Inter-laboratory comparison on the determination of the hydrophobicity index of nanomaterials through an affinity measurement.
Date: 2023-06-13
Institutions: Joint Research Centre
Subjects: biology, chemical product, environmental indicator, nanotechnology, research method, research report, technical regulations, testing

ISSN 1831-9424 

Inter-laboratory comparison on the 
determination of the hydrophobicity index 
of nanomaterials through an affinity 
measurement 

Desmet,  C.,  Roncari,  F.,  Gottardo,  S.,  Ponti,  J., 
Riego Sintes, J., Colpo, P., Valsesia, A. 

2023 

EUR 31542 EN 

 
 
 
 
This publication is a Technical report by the Joint Research Centre (JRC), the European Commission’s science and knowledge service. It 
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Contact information  
European Commission -Joint Research Centre  
Directorate Health and Food 
Via E. Fermi, 2749, TP 125  
I-21027 Ispra (VA), Italy  
Email: JRC-NANOTECHNOLOGY@ec.europa.eu 

EU Science Hub 
https://joint-research-centre.ec.europa.eu 

JRC133937 

EUR 31542 EN 

PDF 

ISBN 978-92-68-04186-4 

ISSN 1831-9424 

doi:10.2760/486603  KJ-NA-31-542-EN-N 

Luxembourg: Publications Office of the European Union, 2023   

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How to cite this report: Desmet, C., Roncari, F., Gottardo, S., Ponti, J., Riego Sintes, J., Colpo, P., Valsesia, A., Inter-laboratory comparison on 
the  determination  of  the  hydrophobicity  index  of  nanomaterials  through  an  affinity  measurement.  Publications  Office  of  the  European 
Union, Luxembourg, 2023, doi:10.2760/486603, JRC133937. 

 
 
 
 
 
 
 
 
 
 
 
 
 
        
 
 
 
 
 
 
 
 
 
 
 
 
Contents  

Abstract ....................................................................................................................................................................................................................................................................... 2 

Acknowledgements .......................................................................................................................................................................................................................................... 3 

Executive summary .......................................................................................................................................................................................................................................... 4 

1 

Introduction..................................................................................................................................................................................................................................................... 5 

2  Methodology .................................................................................................................................................................................................................................................. 6 

2.1  Test materials ................................................................................................................................................................................................................................. 6 

2.2  Collectors and Samples characterisation ............................................................................................................................................................. 8 

2.3  Samples distribution ................................................................................................................................................................................................................. 8 

3  Ring trial results and considerations ...................................................................................................................................................................................... 9 

3.1  Detection of the sample M1 by DFM ........................................................................................................................................................................ 9 

3.2  Evaluation of the reproducibility of the particles binding experiments ............................................................................... 11 

3.3  Evaluation of the reproducibility of the calculation of the Hydrophobicity index ...................................................... 15 

4  Conclusions .................................................................................................................................................................................................................................................. 17 

References ............................................................................................................................................................................................................................................................. 18 

List of abbreviations and definitions ........................................................................................................................................................................................... 19 

List of figures ..................................................................................................................................................................................................................................................... 20 

List of tables ........................................................................................................................................................................................................................................................ 21 

Annexes .................................................................................................................................................................................................................................................................... 22 

Annex 1. Standard operating procedure (SOP) for the determination of the hydrophobicity index of 
nanomaterials according to the advanced draft test guideline ................................................................................................................ 22 

Annex 2. Preparation of the material samples .......................................................................................................................................................... 33 

i 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
Abstract 

Hydrophobicity  is  a  physico-chemical  property  that  may  influence  the  fate  of  nanomaterials  in  the 
environment  and  biological  matrices.  A  method  to  characterise  the  hydrophobicity  of  nanomaterials  was 
developed at the JRC and proposed as an OECD Test Guideline. In this context, the JRC led an Inter-laboratory 
comparison  (ILC)  aiming  to  assess  the  transferability  of  the  standard  operating  procedure.  The  method  is 
based  on  the  measurement  of  the  affinity  of  nanomaterials  to  engineered  collectors.  Nine  laboratories 
participated to this ILC. The variability of the measurements and the reproducibility of the calculation of the 
Hydrophobicity Index were assessed according to the International Standard ISO 5725-2. Accordingly, with |Z-
scores| < 2 for all the participants, the determination of the Hydrophobicity Index was considered satisfactory. 
The  method  was  adopted  by  the  OECD  Working  Party  of  the  National  Coordinators  of  the  Test  Guidelines 
Programme in April 2023 as Test Guideline 126. 

2 

 
Acknowledgements 

The authors would like to thank all the participant institutions in this inter-laboratory study and acknowledge 
their effort and availability to perform the experimental activities while facing extraordinary challenges and 
restrictions due to the-Covid-19 pandemic. 

—  Food and Drug Administration (FDA), US. Contact persons: Anil Patri and Arjun Sharmah 

—  National Institute of Standard and Technology (NIST), US. Contact persons: Keana Scott and Shawn Chen 

—  Institut  des  Sciences  Analytiques  et  de  Physico-Chimie  pour  l’Environnement  et  les  Matériaux  (IPREM), 

France. Contact persons: Cloé Veclin 

—  Badische Anilin- und Soda-Fabrik (BASF), Germany. Contact person: Sabine Hirth 

—  International Iberian Nanotechnology Laboratory (INL), Portugal. Contact person: Elisabete Fernandes 

—  Istituto Italiano di Tecnologia (IIT), Italy. Contact persons: Despina Fragouli, Katarzyna Dziza and Valentina 

Tolardo 

—  University of Birmingham (BHAM), School of Geography,  Earth and Environmental Sciences,  UK. Contact 

persons: Eugenia Valsami-Jones and Nathan Langford 

—  Institute of Electronic Structure and Laser, Foundation for Research and Technology Hellas (IESL/FORTH), 

Greece. Contact persons: Emmanuel Stratakis and Kostantinos Brintakis 

—  The  authors  would  also  like  to  acknowledge  Ana  Ruiz  Moreno  (JRC)  for  the  preparation  of  the  size 

calibrant used for camera calibration in this inter-laboratory exercise. 

Authors 

Desmet, C., Roncari, F., Gottardo, S., Ponti, J., Riego Sintes, J., Colpo, P., Valsesia, A. 

3 

Executive summary  

The European Commission’s Joint Research Centre (JRC) led the development of an OECD Test Guideline (TG) 
for the determination of the hydrophobicity index of manufactured nanomaterials (NMs) through an affinity 
measurement. To achieve this result, the JRC collaborated with a group of experts from different institutions 
to  prepare,  execute  and  assess  the  entire  project,  including  an  inter-laboratory  comparison  (ILC)  study.  The 
final  version  of  the  TG  was  approved  by  the  OECD  Working  Party  of  the  National  Coordinators  of  the  Test 
Guidelines Programme in April 2023. 

As agreed during the first meeting of the expert group in October 2019, the JRC organised and evaluated an 
ILC  on  the  determination  of  the  Hydrophobicity  index  of  NMs  by  measuring  their  affinity  for  engineered 
collectors. (Desmet, 2017, Valsesia, 2018) in collaboration with eight different laboratories from the US and 
Europe.  The  goal  of  this  ring  trial  was  to  evaluate  the  transferability  of  the  SOP  to  a  generic  dark  field 
microscopy  (DFM)  equipment  (different  acquisition  cameras  and  microscopes),  by  the  assessment  of  the 
reproducibility of the NMs binding experiments and the calculation of the corresponding Hydrophobicity index. 

This ILC was launched in November 2020 and concluded in October 2021. Nine laboratories from the US (2), 
France, Germany, the UK, Greece, Italy, Portugal and the JRC-Ispra participated in this ring trial by analysing a 
minimum of three different materials randomly attributed, out of the five selected. This choice was made to 
save  time  to  each  entity  while  keeping  a  consistent  number  of  results  for  each  material.  In  particular,  one 
material was tested by all the laboratories, while  all the other materials  were tested  by a minimum of five 
participants,  with  three  independent  replicates  for  each  material.  The  following  materials  were  considered: 
naked  polystyrene  and  carboxylate  polystyrene  particles  (commercial  references),  gold  nanoparticles 
stabilised  with  citrate,  hydrophobic  TiO2  nanoparticles  stabilised  with  natural  organic  matter  (from  the  JRC 
Nanomaterials  Repository)  and  the  commercial  food  additive  TiO2  (E171).  All  materials  were  fully 
characterised in the JRC Nanobiotechnology Laboratory before shipment to the ILC participants. 

Despite the novelty of the method and the differences in the used instrumentation, the nine laboratories were 
able to detect the NMs by DFM and reported measurements in triplicate. One material was discarded because 
it  was  not  detectable  by  any  participant  after  shipping,  probably  due  to  stability  issues.  The  data  analysis 
procedure was not assessed in the ILC and was optional for the participants. The JRC determined the binding 
rates  of  the  NMs  on  the  different  collectors  by  analysing  the  image  sequences  of  DFM  acquired  by  the 
participants. The Hydrophobicity index was then calculated as the final result of the test. The reproducibility 
was  considered  satisfactory  according  to  the  International  Standard  ISO  5725-2,  as  all  |Z-scores|  were 
determined to be < 2. 

