aquatic food 

chain has found that these materials can be transferred to rotifers through dietary uptake of ciliated 

protozoans. Although there was transfer across these levels, bioconcentration (accumulation from 

surrounding environment) in the ciliates was limited and no biomagnification (enrichment across trophic 

levels) in the rotifers was detected. This study indicates potential for transfer across food chain levels but 

this would depend on material type and food chain, as it is mostly the case for other studies of chemicals. 

Also Fortner et al. (2005) have observed that fullerene nanoparticles accumulate in microbial cells, in 

worms eating those microbes and possibly in animals higher up the food chain. The bioaccumulation 

assessment should consider the special uptake pathways, such as endocytosis (Iversen et al., 2011).
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C. 2 Test Method Applicability and Dosimetry 

108. Generally, it should be noted that when research and testing involves both ecotoxicology and 

environmental behaviour, degradation, transformation, and especially bioaccumulation, harmonized 

methods for media preparation are recommended. This approach will maximize comparability and 

integration of exposure and effects studies by increasing the probability that nanomaterials have similar 

suspension properties, at least at the initiation of each type of test. There also may be circumstances where 

well-vetted methods for media preparation and material detection exist for fate-related tests, but not for 

ecotoxicity tests. 

C. 2.1. Environmental behaviour 

C. 2.1.1 Methods 

109. It is likely that the OECD test methods for a number of physical-chemical properties for 

environmental distribution are applicable, and their applicability has been assessed (OECD 2009). 

However, there is also a lack of several methods which can address nano-relevant physical-chemical 

endpoints or measurands (see Section V, part A of this document). Furthermore, as with other test methods, 

dosage for the testing and the detection, analysis and quantification of the nanomaterials are the most 

challenging issues. Methods for the characterisation of key properties of nanomaterials have been 

identified in several publications (e.g. Klaine et al., 2008, Hasselov et al., 2008, Tiede et al., 2008). 

Dispersion and solubility

110. Dispersion and possible solubility/transformation of nanomaterials are important properties that 

have been already addressed in the general part of this document (section 3). It is unclear to date to what 

extent can the effects observed be attributed mostly to the soluble form or to a combination of soluble and 

particle form, and to the size of the particulate form or to degradation products such as metal ions from 

metal based nanomaterials (Franklin et al., 2007, Navarro et al., 2008). Although work in this area is 

increasing now, the results will depend on the material under consideration. The OECD assay on water 

solubility (OECD TG 105) may be useful in this context, but many of the carbon based materials (such as 

fullerenes) are so water insoluble that specialised methods are likely to be needed in order to measure or 

estimate solubility. For example, fullerene solubility is usually estimated by measuring solubility in 

alcohols and extrapolating to a zero carbon alcohol, i.e. water (Jafvert and Kulkarni, 2008). On the other 

hand, organic based nanomaterials have the advantage of possibly being labelled with radioactive isotope 

14C, which would allow easy quantitative determination but may not allow detection of the number of the 

particles and characterisation of e.g. agglomeration. 

Water/octanol partitioning

111. The measurement of the KOW (OECD 107, 117, 123) is problematic given that many organic 

nanomaterials have such low water solubility that measuring their concentration in the aqueous phase is not 

a straightforward procedure as nanomaterial are insoluble but they may stay attached in the liquid 

interphase. However, Jafvert and Kulkarni (2008) have studied the octanol-water partition coefficient (log 

KOW) of fullerene C60 with method modifications. 

C. 2.1.2 Dosage and sample preparation for studies on physicochemical properties 

112. The methods presented in Section V, part A on assessing the physicochemical properties of 

nanomaterials could be generally followed for a dispersion. 
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C. 2.2 Degradation and transformation 

C. 2.2.1 Methods for degradation 

Biodegradation

113. In this document biodegradation means degradation of organic substances and material by 

microorganisms resulting ultimately carbon dioxide, water and increase of microbial biomass in aerobic 

conditions. In anaerobic conditions biodegradation may result in formation of methane and other reduced 

substances. 

114. Again, the main technical challenges in degradation and transformation studies are the detection 

and characterisation of nanomaterials in the various environmental media. The existing test protocols (e.g. 

OECD test guidelines) seem to be as appropriate for nanomaterials as for organic substances. 

115. Performing biotic degradation tests for purely inorganic nanomaterials is unnecessaryt. 

Therefore, it is necessary to examine first whether the nanomaterial contains carbon that can be used as an 

energy and nutrient source for microorganisms. Secondly, the physicochemical and compartmentalisation 

properties of the material can provide insight into whether some of the simulation tests are necessary. For 

example, if the material is unlikely to reside in the water column or if it is not soluble in water (e.g. 

fullerenes and carbon nanotubes), any testing in surface water may be unnecessary. However, current 

OECD guidelines for testing of pesticides (e.g. Hydrolysis TG 111, Photolysis TG 316 and others) do not 

give any lower limit for water solubility. According to current guidelines one has to deal with each 

substance no matter how low solubility might be. 

116. OECD methods on biodegradation have been developed and validated principally for assessment 

of organic compounds. Today’s nanomaterials, however, are principally inorganic; indeed even carbonbased nanomaterials tend to be of an inorganic nature. Hence, they will be most probably considered 

persistent against biodegradation and testing for biodegradation is not relevant. In principle, the methods 

measuring carbon dioxide production or oxygen uptake are applicable, but they require large amounts of 

test material. It is also important to consider whether carbon based nanomaterials such as fullerenes and 

nanotubes can be degraded at all under any conditions. However, limited data have indicated that 

fullerenes could be taken up by wood decay fungi, suggesting that the carbon from fullerenes could be 

metabolised (Filley et al., 2005). This is usually assumed for organic polymers, too. However, in 

combination with UV-treatment (simulated sunlight) a certain degradation of polymers can be determined. 

If several conclusive aerobic degradation tests indicate very low or negligible degradation, then other 

aerobic degradation tests will most likely also be negative and it may be useless to proceed with additional 

tests. For example, if the result of a ready biodegradation test is below 10%, then the chances are that the 

results of the biodegradation simulation test in surface water will also be very low and it may be better to 

decide to skip the more elaborate test, and conclude that the substance is not biodegradable. For 

biodegradation screening purposes, whether any biodegradation can happen, modified test systems may 

serve better than standard OECD test guidelines. The test conditions in the inherent biodegradability test 

e.g. TG 302 series are favourable for degradation. One possibility would be to have the OECD TG 310 

headspace ready biodegradability test with CO2 measurement, and enhance the test media and conditions to 

be more favourable for degradation (like in inherent biodegradability tests). In closed system with the 

measurement of carbon dioxide production of TG 310 or TG 301B the test material does not have to be 

soluble (like nanomaterials), and rather small amounts of test material is needed. 

117. Simulation tests for biological degradation in various environmental compartments are also in 

principle applicable, but again the detection of the nanomaterials is the challenge. The possible degradation 

to carbon dioxide (mineralization), integration into biomass or other partition could be followed by labelled 
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test material. The advantage of using labelled substances would be to allow the testing of low 

concentrations and to provide degradation kinetics and mass balance on the fate of the carbon from the 

tested material. However, for mass balance studies radio-labelled nanomaterials can only be used with 

great caution: the label must be distributed uniformly on the nanomaterial. This very complicated issue 

requires further input from radiochemistry experts. Yet, non-uniformly labelled material could still help to 

decide if degradation occurs at all. 

Abiotic degradation

118. Abiotic degradation means degradation of substances by physicochemical activity e.g. UV-light. 

119. Like for biodegradation testing, for hydrolysis testing, the chemical structure of the material and 

whether it contains groups which could be subject to hydrolysis and/or release ions dictate whether this test 

is necessary or appropriate. 

120. In view of the sometimes very long lifetime of nanomaterials in the environment, the 

photodegradation studies might be considered relevant. The OECD TG 316 for photodegradation and 

transformation in water could be an applicable method for this purpose, but again the characterization of 

the nanomaterial changes (specification) is essential. 

C. 2.2.2 Dosage and sample preparation for degradation studies 

121. Similarly to the testing of physicochemical properties or biotic effects, the dispersion methods to 

prepare the samples for degradation studies could include ultra-sonication and/or stirring for long periods 

e.g. weeks. Especially in biodegradation tests measuring carbon dioxide production or oxygen 

consumption, the use of organic solvents is not possible, as remnants of the solvent will interfere with the 

nanomaterial degradation. In the simulation tests using radiolabelled materials, the use of solvent carrier, 

dispersant or detergent could be possible. However, it should be noted that these will affect also 

nanomaterial characteristics. 

122. The detection of biodegradation in standard screening tests is usually followed by measuring the 

carbon dioxide produced or oxygen consumed by the degraders. As carbon based or more organic 

nanomaterials are normally not soluble, the measurement of dissolved organic carbon might not be relevant. 

Of course, in certain test systems the decrease of total amount of carbon could be assessed. In simulation

testing 14C labelling and chemical analysis and characterisation of nanomaterials and possibly particle 

numbers by other methods (e.g. electron microscopy or Field Flow Fractionation analysis with ICP-MS) 

would be the means of detecting the degradation.

C. 2.3 Bioaccumulation 

C. 2.3.1 Methods for bioconcentration and bioaccumulation 

Aquatic studies

123. Many of the possible ideas for exposure for bioaccumulation studies originate from, and are 

informed by effect studies. 

124. For simple organic chemicals, there is an established relationship between octanol-water partition 

coefficient (KOW) and bioaccumulation or bioconcentration factor (BCF). However, this relationship may 

not hold true for many nanomaterials. The studies of Jafvert and Kulkarni (2008) have shown log KOW of 

6.7 for fullerene, and it is hence expected that C60 has high affinity for lipids and organic matter. Despite 

this hydrophobic characteristic, fullerene agglomerates are able to form a stable water suspensions (nC60 or 
ENV/JM/MONO(2012)40 

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aqu/C60; e.g. Isaacson et al., 2011; Scharff et al., 2004). To what extent such agglomerates can be taken up 

by organisms (and thus can bioaccumulate), however, is still uncertain. 

