SIST EN 14031:2021
(Main)Workplace exposure - Quantitative measurement of airborne endotoxins
Workplace exposure - Quantitative measurement of airborne endotoxins
This document specifies methods for the quantitative measurement of airborne endotoxins and gives general requirements for sampling on filters, transportation, storage as well as the analysis of samples.
This document provides also guidelines for the assessment of workplace exposure to airborne endotoxins.
Exposition am Arbeitsplatz - Quantitative Messung von luftgetragenen Endotoxinen
Dieses Dokument legt Verfahren zur quantitativen Messung von luftgetragenen Endotoxinen fest und enthält allgemeine Anforderungen an die Probenahme auf Filtern, den Transport, die Lagerung sowie die Analyse von Proben.
Dieses Dokument stellt außerdem eine Anleitung für die Bestimmung der Exposition gegenüber luftge-tragenen Endotoxinen am Arbeitsplatz bereit.
Exposition sur les lieux de travail - Mesure quantitative des endotoxines aéroportées
Le présent document spécifie des méthodes pour la mesure quantitative des endotoxines en suspension dans l’air et fixe des exigences générales relatives au prélèvement sur filtres, au transport, à la conservation et à l’analyse des échantillons.
Le présent document fournit également des lignes directrices concernant l’évaluation de l’exposition aux endotoxines en suspension dans l’air sur les lieux de travail.
Izpostavljenost na delovnem mestu - Kvantitativno določevanje lebdečih endotoksinov
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 14031:2021
01-september-2021
Nadomešča:
SIST EN 14031:2003
Izpostavljenost na delovnem mestu - Kvantitativno določevanje lebdečih
endotoksinov
Workplace exposure - Quantitative measurement of airborne endotoxins
Exposition am Arbeitsplatz - Quantitative Messung von luftgetragenen Endotoxinen
Exposition sur les lieux de travail - Mesure quantitative des endotoxines aéroportées
Ta slovenski standard je istoveten z: EN 14031:2021
ICS:
13.040.30 Kakovost zraka na delovnem Workplace atmospheres
mestu
SIST EN 14031:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN 14031:2021
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SIST EN 14031:2021
EN 14031
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2021
EUROPÄISCHE NORM
ICS 13.040.30 Supersedes EN 14031:2003
English Version
Workplace exposure - Quantitative measurement of
airborne endotoxins
Exposition sur les lieux de travail - Mesure quantitative Exposition am Arbeitsplatz - Quantitative Messung von
des endotoxines aéroportées luftgetragenen Endotoxinen
This European Standard was approved by CEN on 7 June 2021.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14031:2021 E
worldwide for CEN national Members.
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SIST EN 14031:2021
EN 14031:2021 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Requirements . 6
4.1 Skills needed for sampling . 6
4.2 Sampling . 7
4.2.1 General . 7
4.2.2 Equipment . 7
4.2.3 Filters . 7
4.3 Sample transport . 7
4.4 Sample storage at the laboratory . 8
4.5 Glassware . 8
4.6 Extraction liquids, water and detergents . 8
4.7 Test for contamination . 8
4.8 Sampling documentation . 8
5 Extraction . 9
5.1 General . 9
5.2 Equipment . 9
5.3 Procedure. 9
5.4 Storage conditions . 10
5.5 Documentation of extraction . 10
6 Analytical method . 10
6.1 General . 10
6.2 Equipment . 11
6.3 Procedure. 11
6.3.1 Measurement . 11
6.3.2 Calibration . 11
6.3.3 Calculation . 11
6.4 Validation . 12
6.5 Documentation of analysis . 12
7 Expression of results . 12
8 Precision . 13
9 Test report . 13
Annex A (informative) Properties of endotoxins and sources of occupational exposure . 14
Annex B (informative) Principle of LAL-assays . 17
Annex C (informative) Deposition of endotoxins on inner sampler walls . 19
Bibliography . 20
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SIST EN 14031:2021
EN 14031:2021 (E)
European foreword
This document (EN 14031:2021) has been prepared by Technical Committee CEN/TC 137 “Assessment
of workplace exposure to chemical and biological agents”, the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by January 2022, and conflicting national standards shall
be withdrawn at the latest by January 2022.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 14031:2003.
