SIST-TS CEN/TS 16868:2016
(Main)Ambient air - Sampling and analysis of airborne pollen grains and fungal spores for allergy networks - Volumetric Hirst method
Ambient air - Sampling and analysis of airborne pollen grains and fungal spores for allergy networks - Volumetric Hirst method
This European Standard specifies the procedure to sample continuously and analyse the concentration of airborne pollen grains and fungal spores in ambient air using the volumetric Hirst type sampler [1] [2] [3] (see Annex A).
This European Standard describes both the sampling and the analysis procedures for the purpose of allergy networks. For the other tasks mentioned in the introduction, other specifications may be required.
Außenluft - Probenahme und Analyse luftgetragener Pollen und Pilzsporen für Allergienetzwerke - Volumetrische Hirst-Methode
Air ambiant - Échantillonnage et analyse des grains de pollen et des spores fongiques aériens pour les réseaux aérobiologiques - Méthode volumétrique de Hirst
La présente Norme européenne spécifie le mode opératoire pour l’échantillonnage en continu et l’analyse de la concentration en grains de pollen et spores fongiques aériens dans l’air ambiant en utilisant l’échantillonneur volumétrique de Hirst [1] [2] [3] (voir l’Annexe A).
Elle décrit les modes opératoires d’échantillonnage et d’analyse applicables aux stations à vocation allergologique. Pour les autres objectifs mentionnés dans l’introduction, des spécifications différentes peuvent être requises.
Zunanji zrak - Vzorčenje in analiza cvetnega prahu in trosov gliv v zraku za alergijsko omrežje - Volumetrična Hirstova metoda
Ta evropski standard določa postopek za stalno vzorčenje in analiziranje koncentracije pelodnih zrn in trosov gliv v zunanjem zraku z volumetričnim vzorčevalnikom vrste Hirst [1] [2] [3] (glejte dodatek A).
Ta evropski standard opisuje postopke vzorčenja in tudi analize za namen alergijskih omrežij. Za druge naloge, omenjene v uvodu, se lahko zahteva druge specifikacije.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 16868:2016
01-februar-2016
=XQDQML]UDN9]RUþHQMHLQDQDOL]DFYHWQHJDSUDKXLQWURVRYJOLYY]UDNX]D
DOHUJLMVNRRPUHåMH9ROXPHWULþQD+LUVWRYDPHWRGD
Ambient air - Sampling and analysis of airborne pollen grains and fungal spores for
allergy networks - Volumetric Hirst method
Außenluft - Probenahme und Analyse luftgetragener Pollen und Pilzsporen für
Allergienetzwerke - Volumetrische Hirst-Methode
Air ambiant - Échantillonnage et analyse des grains de pollen et des spores fongiques
aériens pour les réseaux aérobiologiques - Méthode volumétrique de Hirst
Ta slovenski standard je istoveten z: CEN/TS 16868:2015
ICS:
13.040.20 Kakovost okoljskega zraka Ambient atmospheres
SIST-TS CEN/TS 16868:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 16868:2016
CEN/TS 16868
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
November 2015
TECHNISCHE SPEZIFIKATION
ICS 13.040.20
English Version
Ambient air - Sampling and analysis of airborne pollen
grains and fungal spores for allergy networks - Volumetric
Hirst method
Air ambiant - Échantillonnage et analyse des grains de Außenluft - Probenahme und Analyse luftgetragener
pollen et des spores fongiques aériens pour les réseaux Pollen und Pilzsporen für Allergienetzwerke -
aérobiologiques - Méthode volumétrique de Hirst Volumetrische Hirst-Methode
This Technical Specification (CEN/TS) was approved by CEN on 15 September 2015 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16868:2015 E
worldwide for CEN national Members.
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Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 8
5 Sampling . 8
5.1 Equipment . 8
5.1.1 Apparatus . 8
5.1.2 Scrolling speed of support . 11
5.1.3 Impaction support . 11
5.1.4 Wind vane and rain shield . 13
5.1.5 Complete sampling system . 14
5.2 Operating procedure . 14
5.2.1 Preparation of the coating medium [12] . 14
5.2.2 Support preparation . 15
5.2.3 Changing of the drum . 16
6 Analysis . 16
6.1 Equipment required . 16
6.2 Operating procedure . 17
6.2.1 Support . 17
6.2.2 Mounting medium . 17
6.3 Methodology for counting . 18
6.3.1 Glass slide preparation for microscopy analysis for drum tape [13] [14] . 18
6.3.2 Optical microscopy [14] [16] . 20
6.3.3 Identification [15] [16] . 20
6.3.4 Counting method . 20
6.3.5 Data recording . 20
6.3.6 Conversion factor . 21
7 Performance characteristics [10] [11] [17] [18] [19] . 22
7.1 General . 22
7.2 Performance requirements . 22
7.2.1 Repeatability . 22
7.3 Performance recommendations [18] [19] . 22
7.3.1 Reproducibility and accuracy . 22
7.3.2 Sensitivity and specificity. 22
Annex A (informative) Hirst type volumetric trap . 24
Annex B (informative) Pictures of impaction support. 25
Annex C (informative) Safety data sheet . 27
Annex D (informative) Key of determination . 28
Bibliography . 32
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European foreword
This document (CEN/TS 16868:2015) has been prepared by Technical Committee CEN/TC 264 “Air
quality”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
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Introduction
Biological particles (pollen and fungal spores) are present in the air, causing health impacts at various
levels. In Europe, nearly 18 % to 20 % of people suffer from pollinosis due to pollen and/or fungal
spores. Pollen grains and fungal spores are considered in some Member States as an air pollutant as
well as particles suspended in the air (PM , ). In Europe, European Aerobiology Society (EAS) in
10 2,5
coordination with International Association for Aerobiology (IAA) manage problems of sampling,
analysis, quality control, development and information.
