EN 16868:2019

Ambient air - Sampling and analysis of airborne pollen grains and fungal spores for networks related to allergy - Volumetric Hirst method

This document specifies the procedure to sample continuously and to 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) or an even equivalent method assuring comparable data.
This document describes both the sampling and the analysis procedures for the purpose of networks related to allergy. 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

Dieses Dokument legt das Verfahren fest zur kontinuierlichen Probenahme und zur Analyse der Konzentration luftgetragener Pollenkörner und Pilzsporen in der Umgebungsluft mittels eines volumetrischen Sammlers nach dem Hirst-Prinzip [1] [2] [3] (siehe Anhang A) oder einer gleichwertigen, vergleichbare Daten liefernden Methode.
Dieses Dokument beschreibt sowohl die Probenahme als auch die Analyseverfahren für die Zwecke von Allergienetzwerken. Für die anderen in der Einleitung erwähnten Aufgaben können andere Spezifikationen erforderlich sein.

Air ambiant - Échantillonnage et analyse des grains de pollen en suspension dans l'air et des spores fongiques pour les réseaux relatifs à l’allergie - 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 dans l’air ambiant en utilisant l’échantillonneur volumétrique de Hirst [1] [2] [3] (voir l’Annexe A) ou toute autre méthode équivalente garantissant des données comparables.
La présente Norme européenne décrit les modes opératoires d’échantillonnage et d’analyse aux fins des réseaux relatifs à l’allergie. 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 dokument določa postopek za stalno vzorčenje in analiziranje koncentracije cvetnega prahu ter trosov gliv v zunanjem zraku z volumetričnim vzorčevalnikom tipa Hirst. Ta dokument opisuje postopke vzorčenja in tudi analize za namen alergijskih omrežij. Morda so potrebne druge specifikacije. Med izpostavljenostjo pelodnim zrnom in trosom gliv ter onesnaženostjo zraka obstaja sinergija, za katero so potrebne nekatere določene in dodatne informacije, da se lahko zmanjša tveganje za vpliv na zdravje.

General Information

• Status: Published
• Publication Date: 07-May-2019
• Withdrawal Date: 29-Nov-2019
• ICS: 13.040.20 - Ambient atmospheres
• Technical Committee: CEN/TC 264 - Air quality
• Drafting Committee: CEN/TC 264/WG 39 - Sampling and analysis of airborne pollen grains and fungal spores
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 04-Dec-2024
• Completion Date: 14-Apr-2025
• Ref Project: SIST EN 16868:2019 - Ambient air - Sampling and analysis of airbone pollen grains and fungal spores for networks related to allergy - Volumetric Hirst method

Relations

• Replaces: CEN/TS 16868:2015 - Ambient air - Sampling and analysis of airborne pollen grains and fungal spores for allergy networks - Volumetric Hirst method
• Effective Date: 15-May-2019

Overview

EN 16868:2019 is the CEN European Standard that specifies a harmonized procedure for continuous sampling and analysis of airborne pollen grains and fungal spores in ambient air using the volumetric Hirst type sampler (or an equivalent method ensuring comparable data). The standard covers both sampling and laboratory analysis specifically for networks related to allergy and supersedes CEN/TS 16868:2015. It supports reliable pollen forecasting and public‑health oriented aerobiological monitoring.

Key topics and technical requirements

• Scope and principle: Continuous volumetric sampling of biological particles in ambient air for allergy networks.
• Sampling equipment & installation: Requirements for Hirst‑type traps, sampling support and site installation to ensure representative ambient air intake. (Annex A: Hirst trap details.)
• Operating procedures: Preparation of coating medium, mounting tape/drum preparation and replacement, flow‑rate calibration and time discrimination to ensure correct sampling volumes.
• Analysis & microscopy: Slide preparation, mounting media, optical microscopy magnification and identification procedures for pollen and spores (identification key in Annex D).
• Counting methodology: Defined counting routines, conversion factors to express counts as concentration (pollen/spores per cubic metre), and data recording.
• Performance characteristics: Assessment of measurement uncertainty (counting error, sampling efficiency, flow calibration, detection limits, spatial representativity).
• Quality assurance: Site characterization ("passport of sampling site"), analyst competence, intra‑ and interlaboratory assessments, network monitoring management and control procedures.
• Informative annexes: Photographs of impaction supports, MSDS, and identification aids.