The major technical changes between this document and the previous edition are as follows:
a) document title adjusted to the wording used in the Scope;
b) terms and definitions taken over from EN 13098:2019, where appropriate;
c) Clause 4 restructured;
d) requirements for sample storage at the laboratory rewritten;
e) recommendations for extraction of samples revised;
f) storage conditions described more precisely;
g) further requirements on documentation of extraction added;
h reference given to the recombinant Factor C (rFC) method as Note to 6.1;
i) new Table A.1 with examples on working areas with exposure to endotoxin added;
j) new Annex C on deposition of endotoxins on inner sampler walls added;
k) whole document editorially revised.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
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SIST EN 14031:2021
EN 14031:2021 (E)
Introduction
The term 'endotoxin' refers to lipopolysaccharides that are present in the outer membrane of the cell
walls of Gram-negative bacteria. These lipopolysaccharides are a class of pure lipid carbohydrate
molecules (free of protein and other cell wall components) that are held responsible for most of the
biological properties characteristic of bacterial endotoxins.
Endotoxins play an important role in the development of organic dust-related symptoms (for example,
inflammatory reactions in the airways and/or systemic reactions) among workers exposed via
inhalation.
To investigate ill health resulting from occupational exposure to airborne endotoxins it is important
therefore to measure their presence in workplace air accurately. At present, different methods are used
to measure airborne endotoxins. Standardization with respect to sampling, transportation, extraction,
analytical methods and storage of samples or extracts is important in order to obtain accurate and
comparable results and reduce uncertainties in exposure assessment.
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SIST EN 14031:2021
EN 14031:2021 (E)
1 Scope
This document specifies methods for the quantitative measurement of airborne endotoxins and gives
general requirements for sampling on filters, transportation, storage as well as the analysis of samples.
This document provides also guidelines for the assessment of workplace exposure to airborne
endotoxins.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 481, Workplace atmospheres — Size fraction definitions for measurement of airborne particles
EN 1540, Workplace exposure — Terminology
EN ISO 13137, Workplace atmospheres — Pumps for personal sampling of chemical and biological agents
— Requirements and test methods (ISO 13137)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 1540 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
Note 1 to entry: In particular, the following terms used in this document are defined in EN 1540: aerosol,
biological agent, bioaerosol, exposure (by inhalation), inhalable fraction, measuring procedure, personal sampling,
static sampling and workplace.
3.1
control standard endotoxin
CSE
standard that is traceable to the reference standard endotoxin (RSE)
3.2
endotoxin
constituent of the external membrane of Gram-negative bacteria (lipopolysaccharide), consisting of a
complex lipid, lipid A, which is covalently bound to a polysaccharide
Note 1 to entry: “Free endotoxin” is liberated after cell death and by budding from living cells. Lipid A is the
active (toxic) part and is a potent pro-inflammatory substance and can induce febrile, bronchial and other
symptoms in exposed workers. The composition and the toxicity of endotoxin differ between bacteria species.
Note 2 to entry: A brief overview with respect to physical and chemical properties of endotoxins and sources of
occupational exposure is given in Annex A.
[SOURCE: EN 13098:2019, 3.9 – modified, Note 2 to entry added]
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EN 14031:2021 (E)
3.3
endotoxinfree liquid
water or solution containing no measurable endotoxin
Note 1 to entry: Endotoxinfree and pyrogenfree are used interchangeably in the literature.
Note 2 to entry: In practise, less than 0,2 endotoxin units per millilitre is often used as threshold value for
endotoxinfree.
3.4
endotoxin unit
EU
unit standardized against the defined reference material, reference standard endotoxin
[SOURCE: EN 13098:2019, 3.10]
3.5
Limulus Amoebocyte Lysate
LAL
enzymes extracted from the blood cells of the horse shoe crab (Limulus polyphemus) that are activated
by endotoxin and other molecules (glucans etc.)
