Air quality — Determination of the number concentration of airborne inorganic fibres by phase contrast optical microscopy — Membrane filter method

ISO 8672:2014 specifies the determination of the number concentration of airborne inorganic fibres by phase contrast optical microscopy using the membrane filter method in workplace atmospheres, as defined by the counting criteria given in this document.

Qualité de l'air — Détermination de la concentration en nombre de fibres inorganiques en suspension dans l'air par microscopie optique en contraste de phase — Méthode du filtre à membrane

Kakovost zraka - Ugotavljanje številčne koncentracije lebdečih anorganskih vlaken z metodo fazno kontrastne optične mikroskopije - Metoda z membranskim filtrom

Ta mednarodni standard se uporablja za ugotavljanje številčne koncentracije lebdečih anorganskih vlaken s fazno kontrastno optično mikroskopijo z uporabo metode z membranskim filtrom v delovni atmosferi, kot je določeno z merili za štetje v 6.5.4.

General Information

Status
Published
Publication Date
28-Apr-2014
Current Stage
6060 - International Standard published
Start Date
26-Mar-2014
Completion Date
29-Apr-2014

RELATIONS

Buy Standard

Standard
ISO 8672:2014 - Air quality -- Determination of the number concentration of airborne inorganic fibres by phase contrast optical microscopy -- Membrane filter method
English language
31 pages
sale 15% off
Preview
sale 15% off
Preview
Standard
ISO 8672:2015
English language
36 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day

Standards Content (sample)

INTERNATIONAL ISO
STANDARD 8672
Second edition
2014-05-01
Air quality — Determination of the
number concentration of airborne
inorganic fibres by phase contrast
optical microscopy — Membrane filter
method
Qualité de l’air — Détermination de la concentration en nombre de
fibres inorganiques en suspension dans l’air par microscopie optique
en contraste de phase — Méthode du filtre à membrane
Reference number
ISO 8672:2014(E)
ISO 2014
---------------------- Page: 1 ----------------------
ISO 8672:2014(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2014

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior

written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of

the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 8672:2014(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 General method description .................................................................................................................................................................... 2

4.1 Limitations of the method by particle type .................................................................................................................... 2

4.2 Limit of visibility and detection limits ................................................................................................................................ 2

4.3 Apparatus and equipment ............................................................................................................................................................ 3

4.4 Mounting media ..................................................................................................................................................................................... 5

4.5 Quality assurance.................................................................................................................................................................................. 6

5 Sampling ........................................................................................................................................................................................................................ 6

5.1 Flowrate ........................................................................................................................................................................................................ 6

5.2 Acceptable fibre loadings on filters ....................................................................................................................................... 7

5.3 Storage and transport ....................................................................................................................................................................... 7

5.4 Blanks ......... ..................................................................................................................................................................................................... 7

5.5 Sample duration and volume ...................................................................................................................................................... 8

5.6 Sampling strategy and records.................................................................................................................................................. 9

6 Evaluation .................................................................................................................................................................................................................10

6.1 Sample preparation .........................................................................................................................................................................10

6.2 Microscope adjustment principles .....................................................................................................................................11

6.3 Eyepiece graticule calibration .................................................................................................................................................11

6.4 Microscope/counter performance assessment .......................................................................................................11

6.5 Counting and sizing fibres ..........................................................................................................................................................12

6.6 Calculation of fibre concentration .......................................................................................................................................13

7 Sources of errors and uncertainty ..................................................................................................................................................14

7.1 General ........................................................................................................................................................................................................14

7.2 Uncertainty ..............................................................................................................................................................................................14

7.3 Inter-laboratory variability .......................................................................................................................................................16

7.4 Lowering of intra- and inter-laboratory variance .................................................................................................17

8 Test report ................................................................................................................................................................................................................17

Annex A (informative) Slide mounting: acetone-triacetin procedure ............................................................................19

Annex B (informative) Permanent slide preparation: dimethyl formamide - Euparal procedure ...21

Annex C (normative) Eyepiece graticule ........................................................................................................................................................24

Annex D (informative) Measurement of exposed filter area ....................................................................................................27

Annex E (informative) Microscope adjustment procedure ........................................................................................................28

Annex F (informative) Sources .................................................................................................................................................................................29

Bibliography .............................................................................................................................................................................................................................30

© ISO 2014 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 8672:2014(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers

to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 146, Air quality, Subcommittee SC 2, Workplace

atmospheres.

This second edition cancels and replaces the first edition (ISO 8672:1993), which has been technically

revised. This second edition provides additional quality assurance procedures.
iv © ISO 2014 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 8672:2014(E)
Introduction

The concentration of optically visible airborne inorganic fibres can only be defined in terms of the

results obtained with a particular measurement method. Moreover, experience has shown that different

laboratories, using the membrane filter optical counting method, can obtain different results on the

same sample, even when the laboratories appear to be working from a written version of the method

which attempts to specify all variables.