4 

 
1 

Introduction 

Hydrophobicity is a relevant property for understanding the behaviour of nanomaterials or other chemicals in 
terms of safety assessment. In fact, hydrophobicity is currently listed as a key physicochemical parameter in 
the  OECD  Draft  Physico-chemical  Decision  Framework  for  Manufactured  Nanomaterials  (OECD,  2019). 
Hydrophobicity  provides  information  on  the  affinity  of  nanomaterials  to  aquatic  and  terrestrial  organisms 
(including bioaccumulation and persistence) and to human cell surfaces (including entry into the body through 
the  skin  and  lungs  and  subsequent  accumulation  in  or  removal  from  tissues/organs)  and  blood.  An  ISO 
technical  report  (ISO,  2019)  also  mentions  hydrophobicity/hydrophilicity  as  one  of  the  physico-chemical 
properties  determinant  for  toxicokinetic  behaviour  of  nanomaterials,  since  it  has  an  impact  on  surface 
chemistry,  protein  corona  formation  and  accumulation.  It  can  be  used  as  an  indicator  of  the  fate  and 
transport of a chemical in the aquatic environment and as a parameter in environmental exposure models to 
estimate  soil  and  sediment  sorption,  bioavailability,  and  bioconcentration/bioaccumulation  in  aquatic 
organisms.  It  can  also  play  a  role  in  (eco)toxicity  prediction  (e.g.  in  (Q)SAR  modelling)  and  help  assess 
bioaccumulation  potential  (e.g.  for  PBT  assessment)  and  predict  environmental  fate  and  concentrations  in 
exposure models. 

In its nano-specific guidance for the implementation of the REACH regulation, the European Chemicals Agency 
(ECHA) proposes a series of descriptors that may be used to predict the fate and transport of nanomaterials 
in  environmental  and  biological  media.  These  descriptors  are:  'agglomeration,  aggregation,  deposition  and 
attachment'  (ECHA,  2022),  all  of  them  influenced  by  surface  hydrophobicity  (among  other  physicochemical 
properties).  Hydrophobicity  is  also  included  in  the  list  of  potentially  relevant  parameters  to  justify  the 
application of grouping and read-across principles to nanoforms of the same substance under REACH (ECHA, 
2019).  However,  it  is  recognised  that  "analytical  determination  of  the  hydrophobicity  of  nanoforms  is  still 
under  development,  e.g.  sessile  drop  contact  angle,  dye  adsorption"  (ECHA,  2019).  Indeed,  existing 
standardised quantification methods are not suitable for nanomaterials, as suggested by the OECD Working 
Party on Manufactured Nanomaterials (WPMN) (e.g. OECD, 2009, 2014, 2017) and the research community 
(e.g. NANoREG, 2015).  

The JRC (Directorate Health and Food, Technologies for Health Unit, F.2) coordinated the development of  an 
OECD  Test  Guideline  (TG)  for  the  determination  of  the  Hydrophobicity  index  of  nanomaterials  through  an 
affinity  measurement.  The  method  is  based  on  the  measurement  of  the  binding  rates  of  nanomaterials  to 
engineered  hydrophobic  and  hydrophilic  surfaces  (collectors).  It  is  an  alternative  to  the  other  potentially 
applicable techniques. A standard operating procedure was designed by the JRC, in collaboration with a group 
of  experts  from  different  countries,  and  tested  in  several  laboratories  in  an  inter-laboratory  comparison 
exercise, to assess the transferability of the method.  The ILC study reported here was carried out to support 
the adoption of the new OECD TG 126.  

5 

 
 
 
2  Methodology 

The purpose of the inter-laboratory comparison (ILC) was to assess the applicability and transferability of the 
proposed method to determine the hydrophobicity index of NMs by affinity measurements. See Appendix 1 for 
the definition of hydrophobicity index. 

The  planning  of  this  ILC  was  discussed  for  the  first  time  in  an  "Expert  Meeting  to  Develop  an  OECD  Test 
Guideline to Determine Surface Hydrophobicity of Nanomaterials" held at the JRC in Ispra in autumn 2019. In 
January 2020, interested laboratories were contacted to participate in this experimental exercise, which was 
planned  to  begin  in  April  2020.  Due  to  the  Covid-19  outbreak,  the  start  time  was  postponed  and  all 
participants were contacted again in late summer 2020 to confirm their participation. The ring trial effectively 
started in November 2020, with the sending of test samples, standard operating procedure (SOP) and files for 
data treatment. Results were expected in the second quarter of 2021, but due to Covid-19 related issues and 
the defection of some laboratories, an extension was decided as well as involving new laboratories.  The end 
date was moved to the beginning of the fourth quarter. 

Five  material  samples  were  selected  for  the  ILC  based  on  their  ‘presumed’  hydrophobicity.  First,  the  JRC 
developed  the  SOP  and  conducted  experimental  tests  on  the  selected  materials.  The  JRC  sent  test  kits 
containing  samples,  collectors,  camera  setup  calibration  platforms,  memory  sticks  containing  the  SOP,  and 
Excel  files  containing  macros  for  data  reporting  and  analysis  to  all  participating  laboratories.  The  nine 
laboratories that effectively participated in this exercise are listed in Table 1. 

Table 1. Laboratories participating in the ILC 

Participant 

Food and Drug Administration (FDA) 

National Institute of Standard and Technology (NIST) 

Country 

United States 

United States 

Institut  des  Sciences  Analytiques  et  de  Physico-Chimie  pour 
l’Environnement et les Matériaux (IPREM) 

France 

Badische Anilin- und Soda-Fabrik (BASF) 

International Iberian Nanotechnology Laboratory (INL) 

Istituto Italiano di Tecnologia (IIT) 

Germany 

Portugal 

Italy 

Birmingham University (BHAM) 

United Kingdom 

Foundation for Research and Technology-Hellas (FORTH) 

Greece 

Joint Research Centre (JRC) 

European Commission 

2.1  Test materials 

In  order  to  assess  the  applicability  and  the  reproducibility  of  the  proposed  method,  an  initial  list  of  seven 
materials  with  different  characteristics  (i.e.  size,  chemical  composition,  physical  state)  was  selected  for  the 
inter-laboratory exercise. Due to the time constraints, the list was shortened to the five materials described in 
Table 2. 

6 

 
 
 
 
Table 2. Selected materials for the ILC 

Sample 

Type of material 

Mean size diameter (nm) 

Source 

M1 

M2 

M3 

M4 

M5 

Polystyrene (hydrophobic) 

500 (commercial reference) 

Polysciences, Inc. 

Hydrophobic gold NPs in citrate 

70 

JRC laboratory 

Carboxylate Polystyrene (slightly hydrophilic) 

500 (commercial reference) 

Polysciences, Inc. 

Hydrophobic TiO2 Rutile 

21 (primary size) 

JRC repository 

TiO2 Anatase, E171* 

20-300 

Farmalabor 

*Farmalabor technical datasheet 

The JRC sent samples of a minimum of three different materials selected from the five shown in Table 2 to 
each participant laboratory.  The M2 and M3  samples were analysed by five laboratories, M4 and M5 by six 
laboratories and M1, which was also used as a standard control material to assess the collectors’ properties, 
by all the participants. 

Despite  M1  and  M3  are  not  strictly  nanomaterials  (i.e.  diameter  >100  nm),  they  were  chosen  as  control 
samples (one hydrophobic and one hydrophilic) due to their large light scattering intensity, which makes them 
easily  detectable  in  Dark  Field  microscopy  (DFM).  Because  of  the  direct  relationship  between  the  scattering 
property and the type and size of a material, only nanomaterials with  a significant light scattering intensity 
can be selected as control. 

Table 3. Materials distributed to the ILC participants 

Lab. 

M1 

M2 

M3 

M4 

M5 

1 

2 

3 

4 

5 

6 

7 

8 

9 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

x 

The material was dispersed in an aqueous solution of fixed ionic strength (phosphate buffer (PB) 10 mM, pH 
7) to perform the test. Materials M1, M2 and M3 were provided to participants as dispersions while material 
M4 and M5 were provided as powders together with the buffer in which they had to be be dispersed and the 
dispersion protocol. The M4 powder was not dispersible directly in PB, therefore a stabilising agent was added. 

7 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2.2  Collectors and Samples characterisation 

The  collectors’  surfaces  were  characterised  using  several  techniques.  The  thickness  and  refractive  index  of 
each  deposited  layer  were  measured  by  Ellipsometry  (Vase  VUVTM  J.A.  Woollam  Co.,  United  States).  The 
contact angle of the modified glass slides was measured by using a DigidropTM goniometer  (GBX Scientific 
ltd,  France)  with  a  water  droplet.  Finally,  a  control  test  based  on  DFM  with  a  known  commercial  standard 
sample (Polybead® Polystyrene Microspheres 0.5 µm, Polysciences, Inc., United States), was performed. 