For the sampling and analysis of biological particles different methodology and operating procedures
are used.
Sampling equipment is diversified (see Annex A). Analysis is based on optical light microscopy for
identification and counting pollen grains and fungal spores.
Elements and reagents used during sampling and analysis have very specific properties and require to
be handled carefully.
Given the close relationship between aerobiology and other sciences, one of the main aims is that
information on airborne biological-particle counts should be of use in a wide range of disciplines and
fields of application, including aerobiology, biodiversity, agriculture, forestry, phytopathology,
meteorology, climatology, forensic science, bioterrorism, and health (sensitization and allergy).
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1 Scope
This European Standard specifies the procedure to sample continuously and analyse the concentration
of airborne pollen grains and fungal spores in ambient air using the volumetric Hirst type sampler [1]
[2] [3] (see Annex A).
This European Standard describes both the sampling and the analysis procedures for the purpose of
allergy networks. For the other tasks mentioned in the introduction, other specifications may be
required.
2 Normative references
Not applicable.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
accuracy
closeness of agreement between a measured quantity value and a true quantity value of a measurement
3.2
bench
long work table in a workshop or laboratory
3.3
clockwork
mechanism with a spring and toothed gearwheels, used to drive a mechanical clock, toy, or other device
3.4
combined standard measurement uncertainty
obtained using the individual standard measurement uncertainties associated with the input quantities
in a measurement model
3.5
defatted
surface conditions after clearing with a fat removing substance
3.6
drum
cylindrical device for the mounting of a sticky tape
3.7
exine
outer layer of the wall of a spore or pollen grain, also called an exosporium
3.8
eyepiece
lens or combination of lenses in an optical instrument through which the eye views the image formed
by the objective lens or lenses; ocular
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3.9
flow meter
instrument for measuring the flow rate of a fluid in a pipe
3.10
flow rate
amount of fluid (e.g. air) that flows in a given time
3.11
fungal spore
reproductive unit capable of giving rise to a new individual with or without sexual fusion
3.12
hood
metal cover or canopy for a stove, ventilator, etc
3.13
impaction
sampling of airborne particles by inertial separation on any surface (e.g. of an adhesive)
3.14
magnetic stirrer
object or mechanical device used for stirring something
3.15
magnification
magnifying power of an instrument, e.g. this microscope should give a magnification of about x 100
3.16
microscope
optical instrument having a magnifying lens or a combination of lenses for inspecting objects too small
to be seen or too small to be seen distinctly and in detail by the unaided eye
3.17
objectives
optics (in a telescope, microscope, camera, or other optical system), the lens or combination of lenses,
that first receive the rays from the object and form the image in the focal plane of the eyepiece, as in a
microscope, or on a plate or screen as in a camera
Note 1 to entry: Also called object glass, object lens, objective lens.
3.18
orifice
opening or aperture, as of a tube or pipe; a mouthpiece with a slotlike opening on the side of the trap
3.19
particle
minute portion of matter
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3.20
pollen
male gametophyte of flowering plants, consisting of microscopic grain discharged from the anthers
(Angiosperms) or from a male cone (Gymnosperms)
Note 1 to entry: Each grain contains two male gametes (only one can fertilize the female ovule).
Note 2 to entry: Pollen are transported by wind, water, animals (e.g. insects).
3.21
precision
closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions
3.22
repeatability
condition of measurement, out of a set of conditions that includes the same measurement procedure,
same operators, same measuring system, same operating conditions and same location, and replicate
measurements on the same or similar objects over a short period of time
3.23
reproducibility
condition of measurement, out of a set of conditions that includes different locations, operators,
measuring systems, and replicate measurements on the same or similar objects
3.24
sensitivity
in aerobiology, measurement of the proportion of search particle which is correctly identified
3.25
slide
rectangular piece of glass on which an object is mounted or placed for examination under a microscope
3.26
specificity
in aerobiology, measurement of the proportion of non-searched particles which are correctly identified
as different from the searched particles
3.27
standard measurement uncertainty
measurement uncertainty expressed as a standard deviation
3.28
suction
production of a negative pressure by the removal of air to force fluid into a vacant space
3.29
taxon
taxonomic group of any rank, such as a species, family or class
3.30
trap
container or device used to collect something
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3.31
vacuum
space from which the air has been completely or partly removed
3.32
vacuum pump
pump or device by which a partial vacuum can be produced
3.33
wind vane
mechanical device attached to an elevated structure; rotates freely depending on the direction of the
wind
4 Principle
Ambient air is sampled by a volumetric suction system and directed towards a suitably coated sampling
surface through a specific orifice oriented towards the wind; the particles contained in the sampled air
are deposited by impaction on a continuously moving adhesive acceptor surface. The sampling surface
is then examined with an optical microscope in order to identify and count the allergy relevant particles
per area (deposition rates). Using this method allows to count particles and subsequently calculate
concentrations as a daily mean or a 2-hour mean. The low-volume sampler (10 l/min) allows a
continuous sampling for up to seven days [4] [5] [6].