Practical applications and users

EN 16868:2019 is designed for practical use by:

• National and regional pollen monitoring networks and allergy forecast services
• Environmental and public health agencies producing pollen/spore concentration information for allergen avoidance guidance
• Aerobiology laboratories and researchers conducting routine monitoring, trend analysis and comparative studies
• Clinical and immunotherapy services that use pollen data to support treatment decisions
• Manufacturers and calibrators of volumetric samplers ensuring device compliance with network standards

Beyond allergy networks, the standardized method can support applications in biodiversity monitoring, agriculture, forestry, meteorology and forensic aerobiology where comparable airborne pollen/spore data are required.

Related standards & organizations

• Prepared by CEN/TC 264 (Air quality).
• Aligns with guidance from the European Aerobiology Society (EAS) and International Association for Aerobiology (IAA).
• Supersedes CEN/TS 16868:2015.

Keywords: EN 16868:2019, volumetric Hirst method, airborne pollen monitoring, fungal spore sampling, pollen count, aerobiology standards, allergy networks, ambient air sampling.

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2019
Nadomešča:
SIST-TS CEN/TS 16868:2016
Zunanji zrak - Vzorčenje in analiza cvetnega prahu in trosov gliv v zraku za
alergijsko omrežje - Volumetrična Hirstova metoda
Ambient air - Sampling and analysis of airbone pollen grains and fungal spores for
networks related to allergy - 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 relatifs à l'allergie - Méthode volumétrique de Hirst
Ta slovenski standard je istoveten z: EN 16868:2019
ICS:
13.040.20 Kakovost okoljskega zraka Ambient atmospheres
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 16868
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2019
EUROPÄISCHE NORM
ICS 13.040.20 Supersedes CEN/TS 16868:2015
English Version
Ambient air - Sampling and analysis of airborne pollen
grains and fungal spores for networks related to allergy -
Volumetric Hirst method
Air ambiant - Échantillonnage et analyse des grains de Außenluft - Probenahme und Analyse luftgetragener
pollen en suspension dans l'air et des spores fongiques Pollen und Pilzsporen für Allergienetzwerke -
pour les réseaux relatifs à l'allergie - Méthode Volumetrische Hirst-Methode
volumétrique de Hirst
This European Standard was corrected and reissued by the CEN-CENELEC Management Centre on 12 June 2019.

This European Standard was approved by CEN on 8 March 2019.

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, North
Macedonia, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

E UR O P ÄISCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16868:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 10
5 Sampling . 10
5.1 Equipment . 10
5.1.1 Apparatus . 10
5.1.2 Sampling support . 14
5.1.3 Installation conditions . 16
5.2 Operating procedure . 16
5.2.1 Preparation of the coating medium . 16
5.2.2 Support preparation . 17
5.2.3 Changing of the drum . 18
6 Analysis . 18
6.1 Equipment . 18
6.2 Operating procedure . 19
6.2.1 Support . 19
6.2.2 Mounting medium . 19
6.3 Methodology for counting . 19
6.3.1 Glass slide preparation for microscopy analysis for drum tape . 19
6.3.2 Optical microscopy . 21
6.3.3 Identification . 22
6.3.4 Counting method . 22
6.3.5 Data recording . 22
6.3.6 Conversion factor . 23
7 Performance characteristics for pollen and fungal spores counts . 24
7.1 General . 24
7.2 Integrated uncertainty assessment . 24
7.3 Uncertainty from counting error and counting routine. 24
7.4 Measurement uncertainty relating to sampling efficiency . 24
7.5 Measurement uncertainty relating to capture film, adhesive and specimen
preparation . 24
7.6 Measurement uncertainty relating to time discrimination . 25
7.7 Measurement uncertainty related to the detection limit . 25
7.8 Measurement uncertainty in relation to the calibration of the flow rate . 25
7.9 Measurement uncertainty relating to spatial representativity . 25
8 Quality assurance . 25
8.1 General . 25
8.2 Measurement site/trap . 25
8.2.1 Control . 25
8.2.2 Characterization of the site and its ambient conditions (passport of sampling site) . 25
8.2.3 Spatial representativity . 26
8.3 Analyst . 26
8.4 Intra- and interlaboratory quality assessments . 26
8.4.1 General . 26
8.4.2 Repeatability . 26
8.4.3 Reproducibility and accuracy . 26
8.4.4 Sensitivity and specificity . 27
8.5 Network monitoring management . 27
Annex A (informative) Hirst type volumetric trap . 28
Annex B (informative) Pictures of impaction support . 29
Annex C (informative) Material Safety Data Sheets . 31
Annex D (informative) Identification key . 32
Bibliography . 37