[SOURCE: EN 13098:2019, 3.16]
3.6
Limulus Amoebocyte Lysate-assay
LAL-assay
functional assay to measure endotoxin concentrations
Note 1 to entry: The method is highly sensitive and based on the activation of a clotting enzyme present in the
lysate of hemolymph of the horseshoe crab (Limulus polyphemus).
3.7
lipopolysaccharide
LPS
water-soluble and stable molecule composed of lipid and polysaccharide present in Gram-negative
bacteria
Note 1 to entry: The lipid part of LPS is termed 'lipid A' and is essential for the toxic properties of LPS.
Note 2 to entry: The terms 'endotoxin' and 'lipopolysaccharide' are often used interchangeably in scientific
literature regarding occupational health.
3.8
reference standard endotoxin
RSE
purified lipopolysaccharide from Escherichia coli that serves as an international reference standard
4 Requirements
4.1 Skills needed for sampling
The person who takes the sample shall be trained in aseptic techniques to avoid contamination of the
sample during any measurement phase. This includes knowledge about sampling equipment and how
to perform the sampling.
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EN 14031:2021 (E)
4.2 Sampling
4.2.1 General
Measurement of airborne endotoxins should preferably be performed by sampling of the inhalable
fraction of aerosols. Aerosols shall be sampled on a filter using a pump to draw air through the filter.
Personal sampling shall be used for risk evaluation including comparison with exposure guidelines.
Static sampling can be used to identify sources of exposure and to measure background concentrations
of airborne endotoxins.
NOTE Other comparable sampling methods can be used for evaluation of other specific parameters, for
example, to measure endotoxins in different size distributions or particle size fractions, or for short- or long-term
exposure measurement.
4.2.2 Equipment
The pumps used for personal sampling and static sampling shall fulfil the requirements specified in
EN ISO 13137. Personal samplers shall collect the inhalable fraction of aerosols as specified in EN 481.
The filter holder or cassette should be rendered endotoxinfree. Heat-resistant reusuable filter
holder/transportation jars shall be made endotoxinfree by heating up according to 4.5. Transportation
jars that do not meet these requirements shall be cleaned thoroughly so that contamination of the
samples is avoided. Cleaning agents shall be removed by subsequent washing in endotoxinfree water.
Disposable filter holder do not need to be sterilized when it is assumed that they are endotoxinfree due
to the manufacturing process. Possible contamination shall be controlled by examination of blank
samples (see 4.7).
4.2.3 Filters
Binderfree glass fibre filters should be used for sampling of airborne endotoxins because these filters
perform very well regarding their collection efficiency, blank values and recovery. If any other filter is
used the performance of these parameters shall be comparable.
NOTE 1 The type of filter used when sampling endotoxins from the air has a strong influence on the
measurement results. Several studies report higher measured concentrations when glass fibre filters were used.
However, in other studies, concentrations measured with the use of PVC, PC or PTFE filters were equivalent to or
even higher than those measured with glass fibre filters. The effect of the nature of the filter seems to be
multifactorial and probably involves parameters as diverse as the filter material itself (possibility of adsorption of
endotoxins on the filter, interference during analysis), the sampling method, the extraction protocol etc. In the
current state of knowledge, it is difficult to give precise recommendations concerning the choice of filters and
further studies are still necessary for that purpose.
NOTE 2 Several filter materials (for example, polyvinylchloride, polytetrafluoroethylene and polycarbonate)
are commonly used but some materials adsorb endotoxins and thereby give false low values.
4.3 Sample transport
After sampling, filters shall be prepared for transport by sealing in a container or sampler cassette. The
sample shall be transported in conditions that prevent large variations in temperature and the gaining
of moisture. If transport time is above 24 h, or there is risk of large temperature variations, actions to
prevent moisture shall be taken, either by using an isolating container or by including a dehumidifier.