5 Sampling
5.1 Equipment
5.1.1 Apparatus
5.1.1.1 Motorised suction pump
The motorised suction pump shall work 24 hours a day and continuously throughout the year always at
the same flow rate. The power supply may be either mains or battery driven (solar panels). The electric
motor shall be capable of continuous operation.
The suction system, for instance, a vacuum pump, shall have a regular and continuous flow rate. The
flow rate of suction may be adjusted by a flow control valve. The flow rate shall be 10 l/min (±1 l/min).
The flow rate shall be checked at every change of the impaction support with an adapted flow meter (i.e.
the flow meter supplied by the same supplier of the sampler).
The flow meter shall be checked annually or less depending on the historical experience.
Sampling shall always be continuous and stable. The support shall scroll regularly in front of the back
outlet of the orifice; its geometry and the scrolling speed depend on the duration of sampling period
requested.
Example for the sampling area:
— a support of 48 mm (tape) with a speed of 2 mm/h: sampling period = one day;
— a flexible support of 336 mm with a speed of 2 mm/h: sampling period = seven days.
The scrolling speed may be adjusted to other sampling period durations. The trap can work with two
different impaction supports: a drum (see Figure 1a) covered with a coated transparent tape or a glass
slide (see Figure 1b) coated.
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Key
1 lid
2 start reference pointer
3 lock nut
4 orifice start position
5 trapping surface
Figure 1a — The Hirst volumetric trap showing 7-day lid assembly with drum [5, modified]
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Key
1 lid
3 lock nut
6 trapping surface on slide
Figure 1b — The Hirst volumetric trap showing 24-h lid assembly with slide [5, modified]
5.1.1.2 Specific orifice
The orifice shall have the following dimensions (with associated tolerances):
— rectangular opening: 14 mm (±0,1 mm) × 2 mm (±0,1 mm)
— depth of the orifice: > 19 mm
— distance from the inside orifice to the drum without the tape: 0,70 mm (±0,1 m)
The depth allows the non-turbulence of laminar flow and directs the mixture of air and particles
towards the coated support. In consequence, an efficient particle impaction for pollen grains and fungal
spores, induced by the laminar flow, is ensured.
The outlet of the orifice shall be 0,70 mm (±0,1 mm) of the coated support (see Figure 2 – distance A-B).
The distance allows efficient particle impaction for pollen grains and fungal spores. It shall be
controlled with a ruler [7] [8] [9].
The orifice should be directed into the air-stream using a wind vane.
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Key
1 drum
2 orifice
3 cover
A 20,5 mm or 22,5 mm, depending on supplier
B 19,8 mm or 21,8 mm, depending on supplier
C drum diameter 110 mm to 112 mm
A-B 0,7 mm (±0,1 mm)
Figure 2 — Pollen trap (Head of Hirst system) [9, modified]
5.1.2 Scrolling speed of support
The scrolling speed of the support is from 2 mm/h to 14 mm/h (±0,01 mm/h).
It shall be consistent throughout the scrolling period and can be ensured by a clockwork system or by
an electric motor that shall not be stopped during the sampling period.
5.1.3 Impaction support
5.1.3.1 General
Only two options are allowed, both clockwork driven:
— a glass slide (76 mm x 26 mm) on which a transparent tape is fixed (48 mm x 19 mm) coated with
specific reagents (see Figure B.1 in Annex B);
— a 110 mm to 112 mm diameter drum on which a transparent coated tape is wrapped around (see
Figure B.2 in Annex B). The length of this transparent tape is 345 mm (±0,5 mm) to 350 mm
(±0,5 mm).
5.1.3.2 Transparent tape
The transparent tape is coated with an adhesive in order to fix the particles.
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The following requirements shall be fulfilled:
— The transparent tape shall be water-insoluble.
— The thickness of the whole transparent tape shall not be changed over time, and should not be
affected by operational conditions (temperature between – 20 °C to + 60 °C or humidity between
20 % and 100 %).
— It shall be perfectly transparent to allow the passing of microscopic light.
— The length is > 336 mm.
5.1.3.3 Reagents
Two transparent coating products (solubilised in specific solvents or not) are useable: Vaseline
(petroleum jelly) or silicone.
5.1.3.3.1 Vaseline (petroleum jelly)
The main characteristics of Vaseline (petroleum jelly) (n° CAS: [8009 03 8], see Annex C) are the
following:
— odourless;
— colourless;
— viscous liquid;
— boiling point : 68 °C;
— purity > 99 %;
— risk of diarrhoea and other stomach problems if absorbed or in case of repeated exposure.
NOTE Consult Safety Data Sheet or MSDS to obtain special instructions before use.