Because of the unusual operator-dependence of the membrane filter method, it is important to apply

this method with care and use it in conjunction with a quality control scheme. The second edition of this

International Standard provides for additional quality assurance procedures.
© ISO 2014 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 8672:2014(E)
Air quality — Determination of the number concentration
of airborne inorganic fibres by phase contrast optical
microscopy — Membrane filter method
1 Scope

This International Standard specifies the determination of the number concentration of airborne

inorganic fibres by phase contrast optical microscopy using the membrane filter method in workplace

atmospheres, as defined by the counting criteria given in 6.5.4.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 13137, Workplace atmospheres — Pumps for personal sampling of chemical and biological agents —

Requirements and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

NOTE Terms specific to this document are defined, in addition to those found in ASTM Standards D7200-

[6] [7]
12, European Standard EN 1540.
3.1
reference slide

slide prepared from a field sample by the acetone-triacetin method (Annex A) or the dimethyl formamide-

Euparal method (Annex B) with a non-gridded cover slip that is to be used in a long-term quality control

scheme

Note 1 to entry: For the inventory of reference slides, they should be selected from a previous prepared bank

of samples for which the mean and variability have been historically established. They should also comprise of

samples with varying fibre densities, and if available different fibre types. Reference slides should be checked for

filter integrity periodically and replaced if necessary.
3.2
breathing zone
space around the nose and mouth from which a worker’s breath is taken

Note 1 to entry: Technically, the breathing zone corresponds to a hemisphere (generally accepted to be a 30 cm

in radius) extending in front of the human face, centred on the mid-point of a line joining the ears. The base of

the hemisphere is a plane through this line, the top of the head and the larynx. This technical description is not

[7]
applicable when respiratory protective equipment is used.
3.3
countable fibre

any object having a maximum width less than 3 μm, an overall length greater than 5 μm and a length to

width ratio greater than 3:1.
© ISO 2014 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 8672:2014(E)
3.4
occupational exposure limit value

limit of time-weighted average of the concentration of a chemical agent in the air within the breathing

zone of a worker in relation to a specified reference period

Note 1 to entry: Limit values are mostly set for reference periods of 8 h, but can also be set for shorter periods or

concentration excursions. Limit values for gases and vapours are stated in terms independent of temperature and

3 3

air pressure variables in ml/m and in terms dependent on those variables in mg/ m for a temperature of 20 °C

and a pressure of 101,3 kPa. Limit values for airborne particles and mixtures of particles and vapours are given in

mg/m or multiples of that for actual environmental conditions (temperature, pressure) at the workplace. Limit

3 3

values of fibres are given in numbers of fibres/m or number of fibres/cm for actual environmental conditions

[7]
(temperature, pressure) at the workplace.
4 General method description

A sample is collected by drawing a measured quantity of air through a membrane filter by means of a

battery-powered sampling pump. The entire filter or a portion of the filter (wedge) is later transformed

from an opaque membrane into a homogeneous optically transparent specimen. The fibres are then

sized and counted using a phase contrast optical microscope. The result is expressed as fibres per

cubic centimetre of air, calculated from the number of fibres on the filter and the measured volume

of air sampled. The method is applicable for routine sampling and sample evaluation necessary to

assess personal exposure to fibres and implement control measures of their presence in occupational

environments. The method is applicable for routine static sampling and measurement of personal

exposure to fibres.
4.1 Limitations of the method by particle type

This method cannot identify the composition or characteristics of particular fibre types and its use shall

be restricted to workplace atmospheres where the predominant fibre types are inorganic.

The use of this method also has limitations when applied to samples containing platy or acicular particles

and consequently it should not be implemented without prior knowledge of the fibres present in the

workplace atmosphere. There are a variety of analytical methods which can be useful, e.g. polarizing

light microscopy, electron microscopy.
4.2 Limit of visibility and detection limits

This procedure cannot enumerate thin fibres whose width is below the limit of visibilty by phase contrast

optical microscopy. The limit varies according to the refractive index contrast between the fibres and

the mounting medium, and the phase-shift of the microscope. The triacetin mounting medium proposed

in this method has a refractive index of approximately 1,45, and the Euparal mounting medium has a

refractive index of 1,48. In workplace atmospheres, fibres with refractive indices in the range of 1,4 to 1,5

might occur. As the relatively small refractive index difference between these fibres and the mounting

media might not be sufficient for them to be visible, this mounting media might not be appropriate.

Previously published method limitations of 0,2 µm or 0,25 µm width limits are conservative consensus

values. Practical studies have indicated the ability of a microscope properly adjusted to detect chrysotile

[11] [12]

fibres of 0,15 µm width and Amosite fibres of 0,062 5 µm width. These results suggest crocidolite

fibres can be detectable at 0,05 µm width. Fibres with smaller widths can be detected under the electron

microscope, but large differences in results sometimes observed between the two methods are more

likely due to undercounting fine fibres under phase contrast microscopy (PCM) than to the presence

of substantial numbers of fibres that can only be seen under the electron microscope. The quality

assurance procedures in this International Standard are used to identify and resolve several types of

counting errors under PCM.