Conditions during transport shall be documented, including transport time and temperature log.
If the temperature is above 30 °C the filters shall be placed in cooler conditions (for example, by using a
cooling box).
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SIST EN 14031:2021
EN 14031:2021 (E)
4.4 Sample storage at the laboratory
If the sample is not extracted within two weeks after arrival at the laboratory the sample shall be stored
in conditions that prevent large variations in temperature and gain of moisture. Alternatively, for
storage of more than two weeks they should be frozen at a temperature of about – 20 °C or below,
because endotoxins are very stable when frozen. However, samples shall not be frozen and thawed
more than once, as this can affect the detectable endotoxin content of them.
4.5 Glassware
Glassware shall be rendered endotoxinfree, for example, by heating to at least 180 °C for about 4 h.
4.6 Extraction liquids, water and detergents
All liquids used shall be endotoxinfree.
4.7 Test for contamination
To test for contamination at least two filters shall be included in each sampling run as blank samples.
Except for the actual sampling, the filters used as blank samples shall be treated in the same way as the
other filters that are used for sampling.
NOTE If not more than four samples are collected, one blank sample can be seen as sufficient.
4.8 Sampling documentation
The sampling operations carried out shall be documented to obtain comparable and reliable
concentration values of endotoxins.
The sampling documentation shall include at least the following information:
a) name of the organization and person performing the sampling;
b) date of sampling;
c) purpose of sampling;
d) a unique identifier code for the sample and sampling device (for example, sampling pump, used);
e) name and address of the company where the sampling was carried out, or a unique identifier to
preserve confidentiality;
f) workplace description including bioaerosol generating activities;
g) type and name of sampler used;
h) type and name of filter used;
i) type of sampling (personal or static) and in case of personal sampling: name and function of the
person who was involved in the sampling, or a unique identifier to preserve confidentiality; or in
case of static sampling, placement of sampling equipment;
j) positioning of sampling equipment;
k) location of sampling inlet and orientation relative to air movement, where applicable;
l) start and end time of sampling and its duration;
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EN 14031:2021 (E)
m) flow rate, in litres per minute, at least before and after sampling period;
n) sampled volume;
o) sampled health-related fraction (see EN 481);
p) details about sample transport and sample storage;
q) environmental conditions during sampling (temperature, relative humidity, outdoor weather
conditions);
r) other relevant observations.
5 Extraction
5.1 General
Samples should not be extracted unless they can be analyzed immediately. If this is not possible,
extracts may be stored refrigerated overnight. For long-term storage, extracts may be frozen as
specified in 5.4.
5.2 and 5.3 specify the equipment to be used in a recommended method for the extraction of endotoxins
from filters as well as the extraction procedure. If other equipment and methods are used they shall be
demonstrably equivalent in performance to this recommended method.
NOTE For deposition of endotoxins in inner sampler walls see Annex C.
5.2 Equipment
5.2.1 Laboratory shaker, able to shake the sample vigorously in extraction liquid.
5.2.2 Vortex shaker.
5.2.3 Centrifuge, able to centrifuge at 1 000 × G.
5.2.4 Tubes or containers, made of glass or polypropylene, endotoxinfree.
5.3 Procedure
The loaded filter shall be transferred aseptically into a tube or container. Extraction shall be performed
in endotoxinfree water using a method validated to maximize extraction.
NOTE 1 Due to lack of data no recommendation can be given for use of detergents in the extraction procedure.
For filters with a diameter of less than 50 mm a minimum of 5 ml of an extraction liquid should be used,
and a minimum of 10 ml for filters with a diameter of 50 mm or more. In order to obtain maximum
extraction of endotoxin into suspension, samples are rocked/shaken for about 1 h at a speed of at least
−1
2 000 min using the laboratory shaker at room temperature.
Before extraction the pH value should be measured in a subsample of the extraction liquid to ensure
compatibility with the performance of the LAL-assay and to comply with the recommendations given by
the manufacturer of the LAL-assay kit used.