The size measurement of M1, M2, M3, M4 and M5 samples was performed by Dynamic Light Scattering (DLS, 
Zetasizer,  Malvern  Panalytical,  UK).  Size  distribution  and  ζ-potential  were  measured  in  10  mM  phosphate 
buffer  pH  7.  All  the  measurements  were  repeated  several  times  over  a  period  of  2  weeks  in  order  to 
guarantee the stability of the samples. 

2.3  Samples distribution 

The  subsampling  of  the  materials  was  carried  out  in  the  JRC  Nanobiotechnology  Laboratory.  The  test  kit 
(collectors  and  samples),  together  with  the  SOP  and  the  data  reporting  files,  were  sent  to  participants  in 
different periods of 2020/2021, according to their availability to perform the measurements.  

8 

 
3  Ring trial results and considerations 

All  the  participant  laboratories  tested  three  materials.  The  measurements  were  performed  in  three 
independent  replicates  for  the  materials  M1,  M3,  M4  and  M5.  The  material  M2  (citrate  stabilised  gold 
nanoparticles)  was  not  detectable  by  any  of  the  involved  laboratories (except  JRC)  due  to  colloidal  stability 
issues related to the shipment conditions (most probably due to uncontrolled temperature variations). For this 
reason,  the  results  obtained  with  sample  M2  are  not  included  in  the  inter-laboratory  comparison  results 
presented below.  

The objective of the inter-laboratory exercise was to assess the method performance (i.e. transferability) and 
demonstrate  that  the  SOP  is  reproducible  and  robust,  regardless  of  the  DFM  setup  (microscope  trademark, 
light  sources,  optical  configuration,  camera,  etc.).  As  explained  in  the  SOP,  the  data  treatment  was  only 
optional for the participant, and the result obtained from their calculation was not assessed in this ILC. The 
JRC  processed  the  results  of  the  ILC  in  two  main  steps,  as  presented  in  Figure  1.  The  image  sequences 
recorded  in  DFM  and  sent  by  the  participant  were  analysed  to  determine  the  binding  rates  for  the  four 
materials on three collectors (Step 1). The Hydrophobicity index was then calculated (Step 2).  

Figure 1. Calculation flowchart 

The experimental results were assessed for different purposes: 

1.  To demonstrate the  capability of the optical setup to  visualize and detect individual  particle of the 

material M1. 

2.  To measure the  binding  rates of the  materials onto the three different  collectors, which allows the 

assessment of the reproducibility of method.  

3.  To calculate the hydrophobicity index from the experimental data. 

3.1  Detection of the sample M1 by DFM 

The assessment of the particle binding rate on the collector was based on the ability to detect the presence of 
particles by DFM, using the image analysis software Fiji (Schindelin, 2012) with the plugin Trackmate (Tinevez, 
2017).  For  correct  detection  of  individual  particles,  DFM  settings  (light  intensity  and  focus)  and  camera 
parameters (exposure time and gain) must be optimised according to the SOP. 

A single M1 particle is considered detected when light scattered by the particle hits on multiple pixels on the 
camera sensor. The number of pixels and intensity of illumination depend on many factors such as: resolution, 

9 

 
 
 
 
 
 
 
 
sensitivity, dynamic range and dark noise of the camera sensor, irradiance of the light source, quality of optics 
(lens and any intermediate optical components in between), the accuracy and stability of the focusing stage, 
any optical aberrations, and any dust contamination on the surface of the liquid cell. Thus, it was important to 
ensure  that  all  participant  laboratories  were  able  to  generate  similar  and  relevant  “images”  that  allow  to 
detect with appropriate resolution the attached particle. This was done using M1 as reference sample. 

Assuming that the scattering particles are point sources of photon scattering, the real image produced on the 
camera sensor consists of one large spot due to the diffraction limit of light. The image of the particle takes 
the  form  of  a  Point  Spread  Function  (PSF).  At  the  focal  plane,  the  PSF  can  be  fitted  to  a  2D  Gaussian 
distribution (Stallinga, 2010, Zhang, 2007). 

In Figure 2, we show the X-sections of typical M1 particles detected at the collector surface corresponding to 
the  focal  plane,  obtained  by  the  different  participants.  All  X-curves  fit  Gaussian  curves  (MSE  or  chi-square 
>0.6 in all cases), indicating that all DF settings (as described in Table 4) allow detection and resolution 
of a single M1 particle on the surface of the collector according to the suggested SOP. 

Figure 2. X-profile of a single M1 particle obtained by the participants and the relative Gaussian fit  

In the insets, the relative image collected on the camera. 

10 

 
 
Table 4. Summary of the optical setups and the measured resolution of a single M1 particle for each participant 

Participant  Microscope  Configuration 

(Reflection 
/Transmission) 

Camera 
Mpixel) 

(res 

in 

100*FWHM-1 
(pixel-1) 

single 

M1 
particle 
detected 

Lab1 

Lab2 

Lab3 

LEICA 
DMRME 

LEICA 
DM/LM 

OLYMPUS 
BX61 

R 

/ 

/ 

Basler acA4600-10uc 

1.03 

Basler ace 5.1MP Color 

1.06 

OLYMPUS QColor5 CCD 

2.02 

Lab4 

LEICA DMRX 

T 

OLYMPUS UC90 

Lab5 

Lab6 

Lab7 

Lab8 

Lab9 

/ 

/ 

/ 

R 

T 

LEICA 
DM6000 

NIKON 
ECLIPSE 
LV100 ND 

ZEISS 
Axioskop 40 

LEICA 
DM2500M 

NIKON 
ECLIPSE 
E800 

DFC 450 

0.98 

1.27 

Color  Camera  Head  DS-
Fi2 

0.74 

CANON 550D 

1.33 

Thorlabs DCC1545M 

1.15 

SMX-150M-E 

2.5 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

Yes 

A  direct  measurement  of  the  lateral  resolution  can  be  obtained  by  inversing  of  the  Full  Width  at  Half 
Maximum (FWHM) value of the fitted Gaussian curve. This value is reported for all the participants in Table 4. 
The lower the value for FWHM-1 the lower is the resolution of the optical system (Zhang, 2007). The average 
value for FWHM-1 for all the participants is 0.013 ± 0.007 pixel-1. Only one participant showed a value of the 
FWHM-1 close to the standard  deviation limit (in italic in  the table), indicating a poor  resolution of the  used 
optical system. 

3.2  Evaluation of the reproducibility of the particles binding experiments 

To  assess  the  inter-laboratory  reproducibility  of  the  particles  binding  experiments,  the  JRC  provided  the 
participant  labs  with  a  set  of  collectors  and  materials,  previously  characterised  and  tested  in  the  JRC 
laboratory.  

Since  the  most  important  parameter  of  the  collectors  influencing  the  particles  binding  is  the  hydrophobic 
character (together with the surface ζ potential), the water contact angle (WCA) of the different collectors was 
measured. Other quality control parameters such as the thickness of the fluorocarbon layer (PTFE, T0) and the 
layer refractive index were also measured for every batch of collectors produced. The variability of the WCA 
for the different collectors prepared by the JRC and shipped to the different participants is shown in Figure 3. 
The nine batches correspond to the nine laboratories participating to the ILC.  

11 

 
 
 
 
Figure 3. Variability of the WCA for the different collectors 

The variability of the WCA for the collectors has been assessed with Z-score evaluation. Contact angles of all 
the  collectors  produced  showed  an  absolute  value  for  the  Z-score  lower  than  2,  indicating  a  good 
reproducibility  of  the  fabrication  technique.  The  variability  range  does  not  affect  the  binding  rates 
measurements, as the analysis of the results shows no correlation between these variables. 

The particles binding experiments were conducted by the laboratories according to the SOP, included as Annex 
1  to  this  report.  All  experiments  were  performed  in  three  independent  replicates  and  the  binding  rates 
calculated by averaging six areas for each image sequence. The output of the binding experiments is the time 
sequence  of  images  of  the  collector’s surface  showing  the  moving  particles and  the  bound  particles  at  the 
focal  plane.  The sequence of images  is then analysed using a  combination of  Fiji and its TrackMate plugin. 
This tool enables the identification of the particles irreversibly  bound on the collectors as a function of the 
exposure time. The number of binding particles as a function of time  is plotted in the binding curves. These 
curves show a linear dependency on the square root of the time, as predicted by the theory of diffusion of 
particles  dispersion.  The  slope  of  the  binding  curve  on  the  different  collectors  represents  the  velocity  of 
binding vTi and is measured in number of Nanomaterial (NM) bound per unit of time NM/s1/2. 

vTi,  according  to  the  XDLVO  theory  (Donaldson,  2015)  is  directly  influenced  by  the  energy  barrier  occurring 
between the particles and the surface as explained below. 

—  vT0: binding rate on the hydrophobic collector T0 is directly related to the hydrophobicity of the particles 

—  vT5:  binding  rate  on  the  collector  with  opposite  charge  compared  to  the  particles  (T5  for  the  material 

tested in this ILC), where the electrostatic forces are dominant, represents the maximum binding 

12 

 
 
 
 
 
—  vT4: binding rate on the hydrophilic collector with the same charge of the one of the particles  (T4 for the 
material tested in this ILC) represents the binding velocity close to zero in the presence of a high energy 
barrier 

The  values  of  vTi  measured  using  the  images  collected  by  the  different  ILC  participants,  for  the  different 
materials and on the three different collectors are shown in Table 5. 