With the parameters specified in this method, the theoretical lower detection limit for a sample of 480 l

3 3

of air is 0,007 fibres/cm . However, the limit of practical use is often 0,1 fibres/cm or higher. This is

because blank filters can frequently give a reading of several countable fibres per 100 graticule areas.

These “fibres” are contaminants on the filter, or artefacts from the clearing process which have the

2 © ISO 2014 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 8672:2014(E)

appearance of fibres. Neither counting more fields nor increasing sampling duration overcomes the

problem of background dust, when fibres are a minor constituent of the dust cloud. In relatively clean

atmospheres, such as cleaned enclosure after asbestos removal (clearance sampling), the expected fibre

concentration is < 0,01 fibres/cm , larger sample volumes (>480 l) are required to achieve quantifiable

loadings.
4.3 Apparatus and equipment
4.3.1 Sampling equipment

4.3.1.1 Filters. Membrane filters (mixed cellulose ester or cellulose nitrate) of 0,8 to 1,2 µm or less pore

size and a diameter of 25 mm are preferred with, or without printed grids (printed grids can allow the

counter to focus easier on the plane containing the fibres, but the lines of the grid can obstruct all or parts

of the fields of view and interfere with the counting so that these fields must be avoided).

In recent years, problems have been observed with portions of batches of mixed cellulose ester filters,

where the porosity is not evenly developed over the filter. Areas of the filter without porosity can lead

to a high pressure drop resulting in premature pump failure, areas of the filter without fibres deposited,

[13]

and the appearance of cracking in acetone-triacetin mounts. It is necessary to pay attention to the

quality of filters in order to avoid these problems. In addition, each batch of filters should be tested for

fibrous contamination as described in 5.4.
[14]
4.3.1.2 Open-faced filter holder fitted with a protective cowl.

The distance between the cowl opening and the filter plane should be between one and a half times and

two times the internal diameter of the cowl. The internal diameter of the cowl should be at least equal

to the exposed diameter of the filter but not more than 2 mm greater.

The cowl helps to protect the filter from accidental contamination. A conducting cowl is preferred to a

plastic one because of the possible risk of fibre loss due to electrostatic charge. Filter holders and cowls

shall be thoroughly washed before re-use.

Due to the design of the filter support utilized in some filter holders, a supporting pad should be used.

The purpose of this supporting pad is to ensure an even distribution of air passing through the primary

membrane.

4.3.1.3 Sampling pump, capable of giving a smooth flow and having flow set to within ± 5 % of the

required flowrate, and of maintaining this flowrate through the filter to within ±10 % for flowrate 2 l/min

and ±5 % for flowrate > 2 l/min during the period of sampling.

Although some pumps are equipped with pulsation dampers, an external damper might have to be

installed between the pump and the collecting media. Personal sampling pumps shall meet the criteria

for a Type P pump as detailed in ISO 13137.

4.3.1.4 Connecting tubing, constriction-proof and the connections shall be leak-proof.

4.3.2 Microscope equipment

Because microscopes with identical “specifications” can give quite different performances, it is necessary

that the performance of the proposed and existing microscopes be assessed by means of a detection

limit test slide. Provided this criterion is met, small departures from the recommended specifications in

items 4.3.2.4 and 4.3.2.5 are permitted. The necessary specifications are as follows.

4.3.2.1 Light source-Kohler or Kohler type illumination. It is preferable for the illuminator to be

built-in with a variable light intensity control.
© ISO 2014 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 8672:2014(E)

4.3.2.2 Substage assembly, Abbe or achromatic phase contrast condenser incorporated into a

substage unit.

There shall be a means of centering each condenser annulus with respect to the phase plate in the

corresponding objective, and also a means of focusing the condenser.

4.3.2.3 Stage, a built-in mechanical specimen stage fitted with slide clamps and x -y displacement.

4.3.2.4 Objectives, a rotating nose-piece fitted with 10X and 40X parfocal phase contrast achromatic

objectives.

The 40X objective shall have a numerical aperture (NA) between 0,65 and 0,75. It shall have a phase ring

of absorption not less than 65 % and not greater than 85 %.

4.3.2.5 Binocular eyepieces, chosen to give a total magnification of 400X to 500X.

At least one eyepiece shall permit the insertion of a graticule. The compensating and focusing type is

recommended.
[15]

4.3.2.6 Graticule (Walton-Beckett or RIB), the diameter of the graticule in the object plane, when

using the 40X phase objective and an appropriate eyepiece, shall be 100 ± 2 μm.
4.3.3 Accessories

4.3.3.1 Centering telescope or Bertrand lens, for checking that the phase rings in the condenser are

centred with respect to those in the objectives.

4.3.3.2 Green filter, to ensure the best phase contrast conditions because the optics are designed for

this wavelength.
4.3.3.3 Stage micrometer, with 1 mm divided into 0,01 mm divisions.

4.3.3.4 Scalpel holder and Disposal blades, #10 or #22 surgical steel, curved blade.

4.3.3.5 Tweezers, fine point.
4.3.3.6 Acetone vaporizer, to clear mixed cellulose filters.
4.3.3.7 Hypodermic syringe, with 22 gauge needle or disposable micropipette.