Solubilise the Vaseline (petroleum jelly) with toluene (n° CAS [108 88 3], see Annex C). It is also
possible to use unsolubilised Vaseline (petroleum jelly).
The purity of Vaseline (petroleum jelly) and toluene shall be > 99 %.
5.1.3.3.2 Silicone
The main characteristics of silicone (n° CAS [90337 93 2], see Annex C) are the following:
— odourless;
— colourless to white;
— pasty;
— high viscosity stable from – 20 °C to +150 °C;
— flammable over 400 °C;
— irritating to eyes;
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— non-biodegradable.
Solubilise the silicone with a specific solvent (see Annex C).
The physical properties of the adhesive medium remain unaltered at temperatures between – 20 °C and
+ 50 °C, making it suitable for the majority of bioclimatic zones.
5.1.4 Wind vane and rain shield
The wind vane allows permanent rotation of the trap head so that the orifice faces the wind. The rain
shield ensures a weather protection for the orifice (i.e. rainfall) (see Figure 3).
Key
1 wind vane
2 lid assembly
3 rain shield
4 orifice
5 rotation lock
6 motor cover
Figure 3a — Schematic picture of a Hirst seven day volumetric spore sampling system [5,
modified]
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Figure 3b — Photo of a Hirst volumetric spore sampler [Source: RNSA]
5.1.5 Complete sampling system
The complete sampling system (so called “trap”) containing the motor, the vacuum pump, the orifice,
the rotating drum, the wind vane, the clockwork system, the impaction support shall be
— resistant to corrosion;
— well attached (i.e. resistant to wind-blow, …);
— always horizontal (at the head level).
The commercial devices that meet the requirements are presented in Annex A.
For the different purposes, refer to the specific publications.
For allergen monitoring purposes, the following requirements and conditions for sampler positioning of
the trap shall be fulfilled:
— The sampler shall be placed on a readily accessible, flat, horizontal surface. It should be on the roof
of a building, and away from the edge of the building in order to reduce the effects of turbulence.
— Care shall be taken to ensure that adjacent buildings do not screen the sampler or interfere with the
airflow. The sampler should be ideally placed on the roof of a building at more than 2 m from the
edge; the height above ground level depends on the city and on the height of neighbouring
buildings.
— The sampler itself shall be elevated between 100 cm to 150 cm from the roof in order to avoid
turbulence between air layers and possible re-suspension of particles from the roof.
— The sampler shall not be placed in the vicinity of fixed or mobile sources of mass emission of
biological or non-biological particles. Proximity to non-biological particle sources may favour the
massive presence of residues in samples, which considerably hinder identification.
5.2 Operating procedure
5.2.1 Preparation of the coating medium [12]
Prepare the coating medium as follows:
— Vaseline (petroleum jelly (18 g (± 1 g)) and toluene (1l) (purity > 99 %);
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— under the hood, add Vaseline (petroleum jelly) to toluene stirring until completely dispersed and
leave it to stand for about 48 h with periodic shaking to obtain a homogenous fluid solution.
Or use pure Vaseline (petroleum jelly).
Or
— silicone fluid (20 g (±1 g)) and a specific solvent;
— under the hood, add silicone to the solvent stirring until completely dispersed and leave to stand
for about 48 h with periodic shaking to obtain a homogeneous fluid solution;
— spread the solution on the tape with a brush and let it dry under the hood for at least 1 h.
1)
The brush should be a pure Ox-hair brush N°14.i.e.: ref 922 N°14 OMEGA, made in Italy
WARNING — For the use of silicone or Vaseline (petroleum jelly) solutions:
— Keep the transparent tape dust free and always work with closed windows to avoid
contamination.
— Always keep the coating medium under an extractor hood due to the release of solvents
(toxic nature of the solvents).
— Wear protective gloves/clothing/equipment for eye and face protection. Wash thoroughly
after handling.
5.2.2 Support preparation
Fixation of the coated or uncoated tape on the drum.
Be careful to
— always clean the drum or the glass slide with ethyl alcohol (70 %) in order to avoid dust and/or
biological particles;
— fix the coated or uncoated transparent tape on the drum.
If it is an uncoated tape, the tape fixed on the drum shall be coated under the hood if a solvent reagent is
used.
The capture surface shall be covered with a thin homogeneous layer of coating medium in order to
retain the targeted particles.
The support shall be protected from ambient air during transportation from the laboratory to the trap
and return and shall remain in a metal box for protection against shocks (see Figure 4). Conservation at
ambient temperatures shall not exceed 12 months for silicone and one month for Vaseline (petroleum
jelly).
1)
Brush: ref 922 N°14 OMEGA is (are) an example(s) of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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Figure 4 — The drum and its protection box [Source: RNSA]
5.2.3 Changing of the drum
The person in charge of changing the drum shall
— mark the tape with a sharp instrument through the orifice at the end of the registered drum;
— measure and record the flow rate of 10 l/min (±1 l/min);
— remove the drum, clean the orifice and the etancheity gasket;
— wind the clock;
— put in the new drum;
— measure the flow rate; make a note of it and correct if necessary after cleaning the orifice and
checking the sealing;
— note date and exact time (hour and minute) as well as flow rate and initials of the operator;
— mark the tape with a sharp instrument through the orifice at the beginning of the new drum.