13 

 
 
 
Table 5. Inter-laboratory results for the binding rates for the different materials on the different collectors 

VT0 

STD 

Z-
score 

VT4 

STD 

Z-score 

VT5 

STD 

Z-
score 

VT0 

STD 

Z-
score 

VT4 

STD 

Z-
score 

VT5 

STD 

Z-score 

M1 

M3 

Lab1 

0.533 

0.182 

-0.439 

0.340 

0.293 

73.171 

3.527 

0.200 

0.523 

0.967 

0.359 

0.101 

0.060 

0.078 

0.900 

4.727 

3.395 

0.580 

Lab2 

0.410 

0.216 

-0.608 

0.013 

0.006 

0.219 

3.457 

0.202 

0.359 

0.897 

0.491 

-0.040 

0.017 

0.012 

-0.660 

3.610 

0.075 

0.044 

Lab3 

1.027 

0.825 

0.238 

0.020 

0.000 

1.708 

3.263 

0.227 

-0.094 

0.990 

0.295 

0.148 

0.037 

0.025 

0.060 

3.220 

1.876 

-0.143 

Lab4 

1.423 

1.427 

0.783 

0.010 

0.000 

-0.525 

3.183 

0.897 

-0.281 

Lab5 

0.970 

0.759 

0.161 

0.050 

0.020 

8.407 

2.070 

0.394 

-2.886 

Lab6 

0.247 

0.012 

-0.832 

0.010 

0.000 

-0.525 

0.640 

0.468 

-6.233 

Lab7 

0.963 

0.755 

0.151 

0.013 

0.006 

0.219 

3.423 

0.012 

0.281 

1.383 

0.501 

0.939 

0.067 

0.021 

1.140 

3.297 

1.054 

-0.106 

Lab8 

0.803 

0.910 

-0.068 

0.010 

0.000 

-0.525 

2.547 

2.227 

-1.771 

0.347 

0.330 

-1.147 

0.020 

0.017 

-0.540 

2.737 

2.356 

-0.375 

1
4

Lab9 

1.300 

0.087 

0.614 

0.137 

0.170 

27.762 

2.967 

0.397 

-0.788 

M4 

M5 

0.250 

0.036 

-0.077 

0.093 

0.006 

0.829 

1.083 

0.295 

1.024 

0.133 

0.058 

-0.538 

0.037 

0.006 

0.960 

1.967 

0.404 

0.648 

0.253 

0.032 

-0.033 

0.077 

0.107 

0.355 

0.667 

0.050 

-0.209 

0.240 

0.174 

-0.256 

0.017 

0.012 

-0.611 

1.667 

0.084 

0.072 

0.247 

0.136 

-0.121 

0.027 

0.029 

-1.066 

0.477 

0.214 

-0.771 

0.773 

0.593 

1.155 

0.020 

0.017 

-0.349 

2.100 

0.580 

0.904 

0.173 

0.006 

-1.093 

0.035 

0.035 

0.450 

0.440 

0.137 

-0.880 

0.183 

0.006 

-0.406 

0.017 

0.006 

-0.611 

1.590 

0.104 

-0.075 

0.347 

0.040 

1.203 

0.015 

0.015 

-0.213 

1.020 

0.161 

0.836 

0.527 

0.546 

0.503 

0.037 

0.006 

0.960 

1.483 

0.214 

-0.279 

0.163 

0.071 

-0.459 

0.020 

0.010 

-0.349 

0.967 

0.667 

-1.270 

Lab1 

Lab2 

Lab3 

Lab4 

Lab5 

Lab6 

Lab7 

Lab8 

Lab9 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The  statistical  analysis  of  the  ILC  data  was  performed  using  the  dedicated  software  Prolab  (Quodata, 
Dresden), according to the International Standard ISO 5725-2. The basis for scoring of the results is the "Z-
score". The Z-score of all the results from the participating laboratories, including those eliminated as outliers, 
is  calculated  with  the  aid  of  the  outlier-free  mean  value  and  the  outlier-free  standard  deviation.  It  can  be 
regarded as the quality characteristic of the mean value of the individual laboratories. The analysis is based 
upon the following formula: 

Z = (Cjk

* - Ck) / s 

Cjk

* = individual mean value 

Ck  = Total mean value/reference value  

s   = Maximum permissible deviation from the reference value (as a rule 10%)  

A  permissible  deviation  of  10%  is  assumed  for  s  during  analysis  of  the  Proficiency  Testing  (PT)  scheme. 
Where data material exhibits strong scatter the permissible deviation may be increased to up to 20%.  The 
individual results are then evaluated as shown below: 

—  |Z| ≤ 1 Good result  

—  1 ≤ |Z| ≤ 2 Satisfactory result  

—  2 ≤ |Z| ≤ 3 Questionable result  

—  3 ≤ |Z| Extremely questionable result  

A result for which |Z| ≤ 2 is deemed satisfactory, i.e. the PT is deemed passed. Should the Z-score exceed 2, 
review  of  the  analysis  method  employed  is  advisable.  The  Z-score  can  be  used  to  determine  further 
statistically important parameters, which shall not however be considered in any greater detail at this point. 

The large majority of measurements are providing a |Z-score| < 2 for all the tested materials on the T0 and 
T5 collectors, which allows considering the obtained results satisfactory. Only  two measurements (Lab5 and 
Lab6 measurements of M1 on T5) have a higher Z-score which underlines the presence of an error affecting 
the measurement. For example it is possible to relate the underestimation of the binding rate on T5 for M1 
for the Lab6 to a poor resolution of the optical configuration used (as shown in the evaluation of the FWHM 
presented  in  table  4).  Another  explanation  could  be  an  accidental  occurrence,  as  the  lack  of  focus,  or  the 
presence of some interferences on the optical path. The higher Z-scores obtained on the collector T4 for M1 
are not considered relevant since this collector is only used as a negative control for the tested materials (the 
collector’s  surface  being  negatively  charged  in  the  measurement  conditions,  as  the  tested  materials),  and 
provides a very low binding rate. 

As  a  general  comment,  all  the  materials  on  the  different  collectors  behave  according  to  the  expectations 
predicted by the XDLVO theory, with a maximum binding rate on T5, a binding rate close to zero on T4 and a 
variable binding rate on T0, depending on the material’s hydrophobicity.  

3.3  Evaluation  of  the  reproducibility  of  the  calculation  of  the  Hydrophobicity 

index  

The binding rates are used for calculation of the hydrophobicity index Hy. It is defined as the logarithm of the 
ratio between the values of the binding rate on the hydrophobic collector vT0, and on the hydrophilic collector 
inducing electrostatic attractive forces vmax (T5 for negatively charged material as it is generally the case in 
the medium of measurement and T4 for positively charged material). The closer to zero the more hydrophobic 
a NM is considered. 

15 

 
 
 
 
 
  
 
 
The ILC data on the hydrophobicity index for the different materials are shown in Figure 4.  

Figure 4. Inter-laboratory variability charts for the Hydrophobicity index Hy for the different materials  

The  main  conclusion  that  can  be  drawn  from  figure  4  comes  from  the  |Z-scores|,  which  are  all  <  2,  thus 
demonstrating  satisfactory  results  for  the  calculation  of  the  Hydrophobicity  index  for  all  materials  and  all 
participants. It should also be noted that 23 out of the 25 |Z-scores| determined are < 1, which corresponds to 
a good result for the proficiency test according to ISO 5725-2. Moreover, experimental variations such as the 
particles  concentration,  that  could  affect  the  reproducibility  of  the  binding  rates  on  the  different  collectors, 
are not affecting the reproducibility of the Hy since it is calculated from the ratio of the binding rates. This is 
reflected by the absolute values of Hy for the Z-scores < 2 for all materials.   

As  a  general  comment,  according  to  the  Z-score  calculated  for  each  data  produced  by  the  different 
participants, the reproducibility of the binding curves and the relative calculation of the hydrophobicity index is 
considered to be satisfactory.   

Two other important results should be underlined: 

—  all the participants were able to detect the different materials with their microscope and camera setup, 

by following the SOP  

—  no correlation was observed between the WCA (fig 3.) for the collectors and the measured data 

These two points demonstrate that the experimental setup of the SOP is robust. This is reflected in the good 
reproducibility of data according to the statistical evaluation using the Z-scores.  

16 

 
 
 
 
 
4  Conclusions 

Nine  laboratories  participated  in  this  ILC  to  determine  the  hydrophobicity  index  of  nanomaterials  through 
affinity measurements. Despite differences in detection systems (cameras and microscopes), all participants 
were able to detect and resolve particles of materials M1, M3, M4 and M5 following the proposed SOP, while 
one  material  (M2)  was  not  detectable  and  quantifiable  by  the  involved  laboratories  due  to  sample  stability 
issues. Specifically, for sample M1, it is possible in all cases to fit the particle scattering curve with a Gaussian 
curve  (MSE  >  0.6).  Furthermore,  when  analysing  the  mean  FWHM  values  for  the  fitted  Gaussian,  we  found 
that only one participant obtained a value close to the STD limit due to a lack of resolution. 