125. The main challenge once again in testing the bioaccumulation of nanoparticles is their detection 

and characterisation in tissues and body fluids. Radiolabelling could make detection and quantification 

easy but it has also limitations, e.g. the labelled material can behave differently from the non-labelled 

particles (e.g. if a tethered label is used). One possibility could be neutron activation of metal and metal 

oxide nanoparticles (Oughton et al., 2008). This enables both localisation and quantification within tissues 

or organisms. Also more traditional chemistry e.g. ICP-MS analysis for metals could provide valuable 

information on the total amounts of material accumulated by the organism. However, these methods can 

not define whether all the material found originates from nanomaterial. Recently field flow fractionation 

(FFF) techniques have been used in combination with ICP-MS. In this way metal content of individual 

particles can be measured. But first the nanomaterial has to be extracted from the animals and tissues. 

126. Standard BCF testing protocols such as OECD 305 (OECD 1996) may have limitations for 

determining bioaccumulation of nanoparticles. It has been observed for substances dissolved in water that a 

large molecular size (MW > 600, or effectively a diameter size > 0.5 nm) effectively limits direct uptake. It 

is likely that in most cases the large size (1-100 nm) of nanoparticles compared to dissolved molecules 

limits their direct uptake by carrier-mediated transport in fish gills, but uptake by endocytosis cannot be 

excluded (Handy et al., 2008b). Fish dietary BAF testing (Fisk et al., 1998; Stapleton et al., 2004) is 

covered by the revised OECD 305 (adopted 2012). According to this Test Guideline dietary exposure tests 

are recommended for super-lipophilic substances, surfactants as well as complex mixtures. However, the 

spiked food method would also be suitable for testing of poorly soluble large molecules such as 

nanoparticles. It should be noted that the dietary approach yields a biomagnification factor (BMF) rather 

than a bioconcenttraion factor (BCF). Fish do eat diets contaminated with nanomaterials, and toxic effects 

have been observed (Ramsden et al., 2008). However, more data using the harmonised OECD dietary 

protocol, especially for testing nanomaterials, are needed. The selection of the water or dietary exposure 

route should be based on the expected relevance of the gill versus dietary exposure under relevant 

environmental conditions. 

127. The testing results of human health endpoints e.g. toxicokinetics (ADME), if available, should 

also be taken into consideration when generating environmental testing plans for specific nanomaterials. 

Uptake studies from mammalian studies may give valuable basic information of uptake characteristics, 

rates and mechanisms of nanoparticles also for non-mammalian species. 

128. Given the tendency of nanomaterials to agglomerate, and thus their likelihood to end up 

associated with sediments (Klaine et al., 2008), bioaccumulation studies on sediment organisms would be 

especially important. OECD adopted in 2008 a new method TG 315 (OECD 2008a) for the 

bioaccumulation in sediment worms e.g. using Lumbriculus variegatus. This method would be then 

probably relevant to be used in a test battery for risk assessment, as OECD has published recently a 

toxicity test OECD TG 225 (OECD 2008b), based on the same species, which would then also provide 

effects data. 

Soil and terrestrial studies

129. Petersen et al. (2008) have indicated that CNTs were not readily accumulated by the earthworm 

Eisenia foetida with results indicating accumulation factors 2 orders of magnitude smaller than those 

measured for pyrene. Scott-Fordsmand et al. (2008) have detected effects on the reproduction of 

earthworms (Eisenia veneta) when the worms were exposed to double-walled carbon nanotubes in food. 

To assess bioaccumulation of chemicals in earthworms a validated OECD method (TG 317 on 

Bioaccumulation in Terrestrial Oligochaetes) is available. 
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130. Effects of ingested nanosized titanium dioxide on enzymatic activity of terrestrial isopods 

(Porcellio scaber) have been detected by Jemec et al., 2008. The TiO2 nanoparticles were dispersed in 

distilled water with and without sonication and pipetted on to homogenised hazelnut tree leaves. The 

isopods were then fed with the leaves. The particle location and its composition were analysed by 

transmission-electron diffraction pattern. 

131. Lee et al. (2008) detected bioaccumulation of insoluble copper nanoparticles in cells of emerging 

and growing plants when tested on agar plates. The particles were well characterised and the homogenous 

distribution of Cu particles in the agar media was evaluated by SEM. The distribution and accumulation of 

Cu particles in the plant cells was characterised by TEM and energy-dispersive spectroscopy. 

C. 2.3.2 Dosage, exposure and sample preparation for bioaccumulation studies 

132. The methods of sample preparation and dosage for bioconcentration and bioaccumulation studies 

could be similar to those for assessing biotic effects. These might include ultra-sonication, stirring for 

various periods, use of solvents and introducing stabilising agents. There is still limited information to 

prioritise the dispersion methods for bioaccumulation studies, but probably the same preference could be 

valid as for testing biotic effects. The aim would be to achieve a stable and homogenous dispersion over 

the exposure period. Often the smallest particle size would be expected to provide high accumulation but 

the exposure conditions should be always decided depending on the nanomaterial tested and the aim of the 

study. Whatever the method of test material dispersion and dosage, the test media quality (pH, ionic 

strength, NOM concentration) should be as harmonised as possible between comparative studies. It is 

especially important that the conditions and the quality of the media are recorded throughout the study in 

order to enable possible retrospective analysis of the results. 

133. Depending on the test, the exposure to the test nanomaterial could be via water, sediment and 

sediment pore water, soil and soil pore water or ingestion and food. The test design and dose selection 

should consider the relevance of dynamic and energy-dependent uptake processes to ensure that saturation 

does not occur, and the exposure is sufficient for reaching steady-state conditions. For aquatic studies, the 

methods for dosage mentioned above are relevant but e.g. for soil studies the nanomaterial can be 

introduced directly to the media in solid form and homogenised. Care should be taken in homogenisation 

so that the test material is not damaged, and details of homogenisers/velocities should be reported. 

134. In bioconcentration and accumulation studies, both the accumulation and depuration phases are 

important. It must be noted that the nanomaterial could be excreted in a different form from which it was 

taken into the organism. Hence the characterisation methods for the test material are important, not just the 

total amount measured e.g. by total metal content of metallic nanomaterial. 

135. It is unclear to date to what extent the effects observed can be attributed mostly to the soluble 

form or to a combination of soluble and particle form, and to the size of the particulate form or to 

degradation products such as metal ions from metal based nanomaterials (Franklin et al., 2007, Navarro et 

al., 2008). Solubility of silver nanoparticles and the resulting release of silver ions can occur in a variety of 

ways. The particles themselves can harm biota by direct interaction with biological targets. This release of 

ions (silver solubility) can happen outside the organisms and cells in the test media, but it has been 

proposed that the small silver particles can enter cells and organisms and release the ions there (the so 

called Trojan Horse effect). Definitely, the manufactured coatings (e.g. citrates, PVP) and also the natural 

coatings [natural organic material (NOM), protein corona] will affect the release kinetics of the silver ions 

from the particles depending on the environment of the particles. Some release of various metals ions or 

oxides can be expected also from other metallic nanomaterials. 

i) Sample preparation 
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Soil studies

136. Direct mixing of dry nanomaterial into soil has proven to give more homogeneous distribution of 

test material than mixing an aquatic dispersion of nanomaterials into soil. This has been the case at least 

when Ag-nanoparticles were mixed into the OECD artificial test soil (Scott-Forsmand 2011). Systematic 

studies concerning spiking of soil for ecotoxicity tests (natural sandy soil (German reference Soil RefeSol 

01-A spiked with powder using soil and silica sand resp. as carrier; soil / food spiked with aquatic 

dispersion; food spiked with powder) revealed that bioavailability differed between the different 

procedures, but replicates (6 samples) taken per spiking procedure used for chemical analyses revealed 

comparable standard deviations for all spiking procedures for soil. 

Aquatic studies

Water phase 

137. Dispersion of nanomaterial might include stirring, sonication [or preferably energy input in J/L 

(joules/litre)], grinding, use of solvents, and stabilising or dispersing agents. The advantages and 

disadvantages of these methods are outlined in Handy et al. (2011). This suggested that some 

nanomaterials are significantly altered by sonication [or preferably energy input in J/L (joules/litre)] and 

grinding (e.g. carbon nanotubes can be shortened) and that the interaction of solvents with some 

nanomaterials might result in toxic by-products. The method of dispersion will also depend on the specific 

material to be tested and whether or not it has been surface treated. Best scientific judgment should be used 

in selecting the methods, and where there is evidence or an indication that a dispersion method might 

significantly alter toxicity, those effects should be controlled or quantified. The operation conditions before 

and during the test should be described in detail. 

138. It is unlikely that these properties will be stable over renewal periods (or the duration of a test), 

rather, the goal should be to have a repeatable initial test medium, and to monitor property changes with 

sufficient frequency to quantify these changes. In some cases, in order to remove the larger particles, 

filtration with 0.45 μm or 0.22 μm filters is employed (e.g. Ma and Bouchard, 2009). This approach should 

be altered where information is available concerning environmentally relevant particle sizes. This might 

include manufacturer information on specific sizes produced and incorporated into products, and unlikely 

to undergo post-production processes that might alter particle size or distribution. 

139. It is assumed that tests will be often conducted using periodic renewal approaches to avoid 

expense and waste production of the flow through systems. Regardless of nanomaterial dispersion and 

dosage methods, the test media quality (pH, ionic strength, NOM concentration) should be as harmonised 

as far as possible between comparative studies. It is especially important that the conditions and the quality 

of the media are recorded throughout the study in order to enable possible retrospective analysis of the 

results. 

Sediment 

140. Direct mixing of dry nanomaterial into dry sediment has proven to give more homogeneous 

distribution of test material than mixing a water dispersion of nanomaterials in sediment. This has been the 

case when Ag-nanoparticles were mixed into the OECD artificial test soil (Scott-Forsmand 2011) 

Ion release

141. When testing e.g. Ag-nanoparticles the ion solubility has to be taken into account. It is preferable 

to assess this by recent analytical methods measuring the solubilized ions by e.g. ICP-MS. However, in the 
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bioaccumulation studies of nanosilver it can be useful to test silver ions (e.g. silver nitrate) simultaneously 

in order to differentiate the accumulation and especially depuration kinetics of nanoform and ions. 

Nanomaterial detection and characterization in the test media 

Ion release

142. Ionic strength, pH, cation-anion composition, and dissolved organic material (NOM and e.g. 

organism exudates) can all affect agglomeration and degradation of primary particles, the fate of both free 

ions and particles, or their interaction with biological targets (Liu and Hurt 2010). 