6 Analysis
6.1 Equipment required
— microscope;
— magnetic stirrer;
— hot plate or microwave;
— bench;
— utensils to use (tweezers, scalpel);
— winding support;
— cutting rule;
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...
SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 16868:2015
=XQDQML]UDN9]RUþHQMHLQDQDOL]DFYHWQHJDSUDKXY]UDNXLQJOLYLþQHVSRUH]D
DOHUJLMVNRRPUHåMH9ROXPHWULþQD+LUVWRYDPHWRGD
Ambient air - Sampling and analysis of airborne pollen grains and fungal spores for
allergy networks - Volumetric Hirst method
Außenluft - Probenahme und Analyse luftgetragener Pollen und Pilzsporen für
Allergienetzwerke - Volumetrische Hirst-Methode
Air ambiant - Échantillonnage et analyse des grains de pollen et des spores fongiques
aériens pour les réseaux aérobiologiques - Méthode volumétrique de Hirst
Ta slovenski standard je istoveten z: FprCEN/TS 16868
ICS:
13.040.20 Kakovost okoljskega zraka Ambient atmospheres
kSIST-TS FprCEN/TS 16868:2015 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST-TS FprCEN/TS 16868:2015
TECHNICAL SPECIFICATION
FINAL DRAFT
FprCEN/TS 16868
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
May 2015
ICS 13.040.20
English Version
Ambient air - Sampling and analysis of airborne pollen grains
and fungal spores for allergy networks - Volumetric Hirst method
Air ambiant - Échantillonnage et analyse des grains de Außenluft - Probenahme und Analyse luftgetragener Pollen
pollen et des spores fongiques aériens pour les réseaux und Pilzsporen für Allergienetzwerke - Volumetrische Hirst-
aérobiologiques - Méthode volumétrique de Hirst Methode
This draft Technical Specification is submitted to CEN members for formal vote. It has been drawn up by the Technical Committee CEN/TC
264.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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 Technical Specification. It is distributed for review and comments. It is subject to change without notice
and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 16868:2015 E
worldwide for CEN national Members.
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Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 Principle .8
5 Sampling .8
5.1 Equipment .8
5.1.1 Apparatus .8
5.1.2 Scrolling speed of support . 11
5.1.3 Impaction support. 11
5.1.4 Wind vane and rain shield . 13
5.1.5 Complete sampling system . 14
5.2 Operating procedure . 14
5.2.1 Preparation of the coating medium [12] . 14
5.2.2 Support preparation . 15
5.2.3 Changing of the drum . 16
6 Analysis . 16
6.1 Equipment required . 16
6.2 Operating procedure . 17
6.2.1 Support . 17
6.2.2 Mounting medium . 17
6.3 Methodology for counting . 18
6.3.1 Glass slide preparation for microscopy analysis for drum tape [13] [14] . 18
6.3.2 Optical microscopy [14] [16] . 20
6.3.3 Identification [15] [16] . 20
6.3.4 Counting method . 20
6.3.5 Data recording . 20
6.3.6 Conversion factor . 21
7 Performance characteristics [10] [11] [17] [18] [19] . 22
7.1 General . 22
7.2 Performance requirements . 22
7.2.1 Repeatability . 22
7.3 Performance recommendations [18] [19] . 22
7.3.1 Reproducibility and accuracy . 22
7.3.2 Sensitivity and specificity . 23
Annex A (informative) Hirst type volumetric trap . 24
Annex B (informative) Pictures of impaction support . 25
Annex C (informative) Safety data sheet . 27
Annex D (informative) Key of determination . 28
Bibliography . 31
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Foreword
This document (FprCEN/TS 16868:2015) has been prepared by Technical Committee CEN/TC 264 “Air
quality”, the secretariat of which is held by DIN.
This document is currently submitted to the Formal vote.
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Introduction
Biological particles (pollen and fungal spores) are present in the air, causing health impacts at various levels.
In Europe, nearly 18 % to 20 % of people suffer from pollinosis due to pollen and/or fungal spores. Pollen
grains and fungal spores are considered in some Member States as an air pollutant as well as particles
suspended in the air (PM , ). In Europe, European Aerobiology Society (EAS) in coordination with
10 2,5
International Association for Aerobiology (IAA) manage problems of sampling, analysis, quality control,
development and information.
For the sampling and analysis of biological particles different methodology and operating procedures are
used.
Sampling equipment is diversified (see Annex A). Analysis is based on optical light microscopy for
identification and counting pollen grains and fungal spores.
Elements and reagents used during sampling and analysis have very specific properties and require to be
handled carefully.
Given the close relationship between aerobiology and other sciences, one of the main aims is that information
on airborne biological-particle counts should be of use in a wide range of disciplines and fields of application,
including aerobiology, biodiversity, agriculture, forestry, phytopathology, meteorology, climatology, forensic
science, bioterrorism, and health (sensitization and allergy).
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1 Scope
This European Standard specifies the procedure to sample continuously and analyse the concentration of
airborne pollen grains and fungal spores in ambient air using the volumetric Hirst type sampler [1] [2] [3] (see
Annex A).