The  WCA  values  shown  in  Figure  2  emphasize  the  very  good  repeatability  of  the  collector  fabrication 
technique. 

The reproducibility of particle binding experiments for different materials on the three different collectors was 
satisfactory,  as  indicated  by  the  calculated  Z-score  values  (with  two  exceptions  for  M1  on  T5).  All  tested 
materials on the different collectors showed behaviour consistent with the predictions of the XDLVO theory, 
with  maximum  binding  on  T5,  close  to  zero  binding  on  T4  and  variable  binding  on  T0  depending  on  the 
material’s hydrophobicity. 

The reproducibility of the calculation of the hydrophobicity index Hy was also assessed. Data collected were 
satisfactory, with an absolute value for the Z-score < 2. 

We may also conclude that organising training on DFM use and sharing best laboratory practices could help to 
improve unsatisfactory results and optimise the application of the TG for future users. 

17 

 
References 

Desmet,  C.,  Valsesia,  A.,  Oddo,  A.,  Ceccone,  G.,  Spampinato,  V.,  Rossi,  F.,  and  Colpo,  P.,  Characterisation  of 
nanomaterial hydrophobicity using engineered surfaces, Journal of Nanoparticle Research, 19, 117, 2017. 

Donaldson,  S.H.,  Røyne,  A.,  Kristiansen,  K.,  Rapp,  M.V.,  Das,  S.,  Gebbie,  M.A.,  Lee,  D.W.,  Stock,  P.,  Valtiner,  M., 
Israelachvili,  J.,  Developing  a  General  Interaction  Potential  for  Hydrophobic  and  Hydrophilic  Interactions, 
Langmuir, 31, 7, 2015, pp. 2051-2064. 

European Chemical Agency (2019), Guidance on information requirements and chemical safety assessment. 
Appendix R.6-1 for nanoforms applicable to the Guidance on QSARs and Grouping of Chemicals, 2019. 

European Chemical  Agency (2022), Guidance on information requirements and  chemical safety assessment. 
Appendix R7-1 for nanomaterials applicable to Chapter R7a Endpoint specific guidance. Version 4.0, 2022.  

ISO  (2019),  Nanotechnologies  —  Considerations  for  performing  toxicokinetic  studies  with  nanomaterials, 
ISO/TR 22019:2019. 

NANoREG (2015), Experimental evaluation of OECD methods for analysis of physicochemical MNM properties, 
Deliverable 2.3.  

OECD  (2009),  Preliminary  Review  of  OECD  Test  Guidelines  for  their  Applicability  to  Manufactured 
Nanomaterials, OECD Series on the Safety of Manufactured Nanomaterials, No. 15, OECD Publishing Paris. 

OECD  (2014),  Report  of  the  OECD  Expert  meeting  on  the  Physical  Chemical  Properties  of  Manufactured 
Nanomaterials and Test Guidelines, OECD Series on the Safety of Manufactured Nanomaterials, No. 41, OECD 
publishing, Paris. 

OECD  (2017),  OECD  test  guidelines  and  guidance  documents  for  their  applicability  to  nanomaterials: 
preliminary  status  review,  17th  Working  Party  on  Manufactured  Nanomaterials,  10-12  May  2017,  OECD 
publishing, Paris. 

OECD  (2019),  Physical-Chemical  Decision  Framework  to  Inform  Decisions  for  Risk  Assessment  of 
Manufactured  Nanomaterials,  OECD  Series  on  the  Safety  of  Manufactured  Nanomaterials,  No.  90,  OECD 
publishing, Paris. 

Prolab, from Quodata, Dresden. 

Schindelin,  J.,  Arganda-Carreras,  I.,  Frise,  E.,  Kaynig,  V.,  Longair,  M.,  Pietzsch,  T.,  Preibisch,  S.,  Rueden,  C., 
Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P.l and Cardona A., Fiji: 
an open-source platform for biological-image analysis, Nature Methods, 9, 2012, pp. 676–682. 

Stallinga,  S.,  and  Rieger,  B.,  Accuracy  of  the  Gaussian  Point  Spread  Function  model  in  2D  localization 
microscopy, Optics Express, 18, 2010, pp. 24461-24476. 

Tinevez, J.-Y., Perry,  N., Schindelin, J., Hoopes,  G.M., Reynolds, G.D., Laplantine, E., Bednarek, S.Y., Shorte, S.L., 
and Eliceiri K.W., TrackMate: An open and extensible platform for single-particle tracking, Methods, 115, 2017, 
pp. 80-90. 

Valsesia, A., Desmet, C., Ojea-Jiménez, I., Oddo, A., Capomaccio, R., Rossi, F., and Colpo, P., Direct quantification 
of nanoparticle surface hydrophobicity, Communications Chemistry, 2018, pp. 1: 53. 

Van Oss, C. J., Acid–base interfacial interactions in aqueous media, Colloids and Surfaces A: Physicochemical 
and Engineering Aspects, 78, 1993, pp. 1–49. 

Zhang, B., Zerubia, J., and Olivo-Marin, J.C., Gaussian approximations of fluorescence microscope point-spread 
function models, Applied Optics, 46, 2007, pp. 1819-1829. 

18 

 
 
List of abbreviations and definitions 

DF 

DFM 

DLS  

ΔG 

Dark Field 

Dark Field Microscopy 

Dynamic Light Scattering 

Energy barrier 

FWHM 

Full Width at Half Maximum 

ILC 

JRC 

mM 

Inter-Laboratory Comparison 

Joint Research Centre 

millimolar 

MNMs  Manufactured NanoMaterials 

MSE 

Mean Squared Error 

M1 

M2 

M3 

M4 

M5 

polystyrene beads 

gold nanoparticles 

carboxylate polystyrene beads 

hydrophobic TiO2 

hydrophilic TiO2 

NPs 

NanoParticles 

OECD 

Organisation for Economic Co-operation and Development 

PB 

PSF 

Phosphate Buffer 

Point-Spread Function 

PTFE 

Polytetrafluoroethylene 

STD 

SOP 

T0 

T4 

T5 

Standard Deviation 

Standard Operating Procedure 

PTFE-like hydrophobic collector 

hydrophilic and negative collector (PTFE-like + 4 PE layers) 

hydrophilic and positive collector (PTFE-like + 5 PE layers) 

TEM 

Transmission Electron Microscopy 

TG 

v 

Test Guideline 

binding velocity 

WCA 

Water Contact Angle 

XDLVO 

eXtended Derjaguin Landau Van Overbeek 

19 

 
 
 
List of figures 

Figure 1. Calculation flowchart ................................................................................................................................................................................9 

Figure 2. X-profile of a single M1 particle obtained by the participants and the relative Gaussian fit ................. 10 

Figure 3. Variability of the WCA for the different collectors ............................................................................................................. 12 

Figure 4. Inter-laboratory variability charts for the hydrophobicity index Hy for the different materials ........... 16 

20 

 
 
List of tables  

Table 1. Laboratories participating in the ILC .................................................................................................................................................6 

Table 2. Selected materials for the ILC ...............................................................................................................................................................7 

Table 3. Materials distributed to the ILC participants ................................................................................................................................7 

Table 4. Summary of the optical setups and the measured resolution of a single M1 particle for each 
participant ............................................................................................................................................................................................................................ 11 

Table 5. Inter-laboratory results for the binding rates for the different materials on the different collectors 14 

21 

 
 
Annexes 

Annex 1. Standard operating procedure (SOP) for the determination of the hydrophobicity index of 
nanomaterials according to the advanced draft test guideline 

1. OBJECT AND SCOPE 

This  Standard  Operating  Procedure  (SOP)  aims  at  the  determination  of  the  surface  hydrophobicity  of 
manufactured nanomaterials as dispersed in an aqueous solution.  It is intended for the sole use in an inter-
laboratory test exercise to ascertain the transferability and reproducibility of the method described in the Test 
Guideline in preparation, in particular the measurement protocol. The data treatment is also included but will 
not be part of the assessment of the ILC. 

The surface hydrophobicity of the nanoparticles dispersed in water is determined by measuring their binding 
rate to different engineered surfaces (collectors). The method is based on two articles, published by Desmet 
et al. (2017) and Valsesia et al. (2018), where the measurement of the binding rates of the nanoparticles to 
the engineered collectors is used to mathematically derive the polar component of their surface free energy, 
according to the XDLVO (eXtended Derjaguin Landau Van Overbeek) theory. 