143. Methods that have been tested for separation of particles and ions in exposure media include 

dialysis, filtration, and ultracentrifugation. A related approach is to control or manipulate the type and level 

of chelators such as thiosulfate or cysteine, both of which strongly bind silver ions and reduce or eliminate 

their toxic potential. However, these chelating ligands can also increase the rate of oxidation and 

dissolution of Ag particles. Dialysis has not proven to be effective, largely due to adsorption of silver to 

membranes and the duration of testing; this has been demonstrated in mass balance studies where losses to 

membranes have been accounted for. 

144. Filtration and ultracentrifugation are currently being researched. Their effectiveness and specific 

approaches have yet to be widely established, although the ease of use for filtration methods offers 

promising solutions 

Detection in the solid matrix/porous media

145. The biggest challenges in the detection and quantification of nanomaterials from porous media 

e.g. soil or sediments lie in the pre-treatment of the samples. The nanomaterial has to be first extracted 

from the media and then separated from the extracted suspension. However, currently there are no 

harmonized ways of extraction. It seems that for separation of nanomaterial from the suspension 

centrifugation is better that filtration (Gimbert et al., 2006). It should be noted that, whatever these sample 

pre-treatment methods are there will most probably affects the characteristics of the nanomaterial. 

146. The analysis of extracted and separated element, e.g. metals using ICP-MS (Geranio et al., 2009) 

or ICP-OES, is relatively simple. However, it is difficult to differentiate the manufactured material and its 

atoms from the natural background. Questions remain as to whether the isotopic fingerprint of a 

nanomaterial would be different from natural materials. This challenge may be addressed through labelling 

of the metal in nanomaterials by such methods as neutron activation, which provides for separation from 

the background. 

147. With various electron microscopic techniques the NMs and their size and specification (state of 

agglomeration/aggregation) in a sample can be directly observed. However, only a limited number of 

microscopic fields can be screened which yields in statistical uncertainty. Hence, at the moment many 

microscopic methods are more or less qualitative methods (Doucet et al., 2005). To some extent the same 

applies to XRF-methods. 

148. Carbon nanomaterials like fullerenes and CNTs can be analysed from environmental samples by 

LC-MS chromatography (Isaacson et al., 2009) 

149. Field flow fractionation combined with chemical analysis e.g. ICP-MS (Ranville personal 

communication 2011) to measure different amounts and sizes of the nanoparticles. 

Nanomaterial detection in the biota 
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150. Detection of the overall amount of, e.g. 14C labelled material is rather easy in the tissues and in 

the whole organism. In addition, neutron activation of metal and metal oxide nanoparticles (Ag, Co or 

Co3O4 and CeO2) can be an option. These could enable both localisation and quantification within tissues 

or organisms. Of course, traditional chemical analysis ICP-MS for metals and various HPLC based 

techniques can be useful for the measurement of the total amount of the nanomaterial accumulated. But 

again these methods cannot separate accumulated nanomaterial from the bulk material or background 

metal concentrations. Electron microscopy provides means both for the detection and semi-quantitative 

analysis of the materials in the exposure media and inside the organism. 

Note 

151. Refer to Section V, Part C of this document for more in-depth discussion of characterization 

approaches. 

C. 3 References 

Arvidsson, R., Molander, S., Sandén, B. A., and Hassellov, M. (2011), “Challenges in exposure modeling 

of nanoparticles in aquatic environments”, Human and Ecological Risk Assessment 17, (1). 

Baalousha, M., Manciulea, A., Cumberland S., Kendall, K. and Lead, J.R. (2008), “Aggregation and 

surface properties of iron nanoparticles: influence of pH and natural organic matter”, Environmental 

Toxicology and Chemistry 27, pp.1875-1882. 

Boxall, A.B., Chaudhry, Q., Sinclair, C., Jones, A., Aitken, R., Jefferson, B., Watts, C. (2007), Current and 

future predicted environmental exposure to engineered nanoparticles, DEFRA Report, Central 

Science Laboratory, York. 

Chistian, P., Von der Kammer F., Baalousha, M. and Hofmann, T. (2008), “Nanoparticles: structure, 

properties, preparation and behaviour in environmental media” Ecotoxicology 17, pp.326-343. 

Doucet, F.J., Lead, J.R., Maguire, L., Achterberg, E.P., and Millward, G.E. (2005), “Visualisation of 

natural aquatic colloids and particles- a comparison of conventional high vacuum and environmental 

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D. HEALTH EFFECTS, PREPARATION OF A TEST SUBSTANCE AND DOSIMETRY 

152. Material characterization for toxicity (screening) studies is most appropriately considered in the 

context of the studies being undertaken. Requirements for in vitro and in vivo screening studies will differ 

according to the material delivery, route or method. Additionally, understanding human exposures in the 

context of relevant studies might present a further set of characterization requirements. At least three main 

study contexts are proposed to be considered, and characterization recommendations are proposed to be 

considered within these contexts (Oberdörster et al., 2005): 

• Human exposure characterization 

• Characterization of administered material 

• Characterization of as-produced or supplied material 

 Precise and comparable measurement techniques as well as the provision of standards and standard 

operating procedures are a prerequisite for this. One other key component for comparable and high quality 

characterisation is appropriate reference materials. 

153. Currently, standard materials include gold11 (NIST), titanium dioxide 12 , single wall carbon 

nanotubules13 (NIST) and polystyrene nanomaterials as well as quality control material colloidal silica 

(IRMM-30414). These materials have assigned values for particle size distribution by various analytical 

techniques and may be useful for evaluating the influence of dispersion and dosing protocols on 

nanomaterial size, agglomeration state, and dispersability. The EU Commission Joint Research Centre 

(JRC) has established a repository of representative commercial nanomaterials (Joint Research Centre 

(JRC) nanomaterials repository http://ihcp.jrc.ec.europa.eu/our_activities/nanotechnology/nanomaterialsrepository) that are characterised independently to provide confirmed reference samples. 

154. Dosimetry, appropriate dose metrics and the absorption, distribution, metabolism and excretion 

processes are particularly relevant in relation to the actual internal dose. The value of this information for 

setting safety testing triggers and assessment factors should be addressed (Gangwal et al., 2011, 

Oberdörster, 2012 in reply). However, this current version of guidance only deals with dosimetry briefly 

in some sections and overall content related to dosimetry requires expansion in future versions. 

 

11 See https://www-s.nist.gov/srmors/view_detail.cfm?srm=8011, https://wwws.nist.gov/srmors/view_detail.cfm?srm=8012, https://www-s.nist.gov/srmors/view_detail.cfm?srm=8013

12 See https://www-s.nist.gov/srmors/view_detail.cfm?srm=1898

13 See https://www-s.nist.gov/srmors/view_detail.cfm?srm=2483

14 See https://web.jrc.ec.europa.eu/rmcatalogue/detailsrmcatalogue.do?referenceMaterial=I0304%2B%2B%2B%2B%2B%2B
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D.1 Knowledge transfer from environmental chemistry and stock dispersion preparation 

155. The chemistry community has been working on colloids and particle chemistry for many decades, 

and this knowledge has been placed in context for nanomaterials ecotoxicology (Handy et al., 2008 and 

references therein). The generation of nanomaterials may be a general phenomenon for materials oxidised 

and reduced under common environmental conditions (Glover et al., 2011). There is a clear list of abiotic 

factors that can have substantial effects on particle agglomeration (and therefore bioavailability), as 

outlined in Section V on specific considerations. Since these abiotic factors are fundamental properties, 

they should also be considered for the preparation of salines and other media for in vitro on mammalian 

studies. The characterisation suggested (Section IV) and information reporting precisely how a stock 

dispersion was made (volumes, sonication times [or preferably energy input in J/L], etc) should be 

provided. Therefore, a test or a combination of tests to confirm the nanoscale of the particles in dispersion 

is absolutely necessary. Dispersability depends on mechanical treatment, surface chemical modifications 

and particle concentration to be dispersed. 

156. In order to investigate particle size and/or particle size distribution, a combination of 

characterisation techniques (see Table 1 below) including fractioning and analytical methods is suggested, 

especially in the case of a wide range of particle size distribution. A suitable combination could be field 

flow fractionation (FFF) for separation (advantage: covers a wide range of particle sizes) and small angle 

x-ray scattering (SAXS) for actual particle size. Alternatively, small angle neutron scattering, multi angle 

light scattering or dynamic light scattering could be considered for size determination. Chromatographic 

methods (size exclusion, gel permeation) are different suitable fractionation methods; however, stronger 

forces than occurring with field flow fractionation might influence the agglomeration behaviour. A 

suggested combination of methods has been in use for polymer analysis in industry for quite some time. 

157. Agglomeration is a critical factor in testing nanoparticles. Its role on health effects in vitro and in 

vivo has to be considered and cautions need to be taken in order to control clustering in sample preparation 

as what’s been done in environmental testing. However, nanoparticles in health effects testing may behave 

quite differently from particles dispersed in environmental media, depending on the particular route of 

exposure (e.g. inhalation of aerosols). Furthermore, the exposure scenario (which in turn will influence the 

testing design) and its regulatory consequences have to be taken into account. Hence, a "realistic" exposure 

scenario needs to be differentiated from a "worst case" scenario. A “realistic” scenario refers to how 

nanoparticles may actually have been taken up by consumers or at the work place (normally agglomerated). 

A “worst case” scenario means maximum dispersal, with primary size being one major factor in testing 

(e.g. for barrier penetration, bioavailability, toxic effects). To assess the toxic potency of a nanomaterial in 

the environment (e.g. for classification and labelling) may thus differ from deriving a "nano-dust" limit 

value at the work place. Accordingly, the regulatory purpose may also direct sample preparation (see 

Table 2). 

158. A number of analytical techniques may be applicable for measuring nanomaterials in dispersion. 

These techniques are summarized in several review articles (Oberdörster et al., 2005; Powers et al., 2006; 

Powers et al., 2007; Handy et al., 2008; Hassellöv et al., 2008; Sayes and Warheit, 2009, Domingos et al., 

2009, Wilkinson KJ, 2009). 
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Table 1: Characterization techniques 

Refer to Section B of this document for more in-depth discussion of characterization approaches. 