4.3.3.8 Pre-cleaned microscope slides, of approximately 76 mm x 25 mm and 0,8 to 1,0 mm thick.

4.3.3.9 Cover slips (without grids), 22 mm x 22 mm, 0,16 to 0,19 mm thick, e.g. No. 1-1/2 or as

specified by microscope manufacturer. Larger cover slips are necessary to cover a whole 25 mm diameter

filter.

4.3.3.10 Phase contrast test slide, HSE/NPL Mark II or HSE/ULO Mark III where the certificate includes

reference to at least one block of lines that should not be visible (see 6.4).

4.3.3.11 Relocatable cover slips, each cover slip has 2 grids and 2 logos which help to orient the cover

slip.

Each grid has 140 viewing fields, each of which is approximately 100 μm in diameter. The viewing fields

are arranged into 14 columns and 10 rows. With proper orientation, a letter appears on the top and

4 © ISO 2014 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 8672:2014(E)

bottom of each column and a number appears on either side of the rows. Thus, each viewing field is

[9]
identified for relocation.

4.3.3.12 Standard relocatable test slides, prepared from different types of asbestos and inorganic

[9]

fibres with different matrix background by the dimethyl formamide/ Euparal method.

[8]

There should be no fibre migration observed in these slides for more than 5 years. Other clearing and

mounting procedures can be used if no filter migration is observed over the term of use.

They can be prepared from a proficiency test filter from the Proficiency Analytical Testing program

[8][9]

(PAT) of the American Industrial Hygiene Association’s (AIHA) Laboratory Quality Programs.

The filter or filter wedge is cleared and mounted by the dimethyl formamide-Euparal method with a

relocatable gridded cover slip (Annex B). The fibres visible in each grid opening have been identified and

their locations marked on a drawing of each opening. The identity, number and position of each fibre

have been verified by a second counter.
4.3.3.13 Disposal gloves.
4.3.3.14 Thermostat-control hotplate or drying oven.
4.3.3.15 Thermometer, 0 °C to 100 °C.
4.3.4 Reagents
4.3.4.1 Dimethyl formamide, reagent grade.
4.3.4.2 Glazier acetic acid, reagent grade.
4.3.4.3 De-ionized water.
4.3.4.4 Euparal resin.
4.3.4.5 Acetone, reagent grade.
4.3.4.6 Triacetin, reagent grade.
4.3.4.7 Lacquer or nail polish.
4.4 Mounting media

Acetone-triacetin is the mounting medium most often used (see Annex A). However, fibre migration

can occur over time when excess triacetin is used. While this does not affect the analysis of routine

samples and it might not affect the count concentration over time, it does restrict the ability to perform

quality checks by re-examining the same areas. This problem can be controlled by using an appropriate

amount of triacetin. However, the visual quality of the slides made with triacetin also deteriorates in

about 12 months. Therefore, for permanent slides, the dimethyl formamide-Euparal mounting method

(see Annex B) should be used. No fibre migration or visual quality deterioration has been observed in

[8] [9]

slides more than 5 years old. Fibre counts are not affected by using Euparal in place of triacetin,

[16]

and have also been shown to be equivalent to fibre counts using the dimethyl phthalate-diethyl

oxalate method which was used previously for samples that were instrumental in the development of

[16]
risk assessments.
© ISO 2014 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 8672:2014(E)
4.5 Quality assurance

Subjective recognition and counting of fibres results in greater uncertainty compared to other analytical

procedures so that particular attention must be paid to quality control measures. Slides are available,

[8][9]

made from proficiency test filters from the PAT of the AIHA’s Laboratory Quality Programs. Each

slide has been mounted with a permanent Euparal medium and covered with a relocatable gridded cover

slip. The fibres visible in each grid opening have been identified and their locations marked on a drawing

of each opening. The identity, number and position of each fibre has been verified by a second counter.

These slides are referred to in this International Standard as standard relocatable test slides, and they

are applied in various ways to improve and assess the quality of fibre counts. The laboratory should also

maintain an inventory of reference slides, being slides of field sample with varying fibre densities (and,

if available, different fibre types). The labels on the reference slides and standard test slides are changed

periodically so that the counter does not become familiar with the slides. The following quality control

measures shall be applied on each day that field samples are counted:

a) Examination of a standard relocatable test slide. The fibres in each designated field are counted and

the counts referred to the accompanying slide descriptions. Counters shall obtain a discrepancy

score of more than 50 before proceeding (6.4). If a score of 50 or better is not achieved, the counter

should review the slide descriptions to determine the cause and then attempt to rectify the situation

by repeating the microscope set-up or re-training the counter.
b) Counting of a reference slide.

c) Re-counting of 10 % of sample slides. Prior to re-counting, the slide is relabled by a person other

than the counter.

Prior to counting field samples, all counters should be trained in the techniques contained in this

International Standard. Documentation of such training shall be maintained by the counter or laboratory.