2. TERMS AND DEFINITIONS 

For the purpose of this procedure, the following abbreviations and definitions apply: 

DF 

DLS 

Dark Field 

Dynamic Light Scattering 

ΔGmax  

energy barrier  

𝐴𝐵 

𝛾𝑁

g 

kT 

λ 

m 

mS 

MΩ 

mol 

nm 

polar component of the surface free energy of the particle 

gram 

product of Boltzmann constant and temperature 

hydrophobic interaction distance 

metre 

sample mass 

MegaOhm, electric resistance 

mole 

nanometre 

PB        phosphate buffer 

PDDA 

poly(diallyldimethylammonium chloride) 

PE 

PSS 

polyelectrolyte 

poly(sodium 4-styrenesulfonate) 

PTFE 

polytetrafluoroethylene 

ρ 

RMS 

SOP 

T0 

T4 

T5 

density (g/cm3) 

root mean square 

Standard Operating Procedure 

PTFE-like Hydrophobic collector 

Hydrophilic and negative collector (PTFE-like + 4 PE layers) 

Hydrophilic and positive collector (PTFE-like + 5 PE layers) 

22 

 
 
 
 
v 

vmax 

Vcell 

Vref 

wt 

velocity of adsorption 

maximum velocity of adsorption 

sample chamber volume (cm3) 

reference chamber volume (cm3) 

weight percent 

XDLVO   eXtended Derjaguin Landau Van Overbeek 

zi 

particle-collector distance 

3. PRINCIPLE OF THE TEST 

The test enables the quantification of the polar component of the surface free energy of nanomaterials and, 
consequently, of their surface hydrophobicity. This is achieved by measuring the binding rate of the dispersed 
particles to different engineered surfaces called collectors and by transferring these experimental values into 
a system of equations derived from the XDLVO theory for spherical particles (Desmet 2017, Valsesia 2018). 

A collector is characterised by the following properties: 

−  Low surface roughness (Root Mean Square roughness < 5 nm)  

−  A certain value of the polar component of the surface free energy  

−  A certain value of the streaming potential  

The particles are dispersed in an aqueous medium in conditions of colloidal stability (according to  OECD TG 
318).  The  dispersion  is  brought  to  the  collectors  by  means  of  a  liquid  cell  and  allowed  to  approach  to  the 
collector  surface  by  diffusion.  The  number  of  particles  binding  to  the  collector  as  a  function  of  time 
determines their affinity to the collector. The counting of bound particles as a function of time is done by Dark 
Field microscopy and a camera. The Trackmate plugin of Fiji software is then used to automatically detect the 
particles  and  to  build  the  binding  curves.  The  test  returns  the  number  of  particles  binding  as  a  function  of 
time on the different collectors.  

As a first result of the method, these experimental data can be used to qualitatively assess the nanomaterial 
hydrophobicity (Desmet et al. 2017, Valsesia et al. 2018).  As a general indication, the larger the affinity to a 
hydrophobic collector is, the higher the nanomaterial hydrophobicity is. 

The binding rate of the NMs to the different collectors is regulated by the XDLVO forces, where Hydrophobic 
forces are added to the DLVO model) and kinetics limitations imposed by the diffusion of the NMs (16). The 
XDLVO interaction energy between NMs and surface determines the formation of an energy barrier inhibiting 
the binding. The XDLVO theory applies to stable NM dispersions in aqueous samples. 

When the energy barrier is comparable  with the thermal  energy, the NM is able to bind to the  surface in a 
stable thermodynamic state. This happens when Van der Waals or hydrophobic forces can counterbalance the 
electrostatic repulsion. When the electrostatic forces are attractive, the only factor limiting the binding rate of 
the particles to the surface is the transport of the particles. The reaction balance can be written as:  

[𝑁𝑀]   +   [𝑆]   =   [𝑁𝑀 − 𝑆]      

Where  [𝑆]  is  the  binding  site  on  the  surface  and  [𝑁𝑀 − 𝑆]  is  the  complex  nanomaterial-binding  site.  The 
equilibrium constant of the reaction is the proportion of the products and the reactants:  

𝐾𝑒𝑞  =   [𝑁𝑀 − 𝑆]/([𝑁𝑀] + [𝑆])   

The equilibrium constant 𝐾𝑒𝑞 cannot be determined since the reaction occurs only in one direction. Thus, it is 
impossible  to  determine  the  interfacial  Gibbs  free  energy  of  adhesion  using  a  single  surface.  The 
thermodynamic  relation  between  the  Gibbs  free  energy  and  the  equilibrium  constant  is  expressed  by  the 
equation:  

𝛥𝐺0  =   −𝑘𝑇𝑙𝑛𝐾𝑒𝑞   

23 

 
 
  
 
Where 𝑘 is the Boltzmann constant and 𝑇 the temperature. On the other hand, 𝛥𝐺0 is the limiting parameter 
that inhibits the binding of NMs to the surface. According to the Boltzmann distribution the binding rate on a 
surface is: 

𝑣  =  𝑣𝑚𝑎𝑥𝑒−(𝛥𝐺0/𝑘𝑇)  

where  𝑣𝑚𝑎𝑥 is the maximum binding  rate (for a  spontaneous  reaction limited only by the  transport of the 
particles to the surface). Thus, 𝛥𝐺0/𝑘𝑇 is:  

−𝛥𝐺0/𝑘𝑇  =  𝑙𝑛(𝑣/𝑣𝑚𝑎𝑥)   

A Hydrophobicity index (𝐻𝑦) is defined, based on the affinity of the material for the hydrophobic collector as 
compared  to  a  hydrophilic  one  which  favours  the  electrostatic  attraction  forces  (the  reference  collector  on 
which the binding velocity is maximum). 𝐻𝑦 can be expressed as:   

𝐻𝑦  =  𝑙𝑜𝑔(𝑣𝐻𝑦/𝑣𝑚𝑎𝑥)  

𝐻𝑦  is  a  direct  measurement  of  the  tendency  of  the  NM  to  bind  to  the  hydrophobic  collector,  rather  than 
staying in the aqueous phase, which is directly related to the NMs hydrophobicity character. 

In  the  method,  𝑣𝐻𝑦  is  the  binding  rate  on  the  hydrophobic  collector,  which  is  directly  related  to  the 
hydrophobicity of the NM, i.e., where binding is driven by hydrophobic interactions between the NMs and the 
collector.  𝑣𝑚𝑎𝑥  is  measured  on  the  collector  on  which  the  binding  rate  is  maximised  by  favouring 
electrostatic  interactions.  NMs  being  in  most  cases  negatively  charged  in  the  measurement  conditions,  the 
binding rate to the hydrophilic positively charged collector is only limited by the NMs transport to the surface. 
For positively charged NMs the binding rate on the hydrophilic negatively charged collector is used instead. 

Since  𝑣𝐻𝑦/𝑣𝑚𝑎𝑥  is  always  <  1,  the  𝐻𝑦  values  are  always  <  0. 𝐻𝑦  values  close  to  zero,  indicate  a  high 
affinity to the hydrophobic collector (as show in figure 2), i.e., particles behaving as hydrophobic chemicals. As 
an  indication,  a  NM  is  considered  hydrophobic  when  the  value  of  this  index  is  between  0  and  -1,  while  a 
hydrophilic  one  shows  values  lower  than  -1.  The  closer  to  0  the  𝐻𝑦  is,  the  more  hydrophobic  the  NM  is.

24 

 
 
Figure 2. Principle of the method for the quantification of the hydrophobicity index of NMs. The NMs in stable 
dispersion are allowed to approach the collector by diffusion. (a). The energy balance described by the XDLVO 
theory drives the stable binding of the hydrophobic NM to the hydrophobic collector (situation in column 1), 
while  the  hydrophilic  NM  is  repelled  (situation  in  column  2)  (b)  The  square  root  of  the  number  of  bound 
particles is presented as a function of the square root of time. The binding rate on the hydrophobic collector 
𝑣𝐻𝑦 is plotted for the NMs with respect to the maximum binding rate 𝑣𝑚𝑎𝑥 occurring when the electrostatic 
forces are dominating. The closer the slope for 𝑣𝐻𝑦 is to 𝑣𝑚𝑎𝑥, the more hydrophobic a particle is. 

4. SAMPLES, REAGENTS, EQUIPMENT AND MATERIALS 

Nanomaterial samples 

Samples  of  nanomaterial  dispersions  will  be  sent  by  the  JRC  to  the  participating  laboratories.  Material  M1 
(control sample) will be sent to all the participants, while the other four materials will be distributed randomly 
in order to have a desirable number of results for each material. 

Material needed for binding rate measurement  

—  3 collectors, one of each type 

—  Nanomaterial samples, dispersed in Phosphate Buffer (PB), 10 mM, pH 7 

—  Vortex 

—  Calibrated pipettes (0.02 mL volume) 

—  Microscope  with  Dark  Field  configuration  (either  in  reflection  of  transmission  mode),  equipped  with  a 

camera and a 10x objective 

25 

 
 
 
 
 
 
 
 
 
5. SAMPLE PREPARATION 

The  JRC  will  provide  ready-to-use  collectors.  They  will  come  numbered  for  traceability  and  protected  in 
individual re-sealable plastic bags.  

—  Collector 1: T0, fluorocarbon coated glass slide, hydrophobic  

—  Collector 2: T4, PTFE coated glass slide + 4 polyelectrolyte layers, hydrophilic and negatively charged 

—  Collector 3: T5, PTFE coated glass slide + 5 polyelectrolyte layers, hydrophilic and positively charged 

Nanomaterial preparation 

Dispersion of the nanomaterial 

To  perform  the  test,  the  material  has  to  be  dispersed  in  an  aqueous  solution  of  fixed  ionic  strength.  It  is 
recommended to use a dispersion protocol that guarantees stability of the colloidal dispersion (according to 
OECD  TG  318)  without  modification  of  the  surface  properties  and  chemical  nature  (e.g.  coating)  of  the 
particles. Material 1, 2 and 3 are provided as dispersions. Material 4 and 5 are provided as a powder together 
with the buffer in which it should be dispersed. 