Properties of as-produced nanoparticles (nanoparticle powder) 

Size, size distribution, shape Scanning electron microscopy (SEM) 

Transmission electron microscopy (TEM) 

Atomic force microscopy (AFM) 

X-ray diffraction (XRD) for crystalline 

nanoparticles 

Differential mobility analysis 

Crystallinity, crystal structure X-ray diffraction (XRD) 

Electron diffraction in a transmission electron 

microscope(ED) 

Chemical composition and purity 

Of a nanoparticle ensemble (powder sample) 

Chemical properties of single nanoparticles 

Inductively-coupled mass spectroscopy (ICPMS) 

Inductively-coupled plasma atomic emission 

spectroscopy (ICP-AES) 

Atom-absorption spectroscopy (AAS) 

X-ray fluorescence spectroscopy (XRF) 

X-ray photoemission spectroscopy (XPS) 

Time-of-flight secondary ion mass spectroscopy 

(TOF-SIMS) 

Ultraviolet-visible spectroscopy (UV-Vis) 

Fourier-transform infrared spectroscopy (FTIR) 

Energy-dispersive (wave-length) dispersive Xray spectroscopy in an electron microscope 

Surface chemistry and surface reactivity X-ray photoemission spectroscopy (XPS) 

Electron spin resonance (ESR) 

Auger electron spectroscopy (AES) 

Surface area 

as indicator for agglomeration 

Isothermal gas adsorption/BET 

Properties of nanoparticles in liquid suspension 

Size distribution Dynamic light scattering (DLS) - 

Analytical ultracentrifugation (AUC) 

Cryo-transmission electron microscopy (cryoTEM) 

Field flow fractionation (FFF) combined with 

SAXS 

Surface charge, surface potential (yields also 

information on agglomeration state) 

Zeta potential by light-scattering electrophoresis 

Radical species Electron spin resonance (ESR) 

Solubility (of a material in a liquid) Conductance 

Visual conformation over time 

Surface activity Photocatalytic degradation 

Vitamin C assay 

Tracking of single autofluorescent or 

fluorophore-functionalized nanoparticles in 

liquid suspension 

Fluorescence microscopy 

Sedimentation of nanoparticles Computationel dosimetry [3] 

Protein corona of single nanoparticles Fluorescence correlation spectroscopy (FCS) [4] 
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Nanoparticles on the surface of cells (in-vitro experiments) 

Averaging techniques, i.e. techniques without spatial resolution, like ICP-MS and others without 

spatial resolution do not allow distinction between nanoparticles on cell surfaces and in cells 

Number density, size distribution Liquid atomic force microscopy (AFM) 

(Environmental) Scanning electron microscopy 

(ESEM) 

Chemical composition and purity of single 

nanoparticles 

Energy dispersive X-ray analysis in a scanning 

electron microscope (EDXS) 

Aggregation of nanoparticles Fluorescence-activated cell sorting (FACS) [5] 

Nanoparticles in tissues and cells 

Averaging techniques, i.e. techniques without spatial resolution, like ICP-MS and others without 

spatial resolution do not allow distinction between nanoparticles on cell surfaces and in cells 

Number density, size distribution and location of 

nanoparticles (spatially resolving techniques) 

Fluorescence microscopy 

Confocal light microscopy 

X-ray fluorescence imaging (XFM) 

Liquid atomic force microscopy (AFM) 

(Environmental) Scanning electron microscopy 

(ESEM) 

Magnetic resonance imaging (MRI) of superparamagnetic nanoparticles 

Chemical composition and purity 

Total mass of nanoparticles 

(Usage of radioactively labelled nanomaterials 

avoids effects of potential contamination from 

culture vessels etc.) 

Inductively-coupled mass spectroscopy (ICPMS) 

D.2 Recommended control measurements on test dispersions and test vehicles during experiments 

159. The following recommended measurements are in addition to routine checks on pH, temperature 

etc (see also section IV of this document): 

Exposure concentrations for test dispersions:

i) Confirm initial exposure concentration where techniques are currently available e.g. the 

concentration of dissolved metal and metal oxide nanomaterials. When ion release is expected, 

such as for ZnO, CuO and other metal and metal oxide nanomaterials, the measured ion 

concentration in the dispersant is also desired. This will aid distinguishing nano-specific effects 

and substance-specific effects. 

This is problematic for carbon-based materials as the methodologies are not necessarily sensitive 

enough to measure environmentally relevant low microgram concentrations in water and the 

background levels of dissolved organic carbon (DOC) may overwhelm the actual concentration 

of carbon based nanomaterials. In salines containing protein (e.g. Bovine Serum Albumin) any 

carbon measurement is likely to be overwhelmed. 

Maintain a consistent exposure concentration by changing the test media if needed (as would be the 

case if a semi-static test method is used). The frequency of media changes may need to be derived 

empirically, but target exposure concentrations should be maintained. Flow through methods can create a 
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waste disposal problem and semi-static methods reduce this and the occupational exposure risk. 

Nevertheless, in particular for in vitro methods, attention needs to be paid to possible detachment of cells 

and to possible removal of cells grown in suspension during test media change and the possibility of waste 

induced occupational exposure risk. Inhalation studies present additional challenges of measuring dose 

over time, and require both on-line and off-line analysis. 

Particle number, size distribution and agglomeration: The characteristaion of these properties in the 

test dispersions is a fundamental problem (see collision theory in Handy et al., 2008). It must be accepted 

that agglomeration and dispersion will vary with each concentration in the test system. The total surface 

area available to the organisms will therefore not be the same at each concentration in the test design. 

Similarly, the total surface area and number concentration available to the organisms might not follow the 

predicted, mathematical dependency on the doses in the test design, but will be also influenced by 

dispersion taking place at each dose level. The rate of agglomeration/aggregation in culture media should 

be also checked over time especially for treatments longer than 24h. It is a huge amount of work to 

measure these changes in every test vessel or test vehicle and in each biological system like tissue, cell, 

cell type, organelle type, nucleus, and one may not have control of this because of different rates of ligand 

secretion by the organisms at each dose (e.g. mucus production). The stability of any nanoparticle 

suspension over time should be determined using techniques such as dynamic light scattering, differential 

centrifugal sedimentation, modified light microscopy, ultraviolet–visible spectroscopy (i.e., UV-Vis). This 

might be especially important in mammalian studies in vitro where saline media are re-circulated or reused, and cellular secretions inevitably build up in the media. This is also very important for in vivo studies 

using inhalation route of exposure and direct delivery techniques. In order to control the particle 

agglomeration behaviour absolute quantification of the particle number or determination of agglomerate 

size, using appropriate methods, is necessary. Real dose may also depend on (a) sedimentation effects 

(size-dependent fractionation may result from sedimentation), or (b) interaction of particles with vessel 

wall for different materials (e.g. glass or plastic). Permanent online analysis of dispersion is recommended 

to control dispersion stability. The surface chemistry of the particles should also be investigated as 

dispersion behaviour strongly depends on the chemical surface properties of the particles. 

It is anticipated that it might not be possible to strictly adhere to some OECD Guidelines for in 

vivo tests requiring specifically high mass concentrations to be tested, because of limited 

dispersability of some nanomaterial. In such cases it is recommended to test a dose range up to 

the dispersability limit. 

Control of vehicle (including dispersant, if used) effects by including vehicle controls- this would be 

the same as any other experiment. These controls must be included. However, the dogmatic approach of 

standardising dispersant levels in all treatments should be avoided. Excess dispersant can change particle 

shape (see discussion in Smith et al., 2007), and so some thought needs to be given as to whether such an 

excess is appropriate or not, or if individual dispersant controls are needed for each dose (as this poses an 

ethical dilemma regarding reduction of animal usage, wherever possible, existing data should be used). If 

dispersion cannot be reasonably achieved, then some standard protocol of sonication or mixing, 

"immediately" before dosing may be required to at least give a known amount of nanomaterial /unit 

volume of dispersant. Any deviation from the standard sonication protocol should be carefully reported in 

terms of duration and energy input. A certain degree of uncertainty would be unavoidable in this case. 

160. It is important to note that characterisation of a nanomaterial in suspension is just one component 

of an overall characterisation strategy for toxicity testing. Various groups (e.g. governments, treaty-based 

organizations, standards development organizations, research consortia) have created lists of 

characterisation requirements for understanding exposure and toxicity assessments, including requirements 

for minimum characterization of a material as produced or supplied, as administered to a test subject, and 
ENV/JM/MONO(2012)40 

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after administration (see, for example, Bouwmeester et al., 2011; Boverhof and David, 2010; Warheit, 

2008; Oberdörster et al., 2005, EFSA Journal 9, 2011). 

D.3 Special considerations for vehicles e.g. physiological media used in cell-free, cell-based, and 

mammalian studies 

161. In addition to the details on stock dispersions above, the high ionic strength of buffer salt 

solutions may present specific problems for dosimetry, including instantaneous agglomeration of the test 

material. The reason for using these salt solutions in the first place is to match the ionic environment (e.g. 

ionic strength, pH) in e.g. intravenous administration. The use of buffers therefore cannot be avoided. It 

may be better to make stock dispersions in ultrapure water, if the nanomaterial is dispersible in water, and 

then disperse smaller volumes in the saline or other buffers. Or if small volumes are used, consider dosing 

the nanomaterials in ultrapure water if that has proven to cause little disturbance of homeostasis and results 

in a good dispersion. This can be controlled by taken along ultrapure water as a vehicle control and 

compare the vehicle to no treatment at all. If this second step is taken, then all the characterisation may 

need to be done again for the saline or other buffers used. It may also be helpful to add dispersion agents 

(such as PEG, Tween, Triton or other similar surfactants or DMSO) to the saline to improve 

dispensability/dispersion handling of the test material. However, this must be fully justified for practical 

reasons (i.e., impossible to handle the nanomaterial in saline phase without it), reflected in the dispersant 

controls, and with some appreciation of how the dispersing agent is working (e.g. coating the surface of the 

nanomaterial). Dispersant similar to what would be found in the target tissue, such as bronchoalveolar 

lavage fluid (BALF) or mimic BALF (Porter et al., 2008) for inhalation studies should be considered first. 

Tween, triton, and other similar products should be obtained at the best available analytical grade to 

minimise spurious effects of contaminants in these products on the nanomaterial chemistry. In addition the 

inherent toxicity of the dispersion agent has to be kept in mind, in order to avoid high toxicity (Zhu, S. et 

al., 2006, Monteiro-Riviere, N. and Tran, L. 2007), which might interfere the interpretation of studies even 

if appropriate vehicle or blank controls are used. 