NOTE 2 The pH value of bioaerol extracts is normally in the range from 6 to 8 which usually complies with the
recommendations given by manufacturers of LAL-assay kits.
Samples shall be centrifuged for about 15 min at about 1 000 × G. Supernatant shall be collected, well
mixed and divided into aliquots of extracts.
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EN 14031:2021 (E)
5.4 Storage conditions
If aliquots of extracts need to be stored they shall either be stored overnight at about 4 °C to 8 °C or be
stored frozen at about – 20 °C or below for long-term storage. Extracts can be stored frozen in tightly
sealed tubes for longer time periods (several years). An extract shall not be repeatedly frozen and
thawed as this can affect the measurable endotoxin concentration in the fluid. Thawed samples should
be mixed using a Vortex shaker before analysed.
Results from the analysis of previously frozen samples shall not be compared with results from samples
analysed immediately after sampling. It shall be documented that analysis has been done on previously
frozen samples.
5.5 Documentation of extraction
The documentation of extraction shall describe the extraction procedure (see 5.2, 5.3 and 5.4) and also
document at least the following information:
a) name of the organization;
b) unique identifier codes for the samples;
c) arrival time of the samples in the laboratory;
d) storage conditions before and after extraction;
e) name of technician performing the extraction;
f) date of extraction;
g) extraction protocol, for example, volume(s) and media used;
h) number of aliquots per sample and volume per aliquot;
i) other relevant observations made by the technician, e.g. colouring of extracts.
6 Analytical method
6.1 General
Kinetic chromogenic LAL-assays shall be used for the detection and quantitative measurement of
endotoxins in the workplace atmosphere. If other equipment and methods are used they shall be
proved to have equal performance as this recommended method.
NOTE The recombinant Factor C (rFC) method is a non-animal-derived reagent assay used to detect bacterial
endotoxins in pharmaceutical products. The rFC method is studied and used increasingly and represents an
alternative for the LAL method. It is based on the detection of endotoxins by fluorimetry when the endotoxin
activated synthetic recombinant Factor C enzyme cleaves a synthetic fluorogenic substrate. Other methods have
been described for determination of chemical markers for endotoxin (for example, GCMS, HPLC), but are not
routinely applied.
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EN 14031:2021 (E)
6.2 Equipment
6.2.1 Photometer, capable of evaluating 96 well plates and be set at a constant temperature of
(37 ± 1) °C for each well.
6.2.2 Computer and software, to record the kinetic readings and to calculate the endotoxin
concentrations.
NOTE Two distinctly different approaches exist to obtain the endotoxin concentration using a kinetic assay.
The first is to calculate the concentration based on the maximum velocity (V ) of the reaction. The reaction is
max
followed over a prolonged time to facilitate this process. The second option is to calculate the concentration on the
basis of only one measurement point, the time elapsed until a certain increase of optical density/refractivity n is
reached (most often n = 0,2). The first approach (V -method) is less sensitive to small analytical irregularities
max
which can be induced by little air bubbles in the wells.
6.2.3 Vortex shaker.
6.3 Procedure
6.3.1 Measurement
Kinetic chromogenic LAL-assays shall be used.
NOTE For the principle of LAL-assays see Annex B.
The samples and LPS standard(s) shall be shaken vigorously for at least 1 min at a speed of at least
−1
2 000 min using a vortex shaker. This is important because aqueous endotoxins tend to adsorb on
surfaces and form agglomerates. Subsequently, the samples (diluted, if necessary, see 6.4) and LPS
standard(s) shall be mixed with an equal volume of LAL reagent and incubated at (37 ± 1) °C. Kinetic
readings shall be recorded automatically each 30 s for a period of about 50 min by a computer such that
the maximum velocity of the kinetic reaction (V ) can be used as an estimate of the endotoxin
max
concentration. The reaction time required for the absorbance to increase 0,2 absorbance units can also
be used as an estimate for the endotoxin concentration.