Characterisation of the nanomaterial 

The  obtained  dispersion  solutions  have  been  used  for  the  characterisation  of  the  NM  before  sending.  Size 
distribution  and  ζ-potential  were  measured  through  Dynamic  Light  Scattering  and  Transmission  Electron 
Microscopy. For comparison  purposes,  aliquots of all  samples have  been tested at the JRC prior  to sending, 
using the proposed method for hydrophobicity determination. 

6. OPERATING PROCEDURE 

You will use Fiji for the different steps of the procedure. If Fiji is not installed on your computer, you can find 
it in the USB drive, in the Tools for data treatment folder. To install it, go to the Fiji.app folder and double click 
on Fiji-win64.exe. 

Measurement of the nanomaterial binding rate to the collectors 

Calibration of Fiji 

A calibration of both the microscope’s camera and Fiji must be done before performing the experiments with 
the material. First, the Calibration chip is used to calibrate Fiji for further image treatment as described in the 
following steps.  

a.  Place the chip under the 10x objective of a DF microscope connected to a camera. When using a 
transparent substrate (e.g. glass), both transmission and reflection mode are possible depending on 
the highest contrast obtained. To use the microscope, please refer to the equipment user's manual.   

b.  Adjust  the  focus  on  the  centre  of  the  chip  with  the  10x  objective  and  save  an  image  as 

“Size_Calibrant” in Tiff format in the Calibration folder.  

c.  Open Fiji and in the File menu choose Open and select your Size_Calibrant image.  

d.  Draw a line using the Straight tool, of the same length as the square as shown in figure 2.  

e. 

In the  Analyze menu, select  Set Scale and fill in the boxes “Known Distance” =  135.00, “Unit of 
length” = um, “Global” = ticked.  

26 

 
 
 
 
 
 
 
 
 
 
Fiji  is  now  calibrated  for  all  the  images  and  must  be  kept  open  (if  closed,  the  calibration  must  be 
repeated). Alternatively, if your camera provides a calibration bar, it can be used for calibration.   

Figure 2. Size calibration in Fiji  

Setting up of the microscope and camera 

To set  up the microscope and  camera, the T0 collector is used.  A line  has been drawn in the middle of the 
slide on the whole length, crossing all the channels. This line will be used to adjust the focus on the surface. 
The first channel on the left side is sealed and must not be opened. It contains particles bound to the surface 
of  the  collector  and  serves  as a  calibration  specimen  for  setting  up  the  parameters  of  the  microscope  and 
camera  (coarse  focus,  illumination  intensity,  exposure  time,  contrast,  etc.).    In  order  to  check  the  quality  of 
your set up, follow the steps described below 

a.  Position the first channel under the objective and adjust the focus on the line in the middle.  

b.  Move  the  microscope  stage  to  position  the  line  at  the  top  or  bottom  of  the  field  of  view  and  re-
adjust the focus on the bright dots, which are particles bound to the surface. The optical zoom of 
the camera software can be used for a finer adjustment of the focus.  

c.  Optimize  light  illumination  of  the  microscope  and  exposure  time  of  the  camera.  Disable  all 
automatic adjustments of image quality from the camera software (only adjust the exposure time, 
example of parameters used with the camera software of JRC are provided in the USB drive in the 
Example  folder).  When  satisfied  with  the  quality  (you  can  observe  clear  bright  spots  on  a  dark 
background), register an image as “Setup Evaluation” in Tiff format and save it in the  Calibration 
folder. You can proceed to the evaluation of the quality of your image.  

d.  Use Fiji (already opened in the previous calibration step) to  open the image: File menu, Open your 
Setup  Evaluation  image.  The  image  should  be  sized  in  µm  x  µm  as  shown  in    figure  3  (if  not,  the 
calibration  step  described  above  was  not  saved  and  should  be  repeated  on  the  previously  saved 
Calibration image).  

e.  Transform the image format: in the Image menu, Type, select 8-bit.  

f.  Zoom on an inter-particles area, select the area (pay attention not to include any particle) with 
the Rectangle tool and specify a size of 10 x 10 µm2 as in the figure below using the  Edit menu, 
Selection  Specify.  

27 

 
 
 
 
 
g.  Acquire the histogram of the selection using the Analyze menu, Histogram. 

h.  Open the Setup Evaluation excel file and annotate background Mean and StdDev values. 

i. 

Zoom on one of the closest particle and select its centre with the Rectangle tool, specify a size 
of 1 x 1 µm2 using the Edit menu, Selection  Specify.  

j.  Acquire the histogram of the selection as previously done and annotate Mean value in the Setup 
Evaluation  excel  file.  Your  setup  (microscope  and  camera)  is  satisfactory  if  the  result  given  in  the 
Setup Evaluation Excel file is >10.  

If  quality  check  is  not  satisfactory,  please  contact  us  before  proceeding  with  the  rest  of  the 
experiments. 

Figure 3. Evaluation of the Microscope and Camera set up in Fiji  

Dark Field measurement of the nanomaterial binding rate to the collectors 

Once  the  microscope  and  camera  parameters  are  set,  they  should  not  be  modified  for  the  rest  of  the 
measurements. Only adjustment of the focus between one channel and the other is allowed. It is advised to 
work with the channels from left to right to avoid any confusion, and to mark used channels in proximity of 
the inlet at the end of each measurement. Follow the steps described below. 

a.  Before  starting  a  measurement,  open  the  camera  software  and  create  a  folder  to  save  the 
images.  It  is  recommended  to  create  one  folder  for  each  measurement:  one  main  folder  for  the 
particle  type  (numbered  with  the  replicate,  e.  g.  M1-r1),  and  inside  three  folders  named  with  the 
collector  type  (T0,  T4  and  T5).  The  path  to  the  folder  should  then  be  in  the  format:  …\ILC 
Hydrophobicity Results\Sample code\Sample code-replicate number\Collector type.  

b.  Set up the camera to record 1 image every 30 s for 12 min, for a total of 25 images.  

c.  First, adjust the focus on the central line of the left channel, then place the channel chosen 

for the experiment under the objective.  

d.  Take  the  collector  from  the  microscope  stage  paying  attention  not  to  move  the  stage  (if  it  is 
necessary to modify the horizontal and vertical position of the stage to get the collector, the original 
position in focus must be recorded to reach it back quickly).  

e.  Vortex the particles dispersion for 30 sec and fill the selected channel with 20 µL of sample 
with  a  micropipette.  For  an  optimal  channel  filling,  it  is  important  to  place  the  tip  of  the 
micropipette  at  the  vertical  (90°  from  the  surface)  inside  the  loading  well  and  lean  on  the 

28 

 
 
 
 
 
 
well’s bottom. The user should be able to see by naked eyes the liquid flowing inside the cell until 
coming to the surface of the outlet. If the loading well seems to be filled and not the channel, the 
liquid  can  be  pipetted  back  and  injected  again  carefully  following  the  instructions  on  tip  position. 
Verify that no air bubble is trapped inside the channel. When the sample reach the outlet, the liquid 
should  be  slightly  taken  back  without  drying  the  surface,  the  injection  is then  pursued  until  all  the 
sample is injected, and the tip removed.  

f.  Place the collector back on the microscope stage, at the original position. Adjust the position of 
the stage  so  that  the  line  is visible  at  the  bottom of  the  field  of  view,  and  re-adjust  finely  the 
focus (you can use the zoom on the camera software to be well focused). If available, the autofocus 
option of the microscope can be used.  

g.  Start recording the images (frame recording rate: 30 s, acquisition duration: 12 min,  total number 
of images: 25. The 25 images are automatically saved in the chosen folder. Remember to mark the 
used  channel  at  the  end  of  the  measurement.  For  a  good  reproducibility  of  the  measurement,  the 
time between injection of the sample and the registration start should be kept as short as possible 
(possibly < 1 minute). 

h.  For the next measurement on the same collector, move the stage horizontally along the central line 
to  bring  the  next  channel  under  the  objective.  The  line  will  be  out  of  focus  because  of  the 
different media in the channels (in air or in liquid). When the new sample will be injected, the images 
on the screen should be close to focus and adjusted again finely after zooming on the middle line.  

i.  Repeat the sample filling operation following the same procedure.  

j.  When  measuring  on  a  different  collector,  repeat  the  quality  check  on  the  first  channel  on  the  left 

side. 

The same procedure is followed for each measurement. If possible, the sample should be tested on the three 
different collectors on the same day. The measurement (on the three collectors) is then performed in triplicate 
for each  sample (9 measurements in total per sample). In each Sample Folder, the  user should then obtain 
three folders for the replicates (r1, r2, r3) and inside each of them, three folders for  the different surfaces 
(T0, T4 and T5) each containing 25 images as presented in figure 4. 