162. Physiological salt solutions and culture media for use in in-vitro studies with mammalian cells or 

tissues are often gassed with high concentrations of oxygen and carbon dioxide (e.g. 95 % O2: 5 % CO2), 

and of course may be used at body temperature (37o

C). It is important to note that these conditions have 

not been employed in agglomeration chemistry studies/environmental colloid chemistry. Currently it is 

assumed that these conditions used in mammalian studies in the above-mentioned systems will not alter the 

chemistry. This assumption may need to be revised when results of research on the nanomaterial chemistry 

in 5% CO2 at body temperature are available. However, in the meanwhile, it should be reported whether 

the particle characterisation was done in gassed or air-equilibrated saline, and at which temperature. It is 

noted that the level of oxygen affects levels of metabolising enzymes. Use of such high oxygen levels are 

not recommended in cell culture protocols, where use ambient levels, i.e., 21% O2 are common practice. 

163. Physiological salt solutions and culture media also contain additional substances that are specific 

to different types of test, e.g. the use of lipopolysaccharide (LPS) as an immune activator (i.e. adjuvant), or 

the addition of metabolic inhibitors in ADME15 studies. It must be stated whether characterisation was 

done before or after adding these extra substances, and preferably with some checks to show this does not 

have an appreciable effect on particle dispersion and toxicity. 

 

15 ADME is an acronym in pharmacokinetics and pharmacology for absorption, distribution, metabolism, and 

excretion, and describes the disposition of a test substance within an organism. The four criteria all 

influence the test substance levels and kinetics of test substance exposure to the tissues and hence influence 

the performance and pharmacological activity of the test substance. 
 ENV/JM/MONO(2012)40 

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164. Protocols for the preparation of stable nanoscale titanium dioxide solutions in biologic media are 

available. One protocol uses a stock solution prepared by ultrasonication methods (Taurozzi et al., 2011a) 

followed by addition of the stock solution into either Phosphate Buffered Saline (PBS) and Dulbecco's 

Modified Eagle Medium containing 10% fetal bovine serum (DMEM-FBS) (Taurozzi et al., 2011b). 

Solutions of nanoscale titanium dioxide prepared according to this protocol were found to be stable under 

incubation conditions for up to 48 hours. A second technique involved stabilising nanoscale titanium 

dioxide with citrate (Ramirez-Garcia, 2011). These suspensions were shown to be stable in cell medium 

with up to 10% protein for up to 25 hours at 37 °C. 

D.4 Routes of delivery and the behaviour of nanomaterials dispersions in mammalian studies 

165. Mammalian tests can involve inhalation/respiratory tract, oral or dermal route of exposure, and 

some consideration of the physical behaviour of the test material is needed for each route. The following 

delivery methods have been employed: 

i. Respiratory tract route: 

Aspiration or instillation of nanoparticle suspensions in salines. 

Exposure to nanomaterials in air or gas phase (inhalation studies for which there is no need to 

produce nanoparticles dispersions in physiological salines but in suitable low toxicity vehicles, if 

any, which prevent agglomeration as much as possible). ). Exposure by inhalation of an aerosol 

of nano-sized particles: particles may be aggregated and/or agglomerated into (much) larger 

particles and should be assessed. 

The selection of a vehicle favouring the dispersion of nanoparticles (e.g. phosphate buffer) can 

address adequately a hazard-driven testing approach, whereas the dry dispersion technique is 

more risk-driven (workplace scenario); both approaches are justified and can be selected for the 

intended purpose. 

ii. Oral route: dosing of saline via gavage (acute or repeated toxicity testing). Phosphate buffer 

saline (PBS) may also be used to achieve better dispersion of the nanomaterial 

iii. Dermal route: External application of salines, emulsions (e.g. ZnO nanoparticles in corn oil for a 

stable suspension), or creams 

iv. Injection route: Test substance in appropriate buffered solution (ADME studies for example). 

166. For a correct interpretation of biological effects after in vivo exposure, at least qualitative 

evidence should be provided that nanomaterial reached (or not) the organ under investigation (Blank et al.). 

Where techniques are available, some attempts could be made to measure effective mass doses to target 

organs, for instance, measuring metal concentrations in case of metal nanoparticles. . In inhalation studies, 

the aerodynamic data of the aerosol (mass median aerodynamic diameter 16 - MMAD and aerosol 

concentration) allow a prediction of the target mass doses using reliable models (MPPD model), other 

models are based on the CMD and a measure of the distribution shape, as often aerosols have wide particle 

size distributions. Alternatively, chemical analysis and in special cases radio labelling can be used to 

determine actual mass doses. In addition, body weights should be monitored throughout the experiments 

 

16 An important distinction is the difference between the geometry of nanomaterials and the aerodynamic diameter. 

The latter is important for the estimating where the dose will be deposited, the former may be important for 

the interaction between a nanomaterial and a biological system (cells). 
ENV/JM/MONO(2012)40 

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and doses should be adjusted with body weights in order to maintain constant dosing. It is possible, 

however, that in some cases the important dose metric might be surface area or the particle number 

deposited in the lung. 

D.4.1 Respiratory tract exposures 

167. Although much research on nanoparticle toxicity have used saline to deliver test material to the 

lung via intra-tracheal instillation (Pauluhn, 2009) or aspiration, inhalation is the physiological process 

during which (nano)particles are deposited in the respiratory tract including the nose and lungs, allowing 

for a slow build up of the dose and for normal clearance processes to occur. In addition, it should be noted 

that intratracheal instillation or aspiration are not considered in the OECD Test Guidelines and Guidance 

Documents for the inhalation route but there may be exemptions e.g. due to costs or time where instillation 

or aspiration is used. If this is the case, it needs to be emphasised that use of these methods may be 

acceptable to evaluate the relative toxicity of the test material when backed up by an appropriate and 

representative inhalation study. In addition, these methods should be supported by quality assurance testing 

including reproducible delivery of the nanoparticle suspension to the lungs such that nanoparticle 

deposition on the airway epithelium can be distinguished from that of the alveolar epithelium. Fewer 

studies have used inhalation exposure (i.e. breathing of particles dispersed in the air of inhalation 

atmospheres). A comparison of advantages and limitations of different exposure methods to respiratory 

tract are presented in available papers including (Madl and Pinkerton, 2009). Documented in vitro to in 

vivo data correlation can be seen in several publications and could be considered in place of aspiration and 

instillation (Geiser et al., 2005), (Rothen-Rutishauser B et al., 2008), (Jones and Grainger, 2009). 

168. Overall, inhalation of aerosols, gases or vapours is the first-choice route of exposure prescribed in 

standard OECD Test Guidelines and this is the only way to determine the NOEL for the airborne 

concentration of suspended (nano)particle dust (Ji JH, et al., 2007). In fact, the gold standard is inhalation 

studies using aerosolized engineered nanoparticles and the performance of these studies should be given 

careful consideration. In inhalation studies, it is important to consider not only toxicity but also potential 

exposure data and interaction of nanoparticles (mechanisms) with fluids, cells and tissue to allow for full 

characterisation of a biological response as related to Physical-Chemical properties of the nanomaterials 

(Ma-Hock et al., 2009), (Stebounoval et al., 2011). Ideally, they would include quantitative data on organ 

burden and distribution (see below). This in turn may be used to help select out nanomaterials with the 

lowest risk for given applications and help to identify control materials. The OECD repeated-dose 

inhalation study (28 days or 90 days) or possibly longer-term studies, which should include specific 

investigations (e.g. lung toxicity and cardiovascular effects), in addition to a complete histopathology, have 

to be preferred over the acute studies, due to the kinetics of deposition of particles in the lungs and the 

possible progressive building up of dose. However, determination of the administered nanoparticle dose is 

complicated and its estimation requires careful monitoring of breathing, of the aerosol parameters and of 

tissue analysis (SCENIHR, 2007). The toxicity of particulates in general and nano-structures in particular 

depends on their differences in the displacement volumes and the earlier attainment of lung overload. 

Particokinetics determines the toxicodynamics in that it influences the distribution patterns in the whole 

organism and the dose at the target site (Solomon et al.). Therefore, inhalation studies need to be designed 

to verify the kinetic threshold from homeostasis to adversity (Geiser et al.). This can be done using 

radiolabelled materials, chemical elemental analysis to determine organ concentrations and transmission 

electron microscopy. Retention and clearance also have to be considered in repeat dose studies. Overall, 

due to many (complex) variables associated with exposure, lung dosimetry needs to focus on cumulative 

lung burdens and associated particokinetics. It is important to characterize the materials and the generated 

aerosols by other metrics in addition to mass, e.g. surface and size distribution. The test atmosphere, e.g. 

with nanoparticle dust should be characterised and data (primary particle size, particle size distribution, 

mass concentration and number concentration and surface area) should be reported carefully so that the 

results can be useful for hazard and risk assessment and characterisation. For hazard assessment, aerosol 
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generation of nanoparticles should produce particle size distributions that allow maximum lung burden 

(MMAD <3μm, including agglomerates). Because repeated exposure studies typically use lower 

concentrations than acute studies, emphasis should be given to generating particle size distributions 

amenable to preferentially depositing in the lower respiratory tract, particularly for particles deemed to be 

innocuous and biologically “inert” in terms of their chemical reactivity. For such materials it is 

recommended that appropriate MMADs ranges be used in repeated exposure rodent studies to maximize 

lung exposure. Additional information is available in OECD Guidelines 403, 412, 413 and 436 and the 

associated Guidance Document GD 39 [OECD (2009)]. The OECD workshop (OECD Expert Meeting on 

Inhalation Toxicity; ENV/CHEM/NANO(2011)23) recommended that both OECD Guidance Document 39 

and the TG collection for inhalation toxicity be updated to take into account of nanomaterials testing. 

Accordingly, further guidance is to be expected for the near future. 

169. All technical aspects of inhalation toxicology studies, including the use of the preferred dynamic 

nose-only inhalation systems, are addressed in OECD Guidelines 403, 412, 413 and 436 and the associated 

Guidance Document GD 39 [OECD (2009)]. For inhalatory hazard assessment, particles should have 

characteristics, in terms of their morphology and size distribution, similar to those experienced in human 

exposures (but adjusted for rodent inhalabilty and respirability) and relevant to realistic human exposure 

scenarios. Test concentrations should cover the expected Maximum Tolerated Dose (OECD Expert 

Meeting on Inhalation Toxicity, 2011). However, aggregation and agglomeration seriously hamper 

generation of an aerosol consisting of nano-sized particles from a powder. There is a clear correlation 

between the number of particles in air (per cm3), the residence time and the formation of 

aggregates/agglomerates. So for sample preparation it is preferable to generate the aerosol as close as 

possible to the breathing zone of a test animal. Maximum reasonable effort should be made to aerosolize a 

powder by a venturi (Cheng et al., 1989) and further deagglomeration of the particles in the aerosol with a 

jet-mill (Cheng et al., 1985; Castranova et al., 1996) (Kuhlbusch et al., 2011). Due to agglomeration, 

attainable aerodynamic particle size distributions are generally expected to be in the 0.3 – 3 μm range, but 

aerosols consisting of single nanoparticles cannot be expected. If relevant, aerosols with smaller particles 

can be generated for sufficiently diluted dispersion of nanoparticles (preferable in water; Mahurin and 

Cheng, 2007, Jing-CAST Technology GmbH, 2002) or by vaporisation and subsequent condensation. 