European foreword
This document (EN 16868:2019) has been prepared by Technical Committee CEN/TC 264 “Air quality”,
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 November 2019, and conflicting national standards shall
be withdrawn at the latest by November 2019.
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 CEN/TS 16868:2015.
The main changes with respect to the previous edition are listed below:
a) the title has been changed;
b) modifications have been made to the Introduction, the Scope and Clauses 3, 4, 5 and 6;
c) new paragraphs have been added to Clauses 7 and 8;
d) modifications have been made to all Annexes;
e) Figures D.2 and D.3 have been modified;
f) the Bibliography has been readjusted;
g) editorial changes have been made.
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, Former Yugoslav Republic of Macedonia, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands,
Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
the United Kingdom.
Introduction
Biological particles (pollen and fungal spores) are present in the air, causing health impacts at various
levels. In Europe, a lot of people suffer from pollinosis due to pollen and/or fungal spores (EFA, European
Federation of Allergy and Airways Diseases Patients Association, 2017). 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 International
10,2,5
Association for Aerobiology (IAA) manage the methodology of sampling, analysis, quality control,
development and information.
Persons and institutions involved in pollen forecasting have a scientific and public health responsibility.
A pollen forecast is a guideline for allergen avoidance with a direct influence on pollen allergy sufferers
and their behaviour. Pollen allergy sufferers are in need of such information since pollen allergy affects
their quality of life and pollen and spores are an abundant, environmental allergen. The health state of
pollen allergy sufferers should never be risked due to inadequate forecasts, financial interests or deficient
working routines applied in the fundamental work such as pollen data evaluation and all involved
processes (maintenance of the device, preparation, evaluation, handling and processing of data).
Further pollen data should be included in therapy (immunotherapy at least for one year) to objectify the
benefit of the personal therapy.
For the sampling and analysis of biological particles different methodology and operating procedures are
used.
Information on airborne pollen and spore concentration (counts and analyses) plays an important role
in aerobiology, as well as in other disciplines and fields of application, such as biodiversity, agriculture,
forestry, phytopathology, meteorology, climatology, paleo-ecology/-climatology, forensic science,
bioterrorism and health (sensitization and allergy). The method described in this European Standard is
aimed for the purposes of networks related to allergy. Besides, it may also be useful for other applications
mentioned above.
1 Scope
This document specifies the procedure to sample continuously and to 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) or an even equivalent method assuring comparable data.
This document describes both the sampling and the analysis procedures for the purpose of networks
related to allergy. For the other tasks mentioned in the introduction, other specifications may be required.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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 http://www.iso.org/obp
NOTE For general terms, see [4] [5].
3.1
measurement accuracy
accuracy of measurement
accuracy
closeness of agreement between a measured quantity value and a true quantity value of a measurand
Note 1 to entry: The concept ‘measurement accuracy’ is not a quantity and is not given a numerical quantity
value. A measurement is said to be more accurate when it offers a smaller measurement error.
Note 2 to entry: The term “measurement accuracy” should not be used for measurement trueness and the term
“measurement precision” should not be used for ‘measurement accuracy’, which, however, is related to both these
concepts.
Note 3 to entry: Measurement accuracy is sometimes understood as closeness of agreement between measured
quantity values that are being attributed to the measurand.
[SOURCE: JCGM 200:2012]
3.2
clockwork
mechanism with a spring and toothed gearwheels, used to drive a mechanical clock, toy or other device
3.3
combined standard measurement uncertainty
combined standard uncertainty
standard measurement uncertainty that is obtained using the individual standard measurement
uncertainties associated with the input quantities in a measurement model
Note 1 to entry: In case of correlations of input quantities in a measurement model, covariances must also be
taken into account when calculating the combined standard measurement uncertainty; see also ISO/IEC Guide
98-3:2014 [22].
3.4
defatted
surface conditions after clearing with a fat removing substance
3.5
drum
cylindrical device for the mounting of a sticky tape
3.6
exine
outer wall of pollen grain, also called an exosporium
3.7
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
3.8