6.3.2 Calibration
New batches of LAL-assay kits shall be calibrated in duplicate with at least two analytical blank samples
and eight calibration points for the range from 0,005 EU/ml to 50 EU/ml. For more narrow measuring
ranges the number of calibration points can be less than 8 bu
...
SLOVENSKI STANDARD
oSIST prEN 14031:2020
01-april-2020
Izpostavljenost na delovnem mestu - Kvantitativno določevanje lebdečih
endotoksinov
Workplace exposure - Quantitative measurement of airborne endotoxins
Exposition am Arbeitsplatz - Quantitative Messung von luftgetragenen Endotoxinen
Exposition sur les lieux de travail - Mesure quantitative des endotoxines aéroportées
Ta slovenski standard je istoveten z: prEN 14031
ICS:
13.040.30 Kakovost zraka na delovnem Workplace atmospheres
mestu
oSIST prEN 14031:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 14031:2020
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oSIST prEN 14031:2020
DRAFT
EUROPEAN STANDARD
prEN 14031
NORME EUROPÉENNE
EUROPÄISCHE NORM
March 2020
ICS 13.040.30 Will supersede EN 14031:2003
English Version
Workplace exposure - Quantitative measurement of
airborne endotoxins
Exposition sur les lieux de travail - Mesure quantitative Exposition am Arbeitsplatz - Quantitative Messung von
des endotoxines aéroportées luftgetragenen Endotoxinen
This draft European Standard is submitted to CEN members for parallel enquiry. It has been drawn up by the Technical
Committee CEN/TC 137.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 14031:2020 E
worldwide for CEN national Members.
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oSIST prEN 14031:2020
prEN 14031:2020 (E)
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Requirements . 6
4.1 Operator skills . 6
4.2 Sampling . 7
4.2.1 General . 7
4.2.2 Equipment . 7
4.2.3 Filters . 7
4.3 Sample transport . 7
4.4 Sample storage at the laboratory . 7
4.5 Glassware . 7
4.6 Extraction liquids, water and detergents . 8
4.7 Test for contamination. 8
4.8 Sampling documentation . 8
5 Extraction . 9
5.1 General . 9
5.2 Equipment . 9
5.3 Procedure . 9
5.4 Storage conditions . 9
5.5 Documentation of extraction . 10
6 Analytical method . 10
6.1 General . 10
6.2 Equipment . 10
6.3 Procedure . 11
6.3.1 Measurement . 11
6.3.2 Calibration . 11
6.3.3 Calculation. 11
6.4 Validation . 11
6.5 Documentation of analysis . 11
7 Expression of results . 12
8 Precision . 12
9 Test report . 12
Annex A (informative) Properties of endotoxins and sources of occupational exposure . 14
Annex B (informative) Principle of LAL-assays . 16
Annex C (informative) Deposition of endotoxins on inner sampler walls . 18
Bibliography . 19
2
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oSIST prEN 14031:2020
prEN 14031:2020 (E)
European foreword
This document (prEN 14031:2020) has been prepared by Technical Committee CEN/TC 137
“Assessment of workplace exposure to chemical and biological agents”, the secretariat of which is held
by DIN.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 14031:2003.
The major technical changes between this European Standard and the previous edition are as follows:
a) document title adjusted to the wording used in the Scope;
b) terms and definitions taken over from EN 13098:2019, where appropriate;
c) Clause 4 restructured;
d) requirements for sample storage at the laboratory rewritten;
e) recommendations for extraction of samples revised;
f) storage conditions described more precisely;
g) further requirements on documentation of extraction added;
h) new Table A.1 on examples of different occupational environments where endotoxins are present
added;
i) whole document editorially revised.
3
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oSIST prEN 14031:2020
prEN 14031:2020 (E)
Introduction
The term 'endotoxin' refers to lipopolysaccharides that are present in the outer membrane of the cell
walls of Gram-negative bacteria. These lipopolysaccharides are a class of pure lipid carbohydrate
molecules (free of protein and other cell wall components) that are held responsible for most of the
biological properties characteristic of bacterial endotoxins.