Alternatively, nanoparticle suspensions in suitable vehicles (e.g. PBS) can be used to generate mixed 

interim particles that after deposition in the respiratory tract easily break down to release the insoluble 

nanoparticles. 

Table 2: Examples of Techniques for Generation of Exposure Atmospheres 

Dispersion technique Vehicle Characterisation of exposure 

atmosphere 

Devices 

Risk-oriented approach (agglomerates; >100nm) 

Dry dispersion with 

pressurised air 

Clean air Aerosol concentration (particle 

mass; particle number); MMAD 

Jet mill, venturi, rotating 

brush generator, Wright 

Dust feeder, ceramic 

electrical heater 

Nebulisation of a liquid 

formulation 

e.g. 

phosphate 

buffer 

Aerosol concentration (particle 

mass; particle number); 

MMAD; nanoparticle-specific 

chemical analysis of filter 

samples for determination of 

dose 

 Jet nebuliser 

+CNT: acoustic feeder 

system (subacute, sub 

chronic testing) 

Clean air Aerosol concentration (particle 

mass); MMAD : to be 

calculated based on filter 

samples/SEM 

Membrane system to 

bring the 

individual/respirable 

agglomerates of CNT 
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into the airborne state 

CNT: Nebulisation of a 

liquid formulation 

(acute testing) 

DPPC, BSA, 

Glucose 

solution 

Aerosol concentration (particle 

mass); MMAD : to be 

calculated based on filter 

samples/SEM 

Ultrasonic finger 

facilitating proper 

dispersion; no vortexing 

Hazard-oriented approach (small agglomerates; < 100nm) 

Spark generation 

(abrasion of a metal 

electrode) 

Argon, clean 

air 

Aerosol concentration (particle 

number) ; mean mobility 

diameter 

Electrical mobility 

spectrometer 

170. It is important to distinguish the influence of the geometry of nanomaterials from the 

aerodynamic diameter on the behaviour of the nanomaterials. The latter is important for estimating where 

the dose will be deposited, the former may be important for the interaction between a nanomaterial and a 

biological system (cells). 

171. Dry powder generation methods are to be preferred and attention has to be paid to respirability 

of the aerosols by rats when translating workplace aerosol measures to experiments. Using a pristine 

aerosol, i.e., without alteration of the particle surface, can best mimic the occupational situation. The 

overall aim should be to avoid artefacts where modified surfaces exhibit a modified toxic response in the 

biological test. These large particles can be removed from the test atmosphere upstream of the exposure 

chamber using a pre-selector such as an impactor. Therefore, in general, the aerosol can be characterised 

with the usual instruments, i.e., cascade impactor or other instruments, based on mass and inertial forces. 

However, for the toxicity of nano-sized particles mass based dose metrics may not be optimal and the 

number of particles or the total surface area may be more relevant. Mass based instruments are insensitive 

to nano-sized particles because of their low weight (e.g. 1 million 10 nm particles may have the same 

weight as one 1μm particle). Therefore, if separate nano-sized particles can be expected to be present, mass 

based instruments to characterize the concentration and particle size distribution should have to be 

supplemented with instruments based on counting individual particles (e.g. Scanning Mobility Particle 

Sizer (SMPS), Electrical Low Pressure Impactor (ELPI)). The concentration and the size distribution of 

surface area can be estimated from the particle size number distribution, though such calculations are to be 

interpreted with caution as these approaches can underestimate surface area relative to direct measurement 

by gas adsorption (Weibel et al., 2005). In addition, separate instruments are available to estimate the 

deposited particle surface area per part of the human respiratory system (Weibel et al., 2009). Direct 

measurement of particle surface using gas adsorption (so-called BET surface area) has historically been 

applied to the bulk powder material. However, LeBouf et al. (2011) has reported a method for 

quantification of airborne surface area of nanomaterials for inhalation toxicology exposures, which uses 

gas adsorption. It is also suggested to characterise the morphology of produced particles. In addition, 

separate integrative reading instruments based on diffusive charging of submicrometer particles are 

available to derive “active” or lung-deposited surface area concentrations. The readings are based on 

calibrations as well as some assumption as examples discussed in Asbach et al. (2009). 

172. Prior to the study, the test substance should be characterised comprehensively as recommended in 

previous chapters. 

173. If it is necessary to use a vehicle to generate an appropriate concentration and particle size, water 

or a physiological buffer e.g. PBS should be given preference as these formulation tools can help to deagglomerate the initially bigger agglomerates. Constancy and homogeneity of atmospheric concentrations 

of the tested particles should be ensured. If a vehicle other than water is used, the concentration of the 

vehicle in the atmosphere should be determined by an appropriate method (e.g. gas chromatography). 
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174. The flow of filtered air through the exposure chamber/system should be carefully controlled, 

continuously monitored, and recorded at least hourly during each exposure. Details on this and on the 

exposure chamber conditions can be found in the inhalation toxicity test guidelines including ISO10801 

(2010) Nanotechnologies – Generation of metal nanoparticles for inhalation toxicity testing using the 

evaporation/condensation method. 

175. The nominal concentration is the mass of nanomaterial introduced into the test atmosphere 

generation divided by the total volume of air passed through the inhalation exposure system, and generally 

should not be used to characterise the animals’ exposure since the nominal concentration will be higher 

than the actual concentration in the animal’s breathing zone in test chamber due to factors such as material 

impaction and settling in the particle generator and/or on the surfaces in the test chamber, . 

176. The actual concentration, which is the nanomaterial concentration as sampled from the animals 

breathing zones in an inhalation system should be measured and reported. For non-volatile single 

component nanoparticles, the actual concentrations can, in some cases, be obtained by non-specific 

gravimetric filter analysis. For multi-component aerosols, concentration may also, in some cases, be 

determined by gravimetric analysis. However, this requires analytical data, which demonstrate that the 

composition of airborne material is homogenous and similar to that of the starting material. The range of 

exposure concentrations should possibly cover a range from the Maximum Tolerated Dose (MTD) down to 

doses relevant for human exposures. The MTD is defined as the dose producing signs of toxicity such that 

higher dose levels, based on the same dosing regimen, would be expected to produce lethality. However, 

the concept of Maximum Tolerated Dose (MTD; based on mass) may not apply to nanomaterials, and it is 

recommended to use concentrations that do not exceed 2-3 order of magnitude of a worse case exposure 

scenario based on either mass or particle number per volume of air. Although it is unlikely that overload 

situations can be achieved for particle distribution in the nanometre range due to the fact that high 

concentrations will lead to a shift to the right of the particle size distribution due to aggregation, overload 

situations in rodents need to be avoided. (Levy (1995), Inhal. Toxicol. (2000)). It should be realised that 

for nanoparticle exposure the maximal dose may be limited by the maximum concentration that can be 

obtained in the dispersion solution. 

177. The exposure atmosphere should be held as constant as possible. The methods for monitoring this 

and the allowed deviation ranges are described in the OECD inhalation toxicity test guidelines. 

178. The particle size distribution of aerosols should be determined at least once during the study for 

each concentration level by using an appropriate measurement method. To improve this process the 

determination could be increased to once per day and before and after the study. The total mass 

concentration obtained by particle size analysis should be within reasonable limits of the mass 

concentration obtained during concentration control analysis. In addition to mass concentration, particle 

number concentration may be measured using instruments such as condensation particle counter 

(approximate range: 10 to 1000 nm). Size distribution may be measured using optical particle counters 

(approximate range: 300 to 20,000 nm) to assess presence of agglomerates and/or aggregates of 

nanoparticles and larger diameter particles of nanostructured materials. To further characterise the 

presence of primary nano-particles in the inhalation atmospheres a differential mobility analysing system 

(DMAS) should be used. Some experts have also mentioned condensation particle counting as a suitable 

possibility (CPC) in some cases. 

179. Other exposure techniques involve direct delivery to the respiratory tract in a liquid, so there is a 

need to disperse the nanoparticles for intratracheal instillation or laryngeal aspiration or for delivery to 

other sites, such as peritoneal cavity, skin or gut. A number of approaches have been published for 

preparing salines, including those used for cells/tissues in culture. Some studies (Warheit et al., 2007), 

including influential studies from the US National Institute for Occupational Safety and Health (Shvedova 
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et al., 2005; 2008) have not used any dispersant other than Ca2+ + Mg2+-free phosphate-buffered saline 

(PBS) with sonication. Other techniques for dispersion of nanoparticles in biocompatible fluids for in vitro 

and in vivo studies of the nanoparticle–biology interaction are also emerging (Ramirez-Garcia et al., 2011). 

All mammals have albumin as a ubiquitous protein and it is well conserved in evolutionary terms. 

Dispersal in bovine serum albumin (BSA) has therefore been used for intraperitoneal injection of 

nanotubes (Poland et al., 2008), although some experts have indicated that it would be preferred to use 

serum of the exposed species to avoid risks of immune response. One study has used the first BALF 

(Bronchoalveolar Lavage Fluid) obtained from normal rats to suspend the nanoparticles in before injecting 

back into rats (Sager et al., 2007; 2008). Although BALF is an effective dispersion medium, its utility is 

constrained by several factors. 

180. First, a separate set of naive animals must be used to obtain BALF and as such requires a 

significant economic investment. Secondly, intra- and inter-laboratory variability in BALF is a problem 

due to differences in many factors, including animal handling, anaesthesia, and BAL technique, as well as 

the inherent complex composition of BAL fluid. Some guidance is provided in the OECD Guidance 

Document no 39 on Inhalation Toxicity Testing. Because of these factors, an alternative nanoparticle 

dispersion medium based on BALF was developed. This alternative nanoparticle dispersion medium (DM) 

is essentially a BALF fluid mimic, i.e., Ca2+ and Mg2+-free phosphate buffered saline (PBS) containing 

serum albumin and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), a lung surfactant (Porter et al., 

2008). It is important to note that DM mimics components of the lung alveolar lining fluid important to 

nanoparticle dispersion, i.e., protein and surfactant, but at concentrations much lower than those present in 

lung alveolar lining fluid (Schürch et al., 1990; Gehr et al., 1990). The choice for the right approach highly 

depends on the physicochemical characteristics of the specific nanomaterial and the administration method. 