Training on both chrysotile and Amosite is the minimum requirement for asbestos analysis. Completion

of successful training shall be documented through the analysis of standard relocatable test slides with

a discrepancy score greater than
...

SLOVENSKI STANDARD
SIST ISO 8672:2015
01-marec-2015
1DGRPHãþD
SIST ISO 8672:1996

.DNRYRVW]UDND8JRWDYOMDQMHãWHYLOþQHNRQFHQWUDFLMHOHEGHþLKDQRUJDQVNLKYODNHQ

]PHWRGRID]QRNRQWUDVWQHRSWLþQHPLNURVNRSLMH0HWRGD]PHPEUDQVNLPILOWURP

Air quality - Determination of the number concentration of airborne inorganic fibres by

phase contrast optical microscopy - Membrane filter method

Qualité de l'air - Détermination de la concentration en nombre de fibres inorganiques en

suspension dans l'air par microscopie optique en contraste de phase - Méthode du filtre

à membrane
Ta slovenski standard je istoveten z: ISO 8672:2014
ICS:
13.040.30 Kakovost zraka na delovnem Workplace atmospheres
mestu
SIST ISO 8672:2015 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST ISO 8672:2015
---------------------- Page: 2 ----------------------
SIST ISO 8672:2015
INTERNATIONAL ISO
STANDARD 8672
Second edition
2014-05-01
Air quality — Determination of the
number concentration of airborne
inorganic fibres by phase contrast
optical microscopy — Membrane filter
method
Qualité de l’air — Détermination de la concentration en nombre de
fibres inorganiques en suspension dans l’air par microscopie optique
en contraste de phase — Méthode du filtre à membrane
Reference number
ISO 8672:2014(E)
ISO 2014
---------------------- Page: 3 ----------------------
SIST ISO 8672:2015
ISO 8672:2014(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2014

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior

written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of

the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved
---------------------- Page: 4 ----------------------
SIST ISO 8672:2015
ISO 8672:2014(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 General method description .................................................................................................................................................................... 2

4.1 Limitations of the method by particle type .................................................................................................................... 2

4.2 Limit of visibility and detection limits ................................................................................................................................ 2

4.3 Apparatus and equipment ............................................................................................................................................................ 3

4.4 Mounting media ..................................................................................................................................................................................... 5

4.5 Quality assurance.................................................................................................................................................................................. 6

5 Sampling ........................................................................................................................................................................................................................ 6

5.1 Flowrate ........................................................................................................................................................................................................ 6

5.2 Acceptable fibre loadings on filters ....................................................................................................................................... 7

5.3 Storage and transport ....................................................................................................................................................................... 7

5.4 Blanks ......... ..................................................................................................................................................................................................... 7

5.5 Sample duration and volume ...................................................................................................................................................... 8

5.6 Sampling strategy and records.................................................................................................................................................. 9

6 Evaluation .................................................................................................................................................................................................................10

6.1 Sample preparation .........................................................................................................................................................................10

6.2 Microscope adjustment principles .....................................................................................................................................11

6.3 Eyepiece graticule calibration .................................................................................................................................................11

6.4 Microscope/counter performance assessment .......................................................................................................11

6.5 Counting and sizing fibres ..........................................................................................................................................................12

6.6 Calculation of fibre concentration .......................................................................................................................................13

7 Sources of errors and uncertainty ..................................................................................................................................................14

7.1 General ........................................................................................................................................................................................................14

7.2 Uncertainty ..............................................................................................................................................................................................14

7.3 Inter-laboratory variability .......................................................................................................................................................16

7.4 Lowering of intra- and inter-laboratory variance .................................................................................................17

8 Test report ................................................................................................................................................................................................................17

Annex A (informative) Slide mounting: acetone-triacetin procedure ............................................................................19

Annex B (informative) Permanent slide preparation: dimethyl formamide - Euparal procedure ...21

Annex C (normative) Eyepiece graticule ........................................................................................................................................................24

Annex D (informative) Measurement of exposed filter area ....................................................................................................27

Annex E (informative) Microscope adjustment procedure ........................................................................................................28

Annex F (informative) Sources .................................................................................................................................................................................29

Bibliography .............................................................................................................................................................................................................................30

© ISO 2014 – All rights reserved iii
---------------------- Page: 5 ----------------------
SIST ISO 8672:2015
ISO 8672:2014(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers

to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 146, Air quality, Subcommittee SC 2, Workplace

atmospheres.

This second edition cancels and replaces the first edition (ISO 8672:1993), which has been technically

revised. This second edition provides additional quality assurance procedures.
iv © ISO 2014 – All rights reserved
---------------------- Page: 6 ----------------------
SIST ISO 8672:2015
ISO 8672:2014(E)
Introduction

The concentration of optically visible airborne inorganic fibres can only be defined in terms of the

results obtained with a particular measurement method. Moreover, experience has shown that different

laboratories, using the membrane filter optical counting method, can obtain different results on the

same sample, even when the laboratories appear to be working from a written version of the method

which attempts to specify all variables.