Endotoxins play an important role in the development of organic dust-related symptoms (for example,
inflammatory reactions in the airways and/or systemic reactions) among workers exposed vial
inhalation.
To investigate ill health resulting from occupational exposure to airborne endotoxins it is important
therefore to measure their presence in workplace air accurately. At present, different methods are used
to measure airborne endotoxins. Standardization with respect to sampling, transportation, extraction,
analytical methods and storage of samples or extracts is important in order to obtain accurate and
comparable results and reduce uncertainties in exposure assessment.
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1 Scope
This document specifies methods for the quantitative measurement of airborne endotoxins and gives
general requirements for sampling on filters, transportation, storage as well as the analysis of samples.
This document provides also guidelines for the assessment of workplace exposure to airborne
endotoxins.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 481, Workplace atmospheres — Size fraction definitions for measurement of airborne particles
EN 1540, Workplace exposure — Terminology
EN ISO 13137, Workplace atmospheres — Pumps for personal sampling of chemical and biological agents
- Requirements and test methods (ISO 13137)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 1540 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
Note 1 to entry: In particular, the following terms used in this document are defined in EN 1540: aerosol,
biological agent, bioaerosol, exposure (by inhalation), inhalable fraction, measuring procedure, personal sampling,
static sampling and workplace.
3.1
control standard endotoxin
CSE
standard that is traceable to the reference standard endotoxin (RSE)
3.2
endotoxin
constituent of the external membrane of Gram-negative bacteria (lipopolysaccharide), consisting of a
complex lipid, lipid A, which is covalently bound to a polysaccharide
Note 1 to entry “Free endotoxin” is liberated after cell death and by budding from living cells. Lipid A is the
active (toxic) part and is a potent pro-inflammatory substance and can induce febrile, bronchial and other
symptoms in exposed workers. The composition and the toxicity of endotoxin differ between bacteria species
Note 2 to entry: A brief overview with respect to physical and chemical properties of endotoxins and sources of
occupational exposure is given in Annex A.
[SOURCE: EN 13098:2019, 3.9 – modified, Note 2 to entry added]
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3.3
endotoxinfree liquid
water or solution containing no measurable endotoxin
Note 1 to entry Endotoxinfree and pyrogenfree are used interchangeable in the literature
Note 2 to entry: In practise, less than 0,2 endotoxin units per millilitre is often used as threshold value for
endotoxinfree.
3.4
endotoxin unit
EU
unit standardized against the defined reference material, reference standard endotoxin
[SOURCE: EN 13098:2019, 3.10]
3.5
Limulus Amoebocyte Lysate
LAL
enzymes extracted from the blood cells of the horse shoe crab (Limulus polyphemus) that are activated
by endotoxin and other molecules (glucans etc.)
[SOURCE: EN 13098:2019, 3.16]
3.6
Limulus Amoebocyte Lysate-assay
LAL-assay
functional assay to measure endotoxin concentrations
Note 1 to entry The method is highly sensitive and based on the activation of a clotting enzyme present in the
lysate of hemolymph of the horseshoe crab (Limulus polyphemus).
3.7
lipopolysaccharide
LPS
water-soluble and stable molecule composed of lipid and polysaccharide present in Gram-negative
bacteria
Note 1 to entry The lipid part of LPS is termed 'lipid A' and is essential for the toxic properties of LPS
Note 2 to entry The terms 'endotoxin' and 'lipopolysaccharide' are often used interchangeably in scientific
literature regarding occupational health.
3.8
reference standard endotoxin
RSE
purified lipopolysaccharide from Escherichia coli that serves as an international reference standard
4 Requirements
4.1 Operator skills
The operator shall be trained in aseptic techniques to avoid contamination of the sample during any
measurement phase. This includes knowledge about sampling equipment and how to perform the
sampling.
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4.2 Sampling
4.2.1 General
Measurement of airborne endotoxins should preferably be performed by sampling of the inhalable
fraction of aerosols. Aerosols shall be sampled on a filter using a pump to draw air through the filter.