It should be recognized that every dispersant can have an influence on dispersion and toxicity and pros and 

cons should be weighed. Every dispersant can have an influence on dispersion and toxicity, and pros and 

cons should be weighed. Overall, permanent online control of dispersion state is strongly recommended. It 

is crucial to assure that dispersed particles are nanoscale and that dispersion rate is not lowered over time 

by agglomeration. A number of chemical surfactants have also been used to disperse nanoparticles prior to 

instilling into rat lungs, including Pluronic-F68 (Mangum et al., 2006) and Tween (Warheit et al., 2004). 

181. Aspiration of fine sprays may present some practical problems in terms of blocking spray 

equipment and achieving a precise quality of spray (and dose). The alternative is to use an instillation 

(essentially a gavage-like “injection”) of saline that gently delivers the dose to the bronchi and lung. There 

are disputes about the level of penetration of each direct delivery method into the alveolar region of the 

lung, but the latter direct delivery method has less practical problems, and may become a more pragmatic 

direct delivery method for comparative hazard assessment. 

182. Nevertheless, exposure via instillation is not considered as physiological because it usually 

results in exceedingly high dose and application rate and, since the particles are suspended in saline, the 

lung surface receives particles contained in a liquid, which is likely to affect the defence systems of the 

lung. In addition, instillation produced a less even particle deposition pattern in the lung than the inhalation 

method (Driscoll et al., 2000) and efforts need to be made to disaggregate the nanomaterial suspended in 

vehicle (Driscoll et al., 2001). The advantage of instillation is the administration of a more precise 

nanoparticles dose. Pharyngeal aspiration is a variant of instillation, still with a high dose, but the particles 

are in suspension. In this case, the exposure is to suspension droplets that disperse in the lung more readily 

than with simple instillation. Results with instillation and pharyngeal aspiration are rather similar in terms 

of allowing comparison in toxic potency between particle types. Both can be used for the oropharyngeal 

region down to the sterile alveolar region in the context of screening purposes and for mechanistic studies. 

However, a concern with the pharyngeal aspiration technique for pulmonary exposure, in addition to 

ensuring disaggregation of the suspended material in the vehicle, is the unintentional aspiration of food 

particles from the oral cavity during this procedure (Rao et al. (2003)). There is anecdotal evidence, which 
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is not reported by all laboratories, that alveolar inflammation induced by bacterial rinsing has been an 

undesired effect of pharyngeal aspiration in rats. In the mouse, there are numerous published studies 

demonstrating a lack of alveolar inflammation in vehicle-exposed mice after pharyngeal aspiration. With 

regards to the “unusually high doses to bronchioles”, this may likely be an observation related to lack of 

adequate nanoparticle dispersion and/or morphology of a particular nanoparticle, and not a general effect 

of pharyngeal aspiration. However, neither method can be used to determine NOEL (SCENIHR, 2007). 

183. Data analysis should include information on number of particles, volume of vehicle, particle 

concentration (Krug, H.F. and P. Wick, 2011) and also interpretation of aerosol characteristics, NOAEL, 

risk assessment implications, mode of action and a strategy for dosimetric extrapolation to humans. In 

addition, the inclusion of kinetic data is important. Overall, caution is advised on interpreting results of 

studies in which exposure is made by intratracheal instillation, and these are not recommended as the 

primary source of information but in a WoE (Weight of Evidence) or categorisation approach. 

D.4.2 Oral Exposure 

184. The acute regulatory tests use a gut gavage dosing (instillation into the stomach) of typically 10 

or 20 ml of saline/kg body weight of laboratory animal (rat from 20 ml/kg to 50 ml/kg; mouse 20 ml/kg or 

30 ml/kg. 17This volume is designed to deliver a dose comfortably to the stomach of the animal without 

dilatation of the stomach. Chronic studies of dietary exposure are best performed by feeding the 

nanoparticles in a diet to the animal. Because nanomaterials are known to be carriers of contaminants, it is 

possible that the results of feeding studies will be affected by the food composition. Few published studies 

describe exposure via the digestive tract by dietary intake of NP contaminated food, but the necessity to 

incorporate the nanoparticles into diet means that considerations of aggregation/agglomeration may be 

secondary. 

185. However, the techniques for manufacturing food often include a step where the test substance is 

sprayed into the feed mixture as it is blended, or used as a topcoat on the feed. In either case, a stock 

dispersion would be required (as above) and the aim would be to ensure a uniform spread of the dose in the 

resulting food pellets. Where possible, the dose should be measured in the food produced, along with the 

usual nutritional analysis of the feed. Storage and degradation of the feed may be an issue, especially with 

oxidising NPs (rancid food). The palatability of the food should also be considered. It may also be 

possible that nanoparticles cause secondary toxic effects by reducing the bioavailability or digestibility of 

the feed ingredients by adsorption processes or other effects. Additionally, the chemistry within the 

digestive tract and enterohepatic circulation needs to be considered. The low pH of the stomach is likely to 

have effects on any protein pre-coating and the general effects of the stomach milieu on nanoparticles 

could be to disperse them or agglomerate them, regardless of pre-treatments. The digestive tract is also a 

high ionic strength environment, containing mucus and other soluble proteins and special microbial 

environment. The chemistry is likely to be very complex, and not likely to be easy to predict from theory 

alone, and observational experiments are needed on nanoparticle bioavailability from different food 

matrices. The influence on the amount of bile excreted and enterohepatic circulation also should be 

considered as this can influence the absorbed dose. In-vitro models simulating GI-tract environment can be 

used to evaluate the interaction of nanomaterials with GI-tract liquids. 

186. Administration of nanomaterials via the drinking water has been reported for repeated dose 

toxicity testing (Trouiller et al., Cancer Res 69: 8784-8789, 2009). This method is in general accordance to 

 

17OECD TG407 notes that the volumne should not exceed 1 ml/100g body weight except in the case of aqueous 

solutions where 2 ml/100g body weight may be used. In addition, OECDTG423 notes that in rodent, the 

volume should not normally excveed 1ml/100g of body weight: however in the case of aqueous solutions 

where 2 ml/100g body weight can be considered. 
ENV/JM/MONO(2012)40 

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oral repeated dose toxicity OECD test guidelines (TG 407, 408). However, care has to be taken to prevent 

sedimentation. In addition, Ca, Mg and total hardness of water should be measured and stability of NP 

dispersion should be checked over time (Kim et al., 2010) (Park et al., 2010) and stability should be 

checked. In case of photosensitive or photoactive materials - light exposure, in this instance capsules or 

other oral dosage forms could also be considered. 

D.4.3 Dermal Exposure 

187. Dermal exposure to nanoparticles may occur in the workplace environment or via consumer 

products (e.g. cleaning agents, cosmetics, personal care products, textiles). In consumer products such as 

personal care products and cosmetics, the nanoparticles are usually dispersed in some excipient, such as 

glycerol, which allows the particles to be applied to the skin (Mortensen et al., 2008). With respect to 

textiles, the question is whether the nanoparticles can be leaching out of the product during use (after 

contact with sweat or saliva). It has been argued that, if nanoparticles are considered to resemble 

macromolecules of high molecular weight, skin absorption is considered unlikely. This expectation was 

verified in the Nanoderm project especially for TiO2 or studies with nanoparticles in cosmetic 

formulations (Gamer et al., 2006). Nevertheless, it cannot be excluded that other nanomaterials might 

penetrate the skin (Mortensen, Oberdörster et al., 2008). According to the Scientific Committee on 

Cosmetic Products (SCCP, 2007) nanomaterial constituents may act as penetration enhancers by 

penetrating individually into the stratum corneum and subsequently altering the intercellular lipid lamellae 

within this skin layer. In addition, nanomaterials may serve as a depot for sustained release of dermally 

active compounds. In addition, openings of hair follicles are compatible with particulate dimensions. The 

discussion herein has focused on particle penetration through intact skin barrier; however, if the barrier is 

compromised, penetration may be a factor of four or more compared to intact skin (Filon-Larese et al., 

2009). Therefore, it is not unreasonable to anticipate a size dependent phenomenon, whereby particles 

lodged within the appendageal openings may allow increased diffusion for ingredients. Additionally, 

nanoparticles may have increased substantivity in skin “furrows” and may not be efficiently removed by 

standard cleaning procedures. It has been demonstrated that spherical and elliptical quantum dots penetrate 

the stratum corneum and localize within the epidermal and dermal layers. If the skin is exposed to large 

nanoparticle doses, even small fractions may become important to accumulating secondary target organs. 

188. Methods for performing skin absorption studies are given in OECD Guidelines 427 (in vivo) and 

428 (in vitro) and Guidance document 28 as well as OECD Guidance Notes on Dermal Absorption 156. 

Related relevant information can also be found in ??? (Oberdörster et al., 2005). The state of 

agglomeration is not easily studied in the assessment of skin absorption in vivo. Because of this and 

additional scientific and ethical issues related to the in vivo skin absorption guideline, it is preferable to use 

OECD TG 428 (Skin Absorption: In vitro Method). OECD TG 428 uses human skin samples as a 

component of the skin absorption testing strategy. This in vitro technique is already being applied to 

nanomaterials using human skin in vitro (Mavon et al., 2007; Baroli et al., 2007; Wissing and Mueller 

2002). The skin samples are used in vitro to assess nanoparticle absorption through the skin by using 

Franz-type diffusion cells or the Saarbruecken penetration model. If, despite the previous considerations 

about the preference for the in vitro method (TG 428), the in vivo test is performed (TG 427), it is 

necessary to consider that one assumption in the in vivo dermal test is that the test material has reasonably 

good access to the skin under the fur or hair of the animal. It would be up to the experimenter to ensure this 

is the case. For example, does the test material agglomerate on fur, and not reach the skin in appreciable 

quantities? Does shaving a small area of skin eliminate this problem, or would this add unnecessary skin 

sensibility? This issue may be important given the known inflammatory effects of some particles in 

epithelial tissues. On the other side, when hairy skin is shaved or depilated before treatment, there is an 

additional risk of damage to barrier function exacerbating further the problem of reliably assessing 

nanoparticle absorption (SCCP, 2007). The use of hairless animal models (genetically modified) might 

overcome these practical issues, but would also create a significant ethical issue. Moreover, hairless mouse 
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skin does contain abnormal hair follicles, which may give rise to false-positive results. If a dispersing 

agent or solvent has been used to make the test solution or cream (emulsion), then a solvent control should 

be included to account for any irritating effects of these reagents, or their ability to alter the intrinsic 

permeability of the skin. The precise location on the skin should be stipulated (e.g. ear, precise region on 

the abdomen or thorax) because the thickness and sensitivity of the skin will change at different locations. 