This European Standard describes both the sampling and the analysis procedures for the purpose of allergy
networks. For the other tasks mentioned in the introduction, other specifications may be required.
2 Normative references
Not applicable.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
accuracy
closeness of agreement between a measured quantity value and a true quantity value of a measurement
3.2
bench
long work table in a workshop or laboratory
3.3
clockwork
mechanism with a spring and toothed gearwheels, used to drive a mechanical clock, toy, or other device
3.4
combined standard measurement uncertainty
obtained using the individual standard measurement uncertainties associated with the input quantities in a
measurement model
3.5
defatted
surface conditions after clearing with a fat removing substance
3.6
drum
cylindrical device for the mounting of a sticky tape
3.7
exine
outer layer of the wall of a spore or pollen grain, also called an exosporium
3.8
eyepiece
lens or combination of lenses in an optical instrument through which the eye views the image formed by the
objective lens or lenses; ocular
3.9
flow meter
instrument for measuring the flow rate of a fluid in a pipe
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3.10
flow rate
amount of fluid (e.g. air) that flows in a given time
3.11
fungal spore
reproductive unit capable of giving rise to a new individual with or without sexual fusion
3.12
hood
metal cover or canopy for a stove, ventilator, etc
3.13
impaction
sampling of airborne particles by inertial separation on any surface (e.g. of an adhesive)
3.14
magnetic stirrer
object or mechanical device used for stirring something
3.15
magnification
magnifying power of an instrument, e.g. this microscope should give a magnification of about x 100
3.16
microscope
optical instrument having a magnifying lens or a combination of lenses for inspecting objects too small to be
seen or too small to be seen distinctly and in detail by the unaided eye
3.17
objectives
optics (in a telescope, microscope, camera, or other optical system), the lens or combination of lenses, that
first receive the rays from the object and form the image in the focal plane of the eyepiece, as in a
microscope, or on a plate or screen as in a camera
Note 1 to entry: Also called object glass, object lens, objective lens.
3.18
orifice
opening or aperture, as of a tube or pipe; a mouthpiece with a slotlike opening on the side of the trap
3.19
particle
minute portion of matter
3.20
pollen
male gametophyte of flowering plants, consisting of microscopic grain discharged from the anthers
(Angiosperms) or from a male cone (Gymnosperms)
Note 1 to entry: Each grain contains two male gametes (only one can fertilize the female ovule).
Note 2 to entry: Pollen are transported by wind, water, animals (e.g. insects).
3.21
precision
closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions
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3.22
repeatability
condition of measurement, out of a set of conditions that includes the same measurement procedure, same
operators, same measuring system, same operating conditions and same location, and replicate
measurements on the same or similar objects over a short period of time
3.23
reproducibility
condition of measurement, out of a set of conditions that includes different locations, operators, measuring
systems, and replicate measurements on the same or similar objects
3.24
sensitivity
in aerobiology, measurement of the proportion of search particle which is correctly identified
3.25
slide
rectangular piece of glass on which an object is mounted or placed for examination under a microscope
3.26
specificity
in aerobiology, measurement of the proportion of non-searched particles which are correctly identified as
different from the searched particles
3.27
standard measurement uncertainty
measurement uncertainty expressed as a standard deviation
3.28
suction
production of a negative pressure by the removal of air to force fluid into a vacant space
3.29
taxon
taxonomic group of any rank, such as a species, family or class
3.30
trap
container or device used to collect something
3.31
vacuum
space from which the air has been completely or partly removed
3.32
vacuum pump
pump or device by which a partial vacuum can be produced
3.33
wind vane
mechanical device attached to an elevated structure; rotates freely depending on the direction of the wind
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4 Principle
Ambient air is sampled by a volumetric suction system and directed towards a suitably coated sampling
surface through a specific orifice oriented towards the wind; the particles contained in the sampled air are
deposited by impaction on a continuously moving adhesive acceptor surface. The sampling surface is then
examined with an optical microscope in order to identify and count the allergy relevant particles per area
(deposition rates). Using this method allows to count particles and subsequently calculate concentrations as a
daily mean or a 2-hour mean. The low-volume sampler (10 l/min) allows a continuous sampling for up to
seven days [4] [5] [6].
5 Sampling
5.1 Equipment
5.1.1 Apparatus
5.1.1.1 Motorised suction pump
The motorised suction pump shall work 24 hours a day and continuously throughout the year always at the
same flow rate. The power supply may be either mains or battery driven (solar panels). The electric motor
shall be capable of continuous operation.
The suction system, for instance, a vacuum pump, shall have a regular and continuous flow rate. The flow rate
of suction may be adjusted by a flow control valve. The flow rate shall be 10 l/min (±1 l/min).
The flow rate shall be checked at every change of the impaction support with an adapted flow meter (i.e. the
flow meter supplied by the same supplier of the sampler).
The flow meter shall be checked annually or less depending on the historical experience.
Sampling shall always be continuous and stable. The support shall scroll regularly in front of the back outlet of
the orifice; its geometry and the scrolling speed depend on the duration of sampling period requested.