Because of the unusual operator-dependence of the membrane filter method, it is important to apply

this method with care and use it in conjunction with a quality control scheme. The second edition of this

International Standard provides for additional quality assurance procedures.
© ISO 2014 – All rights reserved v
---------------------- Page: 7 ----------------------
SIST ISO 8672:2015
---------------------- Page: 8 ----------------------
SIST ISO 8672:2015
INTERNATIONAL STANDARD ISO 8672:2014(E)
Air quality — Determination of the number concentration
of airborne inorganic fibres by phase contrast optical
microscopy — Membrane filter method
1 Scope

This International Standard specifies the determination of the number concentration of airborne

inorganic fibres by phase contrast optical microscopy using the membrane filter method in workplace

atmospheres, as defined by the counting criteria given in 6.5.4.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 13137, Workplace atmospheres — Pumps for personal sampling of chemical and biological agents —

Requirements and test methods
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

NOTE Terms specific to this document are defined, in addition to those found in ASTM Standards D7200-

[6] [7]
12, European Standard EN 1540.
3.1
reference slide

slide prepared from a field sample by the acetone-triacetin method (Annex A) or the dimethyl formamide-

Euparal method (Annex B) with a non-gridded cover slip that is to be used in a long-term quality control

scheme

Note 1 to entry: For the inventory of reference slides, they should be selected from a previous prepared bank

of samples for which the mean and variability have been historically established. They should also comprise of

samples with varying fibre densities, and if available different fibre types. Reference slides should be checked for

filter integrity periodically and replaced if necessary.
3.2
breathing zone
space around the nose and mouth from which a worker’s breath is taken

Note 1 to entry: Technically, the breathing zone corresponds to a hemisphere (generally accepted to be a 30 cm

in radius) extending in front of the human face, centred on the mid-point of a line joining the ears. The base of

the hemisphere is a plane through this line, the top of the head and the larynx. This technical description is not

[7]
applicable when respiratory protective equipment is used.
3.3
countable fibre

any object having a maximum width less than 3 μm, an overall length greater than 5 μm and a length to

width ratio greater than 3:1.
© ISO 2014 – All rights reserved 1
---------------------- Page: 9 ----------------------
SIST ISO 8672:2015
ISO 8672:2014(E)
3.4
occupational exposure limit value

limit of time-weighted average of the concentration of a chemical agent in the air within the breathing

zone of a worker in relation to a specified reference period

Note 1 to entry: Limit values are mostly set for reference periods of 8 h, but can also be set for shorter periods or

concentration excursions. Limit values for gases and vapours are stated in terms independent of temperature and

3 3

air pressure variables in ml/m and in terms dependent on those variables in mg/ m for a temperature of 20 °C

and a pressure of 101,3 kPa. Limit values for airborne particles and mixtures of particles and vapours are given in

mg/m or multiples of that for actual environmental conditions (temperature, pressure) at the workplace. Limit

3 3

values of fibres are given in numbers of fibres/m or number of fibres/cm for actual environmental conditions

[7]
(temperature, pressure) at the workplace.
4 General method description

A sample is collected by drawing a measured quantity of air through a membrane filter by means of a

battery-powered sampling pump. The entire filter or a portion of the filter (wedge) is later transformed

from an opaque membrane into a homogeneous optically transparent specimen. The fibres are then

sized and counted using a phase contrast optical microscope. The result is expressed as fibres per

cubic centimetre of air, calculated from the number of fibres on the filter and the measured volume

of air sampled. The method is applicable for routine sampling and sample evaluation necessary to

assess personal exposure to fibres and implement control measures of their presence in occupational

environments. The method is applicable for routine static sampling and measurement of personal

exposure to fibres.
4.1 Limitations of the method by particle type

This method cannot identify the composition or characteristics of particular fibre types and its use shall

be restricted to workplace atmospheres where the predominant fibre types are inorganic.

The use of this method also has limitations when applied to samples containing platy or acicular particles

and consequently it should not be implemented without prior knowledge of the fibres present in the

workplace atmosphere. There are a variety of analytical methods which can be useful, e.g. polarizing

light microscopy, electron microscopy.
4.2 Limit of visibility and detection limits

This procedure cannot enumerate thin fibres whose width is below the limit of visibilty by phase contrast

optical microscopy. The limit varies according to the refractive index contrast between the fibres and

the mounting medium, and the phase-shift of the microscope. The triacetin mounting medium proposed

in this method has a refractive index of approximately 1,45, and the Euparal mounting medium has a

refractive index of 1,48. In workplace atmospheres, fibres with refractive indices in the range of 1,4 to 1,5

might occur. As the relatively small refractive index difference between these fibres and the mounting

media might not be sufficient for them to be visible, this mounting media might not be appropriate.