Personal sampling shall be used for risk evaluation including comparison with exposure guidelines.
Static sampling can be used to identify sources of exposure and to measure background concentrations
of airborne endotoxins.
NOTE Other comparable sampling methods can be used for evaluation of other specific parameters, for
example, to measure endotoxins in different size distributions or particle size fractions, or for short- or long-term
exposure measurement.
4.2.2 Equipment
The pumps used for personal sampling and static sampling shall fulfil the requirements specified in
EN ISO 13137. Personal samplers shall collect the inhalable fraction of aerosols as specified in EN 481.
The filter holder or cassette should be rendered endotoxinfree. Heat-resistant resusuable samplers
shall be treated as indicated in 4.3. Non heat-resistant resusuable samplers shall be cleaned thoroughly
so that contamination of the samples is avoided. Cleaning agents shall be removed by subsequent
washing in endotoxinfree water. Disposable samplers do not need to be sterilized when it is assumed
that the manufacturing process is endotoxinfree. Possible contamination shall be controlled by using
blank filters (see 4.7).
4.2.3 Filters
Binderfree glass fibre filters should be used for sampling of airborne endotoxins because these filters
perform very well regarding their collection efficiency, blank values and recovery. If any other filter is
used the performance of these parameters shall be comparable.
NOTE Several filter materials (for example, polyvinylchloride, polytetrafluoroethylene and polycarbonate)
are commonly used but some materials adsorb endotoxins and thereby give false low values.
4.3 Sample transport
After sampling, filters shall be prepared for transport by sealing in a container or sampler cassette. The
sample shall be transported in conditions that prevent large variations in temperature and the gaining
of moisture. If transport time is above 24 h, or there is risk of large temperature variations, actions to
prevent moisture shall be taken, either by using an isolating container or by including a dehumidifier.
Conditions during transport shall be documented, including transport time and temperature log.
If the temperature is above 30 °C the filters shall be placed in cooler conditions (for example, by using a
cooling box).
4.4 Sample storage at the laboratory
If the sample is not extracted within two weeks after arrival at the laboratory the sample shall be stored
in conditions that prevent large variations in temperature and gain of moisture. Alternatively, for
storage of more than two weeks they should be frozen at a temperature of about –20 °C or below,
because endotoxins are very stable when frozen. However, samples shall not be frozen and thawed
more than once, as this can affect the detectable endotoxin content of them.
4.5 Glassware
Glassware shall be rendered endotoxinfree, for example, by heating to at least 180 °C for about 4 h.
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4.6 Extraction liquids, water and detergents
All liquids used shall be endotoxinfree.
4.7 Test for contamination
To test for contamination a series of at least two blank filters shall be included in each sampling session.
Except for the actual sampling, blank filters shall be treated equal to the other filters that are used for
sampling.
4.8 Sampling documentation
The sampling operations carried out shall be documented to obtain comparable and reliable
concentration values of endotoxins.
The sampling documentation shall include at least the following information:
a) name of the organization and person performing the sampling;
b) date of sampling;
c) purpose of sampling;
d) a unique identifier code for the sample and sampling device (for example, sampling pump, used);
e) name and address of the company where the sampling was carried out, or a unique identifier to
preserve confidentiality;
f) workplace description including bioaerosol generating activities;
g) type and name of sampler used;
h) type and name of filter used;
i) type of sampling (personal or static) and in case of personal sampling: name and function of the
person who was involved in the sampling, or a unique identifier to preserve confidentiality; or in
case of static sampling, placement of sampling equipment;
j) positioning of sampling equipment;
k) location of sampling inlet and orientation relative to air movement, where applicable;
l) start and end time of sampling and its duration;
m) flow rate, in litres per minute, at least before and after sampling period;
n) sampled volume;
o) sampled health-related fraction (see EN 481);
p) details about sample transport and sample storage;
q) environmental conditions during sampling (temperature, relati
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