Example for the sampling area:
— A support of 48 mm (tape) with a speed of 2 mm/h: sampling period = one day;
— A flexible support of 336 mm with a speed of 2 mm/h: sampling period = seven days.
The scrolling speed may be adjusted to other sampling period durations. The trap can work with two different
impaction supports: a drum (see Figure 1a) covered with a coated transparent tape or a glass slide (see
Figure 1b) coated.
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Key
1 lid
2 start reference pointer
3 lock nut
4 orifice start position
5 trapping surface
Figure 1a — The Hirst volumetric trap showing 7-day lid assembly with drum [5, modified]
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Key
1 lid
3 lock nut
6 trapping surface on slide
Figure 1b — The Hirst volumetric trap showing 24-h lid assembly with slide [5, modified]
5.1.1.2 Specific orifice
The orifice shall have the following dimensions (with associated tolerances):
— rectangular opening: 14 mm (±0,1 mm) × 2 mm (±0,1 mm)
— depth of the orifice: > 19 mm
— distance from the inside orifice to the drum without the tape: 0,70 mm (±0,1 m)
The depth allows the non-turbulence of laminar flow and directs the mixture of air and particles towards the
coated support. In consequence, an efficient particle impaction for pollen grains and fungal spores, induced by
the laminar flow, is ensured.
The outlet of the orifice shall be 0,70 mm (±0,1 mm) of the coated support (see Figure 2 – distance A-B). The
distance allows efficient particle impaction for pollen grains and fungal spores. It shall be controlled with a
ruler [7] [8] [9].
The orifice should be directed into the air-stream using a wind vane.
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Key
1 drum
2 orifice
3 cover
A 20,5 mm or 22,5 mm, depending on supplier
B 19,8 mm or 21,8 mm, depending on supplier
C drum diameter 110 mm to 112 mm
A-B 0,7 mm (±0,1 mm)
Figure 2 — Pollen trap (Head of Hirst system) [9, modified]
5.1.2 Scrolling speed of support
The scrolling speed of the support is from 2 mm/h to 14 mm/h (±0,01 mm/h).
It shall be consistent throughout the scrolling period and can be ensured by a clockwork system or by an
electric motor that shall not be stopped during the sampling period.
5.1.3 Impaction support
5.1.3.1 General
Only two options are allowed, both clockwork driven:
— a glass slide (76 mm x 26 mm) on which a transparent tape is fixed (48 mm x 19 mm) coated with specific
reagents (see Figure B.1 in Annex B);
— a 110 mm to 112 mm diameter drum on which a transparent coated tape is wrapped around (see
Figure B.2 in Annex B). The length of this transparent tape is 345 mm (±0,5 mm) to 350 mm (±0,5 mm).
5.1.3.2 Transparent tape
The transparent tape is coated with an adhesive in order to fix the particles.
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The following requirements shall be fulfilled:
— The transparent tape shall be water-insoluble.
— The thickness of the whole transparent tape shall not be changed over time, and should not be affected
by operational conditions (temperature between – 20 °C to + 60 °C or humidity between 20 % and
100 %).
— It shall be perfectly transparent to allow the passing of microscopic light.
— The length is > 336 mm.
5.1.3.3 Reagents
Two transparent coating products (solubilised in specific solvents or not) are useable: Vaseline (petroleum
jelly) or silicone.
5.1.3.3.1 Vaseline (petroleum jelly)
The main characteristics of Vaseline (petroleum jelly) (n° CAS: [8009 03 8], see Annex C) are the following:
— odourless;
— colourless;
— viscous liquid;
— boiling point : 68 °C;
— purity > 99 %;
— risk of diarrhoea and other stomach problems if absorbed or in case of repeated exposure.
NOTE Consult Safety Data Sheet or MSDS to obtain special instructions before use.
Solubilise the Vaseline (petroleum jelly) with toluene (n° CAS [108 88 3], see Annex C). It is also possible to
use unsolubilised Vaseline (petroleum jelly).
The purity of Vaseline (petroleum jelly) and toluene shall be > 99 %.
5.1.3.3.2 Silicone
The main characteristics of silicone (n° CAS [90337 93 2], see Annex C) are the following:
— odourless;
— colourless to white;
— pasty;
— high viscosity stable from – 20 °C to +150 °C;
— flammable over 400 °C;
— irritating to eyes;
— non-biodegradable.
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Solubilise the silicone with a specific solvent (see Annex C).
The physical properties of the adhesive medium remain unaltered at temperatures between – 20 °C and +
50 °C, making it suitable for the majority of bioclimatic zones.
5.1.4 Wind vane and rain shield
The wind vane allows permanent rotation of the trap head so that the orifice faces the wind. The rain shield
ensures a weather protection for the orifice (i.e. rainfall) (see Figure 3).
Key
wind vane
1
2 lid assembly
3 rain shield
4 orifice
5 rotation lock
6 motor cover
Figure 3a — Schematic picture of a Hirst seven day volumetric spore sampling system [5, modified]
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Figure 3b — Photo of a Hirst volumetric spore sampler [Source: RNSA]
5.1.5 Complete sampling system
The complete sampling system (so called “trap”) containing the motor, the vacuum pump, the orifice, the
rotating drum, the wind vane, the clockwork system, the impaction support shall be
— resistant to corrosion;
— well attached (i.e. resistant to wind-blow, …);
— always horizontal (at the head level).