Previously published method limitations of 0,2 µm or 0,25 µm width limits are conservative consensus

values. Practical studies have indicated the ability of a microscope properly adjusted to detect chrysotile

[11] [12]

fibres of 0,15 µm width and Amosite fibres of 0,062 5 µm width. These results suggest crocidolite

fibres can be detectable at 0,05 µm width. Fibres with smaller widths can be detected under the electron

microscope, but large differences in results sometimes observed between the two methods are more

likely due to undercounting fine fibres under phase contrast microscopy (PCM) than to the presence

of substantial numbers of fibres that can only be seen under the electron microscope. The quality

assurance procedures in this International Standard are used to identify and resolve several types of

counting errors under PCM.

With the parameters specified in this method, the theoretical lower detection limit for a sample of 480 l

3 3

of air is 0,007 fibres/cm . However, the limit of practical use is often 0,1 fibres/cm or higher. This is

because blank filters can frequently give a reading of several countable fibres per 100 graticule areas.

These “fibres” are contaminants on the filter, or artefacts from the clearing process which have the

2 © ISO 2014 – All rights reserved
---------------------- Page: 10 ----------------------
SIST ISO 8672:2015
ISO 8672:2014(E)

appearance of fibres. Neither counting more fields nor increasing sampling duration overcomes the

problem of background dust, when fibres are a minor constituent of the dust cloud. In relatively clean

atmospheres, such as cleaned enclosure after asbestos removal (clearance sampling), the expected fibre

concentration is < 0,01 fibres/cm , larger sample volumes (>480 l) are required to achieve quantifiable

loadings.
4.3 Apparatus and equipment
4.3.1 Sampling equipment

4.3.1.1 Filters. Membrane filters (mixed cellulose ester or cellulose nitrate) of 0,8 to 1,2 µm or less pore

size and a diameter of 25 mm are preferred with, or without printed grids (printed grids can allow the

counter to focus easier on the plane containing the fibres, but the lines of the grid can obstruct all or parts

of the fields of view and interfere with the counting so that these fields must be avoided).

In recent years, problems have been observed with portions of batches of mixed cellulose ester filters,

where the porosity is not evenly developed over the filter. Areas of the filter without porosity can lead

to a high pressure drop resulting in premature pump failure, areas of the filter without fibres deposited,

[13]

and the appearance of cracking in acetone-triacetin mounts. It is necessary to pay attention to the

quality of filters in order to avoid these problems. In addition, each batch of filters should be tested for

fibrous contamination as described in 5.4.
[14]
4.3.1.2 Open-faced filter holder fitted with a protective cowl.

The distance between the cowl opening and the filter plane should be between one and a half times and

two times the internal diameter of the cowl. The internal diameter of the cowl should be at least equal

to the exposed diameter of the filter but not more than 2 mm greater.

The cowl helps to protect the filter from accidental contamination. A conducting cowl is preferred to a

plastic one because of the possible risk of fibre loss due to electrostatic charge. Filter holders and cowls

shall be thoroughly washed before re-use.

Due to the design of the filter support utilized in some filter holders, a supporting pad should be used.

The purpose of this supporting pad is to ensure an even distribution of air passing through the primary

membrane.

4.3.1.3 Sampling pump, capable of giving a smooth flow and having flow set to within ± 5 % of the

required flowrate, and of maintaining this flowrate through the filter to within ±10 % for flowrate 2 l/min

and ±5 % for flowrate > 2 l/min during the period of sampling.

Although some pumps are equipped with pulsation dampers, an external damper might have to be

installed between the pump and the collecting media. Personal sampling pumps shall meet the criteria

for a Type P pump as detailed in ISO 13137.

4.3.1.4 Connecting tubing, constriction-proof and the connections shall be leak-proof.

4.3.2 Microscope equipment

Because microscopes with identical “specifications” can give quite different performances, it is necessary

that the performance of the proposed and existing microscopes be assessed by means of a detection

limit test slide. Provided this criterion is met, small departures from the recommended specifications in

items 4.3.2.4 and 4.3.2.5 are permitted. The necessary specifications are as follows.

4.3.2.1 Light source-Kohler or Kohler type illumination. It is preferable for the illuminator to be

built-in with a variable light intensity control.
© ISO 2014 – All rights reserved 3
---------------------- Page: 11 ----------------------
SIST ISO 8672:2015
ISO 8672:2014(E)

4.3.2.2 Substage assembly, Abbe or achromatic phase contrast condenser incorporated into a

substage unit.

There shall be a means of centering each condenser annulus with respect to the phase plate in the

corresponding objective, and also a means of focusing the condenser.

4.3.2.3 Stage, a built-in mechanical specimen stage fitted with slide clamps and x -y displacement.

4.3.2.4 Objectives, a rotating nose-piece fitted with 10X and 40X parfocal phase contrast achromatic

objectives.

The 40X objective shall have a numerical aperture (NA) between 0,65 and 0,75. It shall have a phase ring

of absorption not less than 65 % and not greater than 85 %.

4.3.2.5 Binocular eyepieces, chosen to give a total magnification of 400X to 500X.

At least one eyepiece shall permit the insertion of a graticule. The compensating and focusing type is

recommended.
[15]

4.3.2.6 Graticule (Walton-Beckett or RIB), the diameter of the graticule in the object plane, when

using the 40X phase objective and an appropriate eyepiece, shall be 100 ± 2 μm.
4.3.3 Accessories

4.3.3.1 Centering telescope or Bertrand lens, for checking that the phase rings in the condenser are

centred with respect to those in the objectives.