The same location must be used on all the test animals. Since licking of treated skin might affect exposure, 

location should be chosen taking this issue into account. For the same reason, animals should be 

individually caged. 

189. Delivery of a material to skin is an important consideration for toxicity studies. Generally, a 

substance is dispersed in a vehicle to facilitate homogeneous delivery to the skin. A wide range of vehicles 

have been used in dermal toxicity studies including water, ethanol, toluene, olive oil, petrolatum, propylene 

glycol, acetone/olive oil co-solvents, etc. The effect of vehicle on penetration rate may be dependent upon 

the test substance. For example, Bonnist et al. (2011) reported that penetration rates of cinnamaldehyde 

varied depending on the vehicle. Only one study was identified that investigated the effect of vehicle on 

penetration of nanoparticles. Labouta et al. (2011) studied four model gold nanoparticles of diameter 6 and 

15 nm and reported that the vehicle (toluene or water) had minimal effect on skin penetration. It is 

important to note that nanoparticles that contact skin may not be suspended in liquids such as water or 

toluene rather, upon deposition materials, will be immersed in the aqueous sweat/oily sebum mixture on 

the human skin surface. Filon-Larese et al. (2009) used an artificial human sweat to disperse silver 

nanoparticles for in vitro skin penetration studies, though the effect of vehicle was not investigated in this 

study. Hence, consideration should be given to the choice of vehicle and its biological relevance and to a 

realistic exposure scenario when performing penetration studies. 

190. In a study to investigate the effect of skin barrier integrity on nanoparticle penetration, FilonLarese et al. (2009) lightly scored human donor skin using the tip of a syringe needle. Penetration of silver 

nanoparticles was compared for this damaged skin relative to intact skin using an in vitro Franz cell 

technique. As noted above, penetration of nanoparticles was a factor of four higher through damaged skin 

relative to intact skin. It is important to note that the skin samples used in this study were only lightly 

scored with a syringe, which disrupted the stratum corneum. It is not unreasonable that nanoparticles (or 

their agglomerates) may come into contact with skin that is damaged deeply to the underlying viable 

epidermis (e.g. open cuts or abrasions) and thus easily cross the skin barrier. 

191. The concept of particle penetration through intact skin has been highly controversial. An early 

pilot study by Tan et al. (1996) suggested TiO2 could penetrate intact skin. In a later study, Tinkle et al. 

(2003) observed in vitro that polystyrene latex spheres <1 μm in diameter, when coupled with flexing 

motion, could penetrate intact human skin. However, many subsequent studies, including a study using the 

highly sensitive Zn-65 label (Fraunhofer ITEM 2011, unpublished) have shown, in agreement, that 

penetration of TiO2 and ZnO nanoparticles into skin is not likely (Cross et al., 2007). The reason for the 

observed disagreement among studies may be due to methodological issues. Rouse et al. (2007) 

demonstrated, in a study with quantum dots, that if the dots were functionalized and the skin sample was 

subjected to a flexing motion of skin, the testing material penetrated the skin within several hours. 

However, only limited penetration was observed for non-functionalised quantum dots in the absence of 

skin flexing. This might indicate that quantum dots penetration may occur if skin is subjected to flexing 

motion but only to a limited degree or not at all in the absence of flexing, whether this result is extrapolable 

to other nanomaterial types is not yet clear. 

 192. The ability of particles to penetrate diseased skin has not been studied in depth. No literature on 

the topic could be found; however, it is well known that diseased skin has different barrier properties from 

healthy skin, which could potentially influence penetration. For example, passive diffusion of water 

through the stratum corneum is referred to as trans-epidermal water loss (TEWL) and provides information 
ENV/JM/MONO(2012)40 

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on changes in the stratum corneum water barrier function, which can be used as a marker of skin integrity. 

Generally, TEWL values are higher for diseased skin from conditions such as icthyosis, psoriasis, 

erthoderma, atopic dermatitis, and allergic dermatitis compared to healthy skin (Tagami et al., 2002; 

Giorgini et al., 1996; Lavrijsen et al., 1993; Grice et al., 1967). 

D.4.4 Injection routes 

193. Injections into circulation, tissues, or body cavities are generally used as administration routes in 

ADME studies18.. This is usually done with formulations dispersed in saline, or in the case of a very 

hydrophobic material, in lipophilic vehicles like corn oil. In addition to the considerations on salines above, 

one concern is the behaviour of the nanoparticles in the syringe. Micro bubbles in the syringe can act as 

precipitation surfaces, and so it would be important to avoid creating bubbles by good dispensing skills, 

and also not allow the syringes to sit for too long where micro bubbles may form on the inner surface of 

the syringe. It is also important to consider that the actual dose may depend on nanoparticle interaction 

with the wall material. The gauge of needle should be sufficient to enable a smooth injection without 

blockage of the syringe. It may be that larger needle sizes are needed for some nanomaterials preparations, 

depending on concentration/viscosity. Additionally, we must not exclude the possibility that nanoparticle 

injections may be very painful because the materials are reactive; therefore, animal welfare should be 

paramount. A precautionary anaesthesia may be advised regardless of needle size. 

194. Lack of dermal penetration and hence false negative results have to be taken into account when 

performing regulatory testing such as skin sensitisation. For instance, the local lymph node assay (LLNA, 

OECD TG 429 requires access to dendritic cells in living skin layers. 

D.4.5 Cell cultures and dispersion of NPs in culture media 

195. In vitro techniques with nanomaterials are discussed in more detail in documents prepared for 

SG7 for the OECD Working party. There are a number of in vitro test systems including cytotoxicity tests, 

mutagenesis tests, cell/tissue culture screening assays, and immunotoxicity assays, etc. Most, if not all of 

these test systems, rely on using a saline solution or a much more complex culture medium. It is inevitable 

that test materials will agglomerate in some commonly used culture mediums (Vevers and Jha, 2008) and 

increase the direct contact of the cells with the test material (J.G. Teeguarden et al., 2007). It might be 

possible to use soluble peptides or other organic ligands to act as dispersing agents in cell culture medium. 

While this may be a good idea from the perspective of nanomaterial dispersion, it may be very problematic 

from a biological perspective. Biological systems may see these added materials as “antigens” or the 

material may change the unstirred layer chemistry on the cell membrane that defines how the material 

interacts with the cell surface, or even adhesion of the cells to the culture plate (also used as an end point in 

some tests). Culture media often contain proteins, and it may not be necessary to add a “special” 

dispersing agent. For example foetal bovine serum (FBS) is commonly added to cell culture systems, and 

contains the ubiquitous mammalian protein albumin (bovine serum albumin; BSA) and so it is already 

present at high concentration in cell cultures that contain serum. BSA, because of its zwitterionic nature 

(contains both positive and negative charges), is a useful dispersant that changes the balance of protein in a 

cell culture very little (Bihari et al., 2008; Poland et al., 2008). It is likely that dispersing in a protein like 

BSA will aid in prevention of false positive toxicity engendered by the adsorption of nutrients from the 

culture medium onto nanoparticle surfaces and which may cause toxicity by nutrient depletion (Casey et 

al., 2008, Orts-Gil et al, 2011). Once dispersed in BSA, the nanoparticle surface should be changed/altered 

 

18 In the case of nanomaterials, distribution may differ substantially from inhalation (not only quantitatively with 

respect to time and concentration). For instance, brain entry via the olfactory bulb is bypassed by injections 

as is the passage through different body compartment that may differentially contribute to the corona 

(Oberdörster, Journal of Internal Medicine 267; 89–105, 2010). 
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and much less able to absorb nutrient proteins from solution. Furthermore, interaction with biomolecules 

may mask or modify specific surface properties of environmental nanomaterials or - in turn – 

environmental nanomaterials binding may affect the structure of the biomolecule. 

196. Another issue with using serum or BSA is that such medium components inevitably contain a 

number of unknown ingredients (peptides, fatty acids, sugars etc) that vary with the batch of serum or BSA 

used. It is possible to buy high purity BSA, or chemically defined media that have been manufactured from 

non-animal sources where all the components of the media are known. A dispersion approach in studies 

with lung epithelial cells uses a surfactant lipid found in the lung lining fluid, called 

dipalmitoylphosphatidylcholine (DPPC, Herzog et al., 2008; Wallace et al., 2007) usually as an addition to 

BSA or serum. This is obviously favoured for studies modelling the lung. 

197. Whichever dispersing agent or method is used, a balance must be sought between using a specific 

dispersion method and finding a realistic in vitro system with quality. It can be argued that the effects of 

serum or tissue-specific natural surfactants like DPPC should be accepted as part of the particle behaviour, 

especially since body fluids consist of a myriad of proteins, peptides etc, and additions of serum to culture 

medium would merely give a better reflection of what would happen in vivo. 

198. Though still in a developmental stage, air-liquid interphase (ALI) test systems may provide a 

promising alternative approach to conventional cell culture assays for specific investigations with regard to 

pulmonary effects (Savi et al., 2008, Paur et al., 2011). ALI is based on aerosol exposure of lung cell 

cultures thus preventing unwanted interactions of nanomaterial with media components and allowing a 

more realistic dosing and maybe a better control of particle agglomeration. Characterisation of the aerosol 

in such a system remains important, availability of these systems may be limited, and exposure 

concentrations may be challenging to control 

199. When handling photoactive nanomaterials measures should be taken to prevent light exposure in 

cell culture assays. Above a critical particle size threshold (hydrodynamic diameter > 40 nm) 

sedimentation will predominate over diffusion. Accordingly, a dose normalized to surface area of bottomadherent cells - considering typical incubation periods of up to 24 h - may overestimate the actual cellular 

dose of nanomaterial (and hence particle-cell interactions) that partition primarily via diffusion(Cho et al., 

2011). 

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