The commercial devices that meet the requirements are presented in Annex A.
For the different purposes, refer to the specific publications.
For allergen monitoring purposes, the following requirements and conditions for sampler positioning of the trap
shall be fulfilled:
— The sampler shall be placed on a readily accessible, flat, horizontal surface. It should be on the roof of a
building, and away from the edge of the building in order to reduce the effects of turbulence.
— Care shall be taken to ensure that adjacent buildings do not screen the sampler or interfere with the
airflow. The sampler should be ideally placed on the roof of a building at more than 2 m from the edge;
the height above ground level depends on the city and on the height of neighbouring buildings.
— The sampler itself shall be elevated between 100 cm to 150 cm from the roof in order to avoid turbulence
between air layers and possible re-suspension of particles from the roof.
— The sampler shall not be placed in the vicinity of fixed or mobile sources of mass emission of biological or
non-biological particles. Proximity to non-biological particle sources may favour the massive presence of
residues in samples, which considerably hinder identification.
5.2 Operating procedure
5.2.1 Preparation of the coating medium [12]
Prepare the coating medium as follows:
— Vaseline (petroleum jelly) (18 g ± 1 g) and toluene (1l) (purity > 99 %);
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— under the hood, add Vaseline (petroleum jelly) to toluene stirring until completely dispersed and leave it to
stand for about 48 h with periodic shaking to obtain a homogenous fluid solution.
Or use pure Vaseline (petroleum jelly).
Or
— silicone fluid (20 g (±1 g)) and a specific solvent;
— under the hood, add silicone to the solvent stirring until completely dispersed and leave to stand for about
48 h with periodic shaking to obtain a homogeneous fluid solution;
— spread the solution on the tape with a brush and let it dry under the hood for at least 1 h.
1)
The brush should be a pure Ox-hair brush N°14.i.e.: ref 922 N°14 OMEGA, made in Italy
WARNING — For the use of silicone or Vaseline (petroleum jelly) solutions:
— Keep the transparent tape dust free and always work with closed windows to avoid
contamination.
— Always keep the coating medium under an extractor hood due to the release of solvents (toxic
nature of the solvents).
— Wear protective gloves/clothing/equipment for eye and face protection. Wash thoroughly after
handling.
5.2.2 Support preparation
Fixation of the coated or uncoated tape on the drum.
Be careful to
— always clean the drum or the glass slide with ethyl alcohol (70 %) in order to avoid dust and/or biological
particles;
— fix the coated or uncoated transparent tape on the drum.
If it is an uncoated tape, the tape fixed on the drum shall be coated under the hood if a solvent reagent is
used.
The capture surface shall be covered with a thin homogeneous layer of coating medium in order to retain the
targeted particles.
The support shall be protected from ambient air during transportation from the laboratory to the trap and return
and shall remain in a metal box for protection against shocks (see Figure 4). Conservation at ambient
temperatures shall not exceed 12 months for silicone and one month for Vaseline (petroleum jelly).
1)
Brush: ref 922 N°14 OMEGA is (are) an example(s) of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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Figure 4 — The drum and its protection box [Source: RNSA]
5.2.3 Changing of the drum
The person in charge of changing the drum shall
— mark the tape with a sharp instrument through the orifice at the end of the registered drum;
— measure and record the flow rate of 10 l/min (±1 l/min);
— remove the drum, clean the orifice and the etancheity gasket;
— wind the clock;
— put in the new drum;
— measure the flow rate; make a note of it and correct if necessary after cleaning the orifice and checking
the sealing;
— note date and exact time (hour and minute) as well as flow rate and initials of the operator;
— mark the tape with a sharp instrument through the orifice at the beginning of the new drum.
6 Analysis
6.1 Equipment required
— microscope;
— magnetic stirrer;
— hot plate or microwave;
— bench;
— utensils to use (tweezers, scalpel);
— winding support;
— cutting rule;
— extractor hood;
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— brush;
— glass slide;
— cover glass or cover slip;
— ethyl alcohol (70 %);
— reagents for coating medium;
— reagents for mounting medium;
— transparent tape;
— drum (with its box).
6.2 Operating procedure
6.2.1 Support
6.2.1.1 Slide
The size of the microscope glass slide is ≥ 76 mm × 26 mm.
The requirements for slides are the following:
— clean;
— defatted;
— disposable (single use).
6.2.1.2 Microscope cover glass
The size of the microscope cover glass shall be longer and larger than the size of the fraction of tape which is
under the cover glass.
Properties that shall be respected:
— transparent;
— disposable (single use).
6.2.2 Mounting medium
The reagents used for colouration and fixation of the preparation are the following:
The colouring agents may be fuchsin (N° CAS [632 99 5], see Annex C) or safranine (N° CAS [477-74 6], see
Annex C). The fixation agents may be glycerine/gelatin or simple glycerine.
2) 3)
Specific mixtures containing Gelvatol /Mowiol or other ready to use products may be used.
...
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