4.3.3.2 Green filter, to ensure the best phase contrast conditions because the optics are designed for

this wavelength.
4.3.3.3 Stage micrometer, with 1 mm divided into 0,01 mm divisions.

4.3.3.4 Scalpel holder and Disposal blades, #10 or #22 surgical steel, curved blade.

4.3.3.5 Tweezers, fine point.
4.3.3.6 Acetone vaporizer, to clear mixed cellulose filters.
4.3.3.7 Hypodermic syringe, with 22 gauge needle or disposable micropipette.

4.3.3.8 Pre-cleaned microscope slides, of approximately 76 mm x 25 mm and 0,8 to 1,0 mm thick.

4.3.3.9 Cover slips (without grids), 22 mm x 22 mm, 0,16 to 0,19 mm thick, e.g. No. 1-1/2 or as

specified by microscope manufacturer. Larger cover slips are necessary to cover a whole 25 mm diameter

filter.

4.3.3.10 Phase contrast test slide, HSE/NPL Mark II or HSE/ULO Mark III where the certificate includes

reference to at least one block of lines that should not be visible (see 6.4).

4.3.3.11 Relocatable cover slips, each cover slip has 2 grids and 2 logos which help to orient the cover

slip.

Each grid has 140 viewing fields, each of which is approximately 100 μm in diameter. The viewing fields

are arranged into 14 columns and 10 rows. With proper orientation, a letter appears on the top and

4 © ISO 2014 – All rights reserved
---------------------- Page: 12 ----------------------
SIST ISO 8672:2015
ISO 8672:2014(E)

bottom of each column and a number appears on either side of the rows. Thus, each viewing field is

[9]
identified for relocation.

4.3.3.12 Standard relocatable test slides, prepared from different types of asbestos and inorganic

[9]

fibres with different matrix background by the dimethyl formamide/ Euparal method.

[8]

There should be no fibre migration observed in these slides for more than 5 years. Other clearing and

mounting procedures can be used if no filter migration is observed over the term of use.

They can be prepared from a proficiency test filter from the Proficiency Analytical Testing program

[8][9]

(PAT) of the American Industrial Hygiene Association’s (AIHA) Laboratory Quality Programs.

The filter or filter wedge is cleared and mounted by the dimethyl formamide-Euparal method with a

relocatable gridded cover slip (Annex B). The fibres visible in each grid opening have been identified and

their locations marked on a drawing of each opening. The identity, number and position of each fibre

have been verified by a second counter.
4.3.3.13 Disposal gloves.
4.3.3.14 Thermostat-control hotplate or drying oven.
4.3.3.15 Thermometer, 0 °C to 100 °C.
4.3.4 Reagents
4.3.4.1 Dimethyl formamide, reagent grade.
4.3.4.2 Glazier acetic acid, reagent grade.
4.3.4.3 De-ionized water.
4.3.4.4 Euparal resin.
4.3.4.5 Acetone, reagent grade.
4.3.4.6 Triacetin, reagent grade.
4.3.4.7 Lacquer or nail polish.
4.4 Mounting media

Acetone-triacetin is the mounting medium most often used (see Annex A). However, fibre migration

can occur over time when excess triacetin is used. While this does not affect the analysis of routine

samples and it might not affect the count concentration over time, it does restrict the ability to perform

quality checks by re-examining the same areas. This problem can be controlled by using an appropriate

amount of triacetin. However, the visual quality of the slides made with triacetin also deteriorates in

about 12 months. Therefore, for permanent slides, the dimethyl formamide-Euparal mounting method

(see Annex B) should be used. No fibre migration or visual quality deterioration has been observed in

[8] [9]

slides more than 5 years old. Fibre counts are not affected by using Euparal in place of triacetin,

[16]

and have also been shown to be equivalent to fibre counts using the dimethyl phthalate-diethyl

oxalate method which was used previously for samples that were instrumental in the development of

[16]
risk assessments.
© ISO 2014 – All rights reserved 5
---------------------- Page: 13 ----------------------
SIST ISO 8672:2015
ISO 8672:2014(E)
4.5 Quality assurance

Subjective recognition and counting of fibres results in greater uncertainty compared to other analytical

procedures so that particular attention must be paid to quality control measures. Slides are available,

[8][9]

made from proficiency test filters from the PAT of the AIHA’s Laboratory Quality Programs. Each

slide has been mounted with a permanent Euparal medium and covered with a relocatable gridded cover

slip. The fibres visible in each grid opening have been identified and their locations marked on a drawing

of each opening. The identity, number and position of each fibre has been verified by a second counter.

These slides are referred to in this International Standard as standard relocatable test slides, and they

are applied in various ways to improve and assess the quality of fibre counts. The laboratory should also

maintain an inventory of reference slides, being slides of field sample with varying fibre densities (and,

if available, different fibre types). The labels on the reference slides and stan

...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.