Figure 4. Chart of the result folders to obtain 

Image analysis 

The image analysis is performed with the software  Fiji and its TrackMate plugin. A USB drive containing the 
right  version  of  both  the  software  and  the  plugin  will  be  provided,  together  with  macros  simplifying  the 
analysis and example images. The analysis consists in an automatic detection of the particles for each frame, 
and the tracking of their positions within the sequence of frames. The objects not moving for more than two 
frames in a row counting back from the last frame (meaning that they are present at 12 min of analysis) are 

29 

 
 
 
 
 
counted.  This  automatic  calculation  permits  to  obtain  the  number  of  bound  particles  per  unit  of  time, 
corresponding to the velocity of adsorption of the material on the collector. The macro produces a file that is 
saved and imported in Microsoft Excel ® for data treatment and fitting. The analysis is done on six different 
area for each image sequence for statistical analysis.  

Before starting the analysis, you need to install the macros. To do so, in the Fiji Plugins tab, select Macros 
and Install. Select the .txt file Hydrophobicity in the Tools for data treatment folder of the USB drive and Open. 
You can then follow the steps described below. 

a.  Open the image sequence corresponding to the measurement: Open Fiji, File  Import  Image 
sequence.  Select  the  folder  in  which  the  measurement  was  saved:  …\ILC  Hydrophobicity 
Results\Sample  code\Sample  code-replicate  number\Collector  type.  Double  click  on  the  first  image 
and choose the Sequence Options: Number of images 25 ; starting image 1 ; increment 1 ; scale 
image  100%  ;  file  name  contains  *leave  empty*  ;  tick  Convert  to  8-bit  Grayscale  and  Sort  names 
numerically.  A  sequence  of  25  images  should  open,  named  as  the  folder  in  which  the  images  are 
saved. 

b.  To  process  the  image  stack  for  a  better  detection  of  the  particles,  use  the  macro  in  Plugins   
Macros  InitImage. Saturated pixel % is fixed at 0.3 by default, click OK. If your image contrast is 
not high enough (it depends on the collector and the material tested), re-launch the macro putting a 
higher  value  (from  0.3  to  3%,  you  can  modify  for  higher  values  but  not  lower  than  what  you 
previously put, so use small incrementing steps).  

c.  A  yellow  square  has  appeared  as  well  as  the  ROI  manager.  Position  the  square  manually  for 
particle  and  track  detection.  To  do  so,  double  click  on  the  ROI  selection  and  drag  then  the  yellow 
square in a “clean area”,  where there is no light artefact (use the rectangle tool to be able to click 
and drag the square). Do remember that you will have to do the same six times, placing the square in 
six different areas. It is then recommended to move the square from left to right and top to bottom 
to cover the surface as much as possible.  

d.  Launch  the  second  macro  using  Plugins    Macros    TrackMateAnalysis  to  duplicate  the 
chosen area and start the Tracking. Give a name to the stack: the name of the collector (T0, T4 or T5) 
and a number for this duplicate (to increment for the next area, from 0 to 6), T0-1 for instance. 

e.  A window appears to confirm that Z/T will be swapped, click Yes. The macro will process the images 
and when the computation is over the Log window should display the computation time as the last 
line.  TrackMate  v5.0.2  window  should  appear  on  display  options  with  a  button  Analysis.  On  your 
Treated stack, if particles are present, they should be surrounded by a pink circle, and some colourful 
lines or dot should have appeared inside. You can close the Log window and  look at the stack. If 
the particles and pink circles are moving when changing frame, and the ones immobile present a line 
(or dot) inside, your automatic analysis is done and you can follow directly point g., if not follow 
the steps in f. 

f.  On the TrackMate window presenting Display options, click on the left arrow until you reach the LoG 
detector. Here you can modify the estimated blob diameter and threshold and click  preview. Adjust 
the parameters until you have pink circles around the particles. Click on the right arrow  Next and go 
to the Simple LAP tracker. Adjust the two first parameters, click next and check if the visible particles 
close to immobility present a line or dot. If not, go back left and adjust again the parameters. When 
you  are  satisfied  click  Next  until  you  reach  the  Display  options.  Follow  now  the  procedure  as 
described below.  

g.  Click on the Analysis button and save the Track statistics window: File  Save As  give the 
same name as your stack (e.g. T0-1) and save in the folder corresponding to this replicate (e.g. M1-
r1). You can close the windows with the treated area and all the windows that DO NOT have the Fiji 

30 

 
 
 
 
 
 
icon: the window that you just saved, as well as Links in tracks statistics, spots in tracks statistics, the 
two TrackMate v5.0.2 windows and the stack analysed.  

h.  You  are  now  back  on  your  Fiji  stack  with  the  yellow  square.  Drag  the  square  in  another  clean 
area  and  start  again  point  c.  Repeat  this  step  until  you  have  saved  six  .csv  files  for  this 
measurement in your folder. 

i.  Once six areas have been analysed on the collector, close the main stack window (T0, T4 or T5) and 
repeat the procedure starting back from point a. for the other two collectors. Remember to 
always keep Fiji opened as it contains the size calibration information. If the opened stack or image 
appears as pixel x pixel instead of µm x µm, the calibration step must be repeated. At the end of the 
Image analysis you should have 18 .csv files in your sample-replicate folder: T0-1 to -6, T4-1 to -6, 
and T5-1 to -6.  

Data treatment 

The data treatment and the calculation of the Hydrophobicity Index are not assessed in this ILC. 
The main objective of the ILC is to evaluate the reproducibility of the collection of the binding curves.  

7. DATA REPORTING 

The RESULTS file provided as a template for the Test report should be filled in and sent via JRC-Box after the 
test, together with: 

- 

- 

the images 

the .csv files 

8. ESTIMATED TIME 

Collectors preparation  

Provided ready-to-use by JRC 

Nanomaterial samples  

3 different samples will be provided by the JRC to the participating laboratories.  

Measurement 

Setting up of the microscope before starting the test: 15 min 

Measuring and collecting images: 15 min per sample (3) per collector (3) per replicate (3)  

In total 27 measurements 

  Total 7h 

31 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Image analysis 

Fiji + Trackmate with macros: 10 min per measurement 

  Total 4h30 

TOTAL ESTIMATED TIME: 11h30 

9. REFERENCE DOCUMENTATION 

Desmet et al. (2017), J. Nanopart. Res., 19 : 117 

Valsesia et al. (2018), Comms. Chem., 1 : 53 

Van Oss et al. (1986), J. Colloid Interface Sci., 111 : 378 

Donaldson et al (2015), Langmuir, 31 : 7  

Microscope, equipment user’s manual 

32 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex 2. Preparation of the material samples 

Material 1 and 3 

The  samples  of  material  1  and  3  are  provided  as  dispersions  in  numbered  tubes  ILT-M1xxxxxxxx  and  ILT-
M3xxxxxxxx. Prior to the first measurement, they should be sonicated in a sonication bath at a frequency of 
40 kHz for 2 min. (The most common sonication bath, which do not provide the frequency adjustment, works 
at 40 kHz.) The material should be vortexed for 30 sec just before injection in the fluidic channel, as described 
in the main SOP.   

Material 2 

The material 2 is provided as a concentrated dispersion in the Eppendorf tube ILT-M2. ILT-M2xxxxxx contains 
the buffer for final dilution. 

The procedure to get the final sample is the following: 

A.  Vortex the tube ILT-M2 and sonicate it for 3 min at 40 kHz.  

B.  Pipette 100 µL of ILT-M2 and add it to ILT-M2xxxxxxxx. Sonicate it for 2 min at 40 kHz 

C.  Vortex ILT-M2xxxxxxxx for 30 sec before each injection on the collector. 

Material 4 

The material 4 is provided in powder form. 3 Eppendorf tubes are provided. 

M4-1 contains the material in powder form, the mass is written on the side. M4-2 contains the buffer to use 
to disperse the powder. ILT-M4xxxxxx contains the buffer for final dilution, it will become the sample to inject. 

The procedure to get the final sample is the following: 

A. Pipette the volume of M4-2 to get a 1 mg/mL solution in M4-1 (if M4-1 is 1.3 mg, pipette 1300 µL from 
M4-2). Vortex for 1 min, sonicate for 5 min at 40 kHz 

33 

 
 
 
 
 
 
 
 
B. Vortex for 1 min. Pipette 10 µL from M4-1 and add it to ILT-M4xxxxxxxx. Do not forget to vortex for 30 sec 
before each injection. 

Material 5 

The material 5 is provided in powder form. 3 Eppendorf tubes are provided. 

M5-1 contains the material in powder form, the mass is written on the side. M5-2 contains the buffer to use 
to disperse the powder. ILT-M5xxxxxx contains the buffer for final dilution, it will become the sample to inject. 

The procedure to get the final sample is the following: 

A. Pipette the volume of M5-2 to get a 1 mg/mL solution in M5-1 (if M5-1 is 1.3 mg, pipette 1300 µL from 
M5-2). Vortex for 1 min, sonicate for 5 min at 40 kHz 

B. Vortex for 1 min. Pipette 10 µL from M5-1 and add it to ILT-M5xxxxxxxx. Do not forget to vortex for 30 sec 
before each injection. 

34 

 
 
 
 
  
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