flow meter
instrument for measuring the flow rate of a fluid in a pipe
3.9
flow rate
amount of fluid (air volume) that flows in a given time
3.10
fungal spore
sexual or asexual reproductive unit of fungi, capable of developing a new individual
3.11
hood
metal cover or canopy for a stove, ventilator, etc
3.12
impaction
sampling of airborne particles by inertial separation on any surface (e.g. of an adhesive)
3.13
magnetic stirrer
object or mechanical device used for stirring something
3.14
magnification
magnifying power of an instrument
3.15
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
3.16
objective
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.
3.17
orifice
opening or aperture, as of a tube or pipe; a mouthpiece with a slot-like opening on the side of the trap
3.18
particle
pollen and spores
3.19
pollen
male gametophyte of seed plants (either angiosperms or gymnosperms)
3.20
measurement precision
precision
closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions
Note 1 to entry: Measurement precision is usually expressed numerically by measures of imprecision, such as
standard deviation, variance, or coefficient of variation under the specified conditions of measurement.
Note 2 to entry: The ‘specified conditions’ can be, for example, repeatability conditions of measurement,
intermediate precision conditions of measurement, or reproducibility conditions of measurement
(see ISO 5725-1:1994, [6]).
Note 3 to entry: Measurement precision is used to define measurement repeatability, intermediate measurement
precision, and measurement reproducibility.
Note 4 to entry: Sometimes “measurement precision” is erroneously used to mean measurement accuracy.
[SOURCE: JCGM 200:2012]
3.21
repeatability condition of measurement
repeatability condition
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
Note 1 to entry: A condition of measurement is a repeatability condition only with respect to a specified set of
repeatability conditions.
Note 2 to entry: In chemistry, the term “intra-serial precision condition of measurement” is sometimes used to
designate this concept.
[SOURCE: JCGM 200:2012]
3.22
reproducibility condition of measurement
reproducibility condition
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
Note 1 to entry: The different measuring systems may use different measurement procedures.
Note 2 to entry: A specification should give the conditions changed and unchanged, to the extent practical.
[SOURCE: JCGM 200:2012]
3.23
sensitivity
measurement of the proportion of search particle which is correctly identified
3.24
slide
rectangular piece of glass on which an object is mounted or placed for examination under a microscope
3.25
specificity
measurement of the proportion of non-searched particles which are correctly identified as different from
the searched particles
3.26
standard measurement uncertainty
standard uncertainty of measurement
standard uncertainty
measurement uncertainty expressed as a standard deviation
3.27
taxa
taxonomic groups of any rank, such as a species, genus, family or other rank
3.28
trap
sampling device
3.29
vacuum pump
pump or device by which a partial vacuum can be produced
3.30
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 deposit on the
sampling surface is examined with an optical microscope in order to identify and count the pollen and
fungal spores per area (deposition rates). Using this method allows to calculate concentrations as a daily
mean or an hourly mean. The sampling is usually done at low-volume rate (10 l/min). It allows a
continuous sampling for up to seven days [7] [8] [9].
5 Sampling
5.1 Equipment
5.1.1 Apparatus
The sampling device and its functional principles are shown schematically in Figures 1, 2 and 3.
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, etc.);
— 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.
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).
Key
1 wind vane
2 impact unit
3 rain shield
4 orifice (inlet)
5 screw for flow rate adjustment
6 vacuum pump
(Source: RNSA)
Figure 1 — Schematic figure and picture of a sampling device operating on the Hirst type
impactor – General view
Key
1 orifice (inlet)
2 drum
3 clock
4 connection to vacuum pump
(Source: RNSA)
Figure 2 — Schematic figure of a sampling device operating on the Hirst type impactor –
Schematic view
5.1.1.1 Suction pump
The suction pump works 24 h a day and continuously throughout the year at the same flow rate. The
power supply may be either mains or battery driven (solar panels). The electric motor is capable of
continuous operation.
The suction system is, for instance, a vacuum pump. The flow rate of suction shall be regularly controlled
and adjusted accordingly.
The recommended flow rate is 10 l/min with a maximum permissible deviation of ± 10 % (±1 l/min).
The manufacturer shall ensure the flow specification and provide a flow verification and calibration
procedure that allows the user to ensure compliance with this specification throughout the life of the
trap. The validity of the calibration method recommended with an error of less than 10 % in the accuracy
of flow rate between calibration and operation shall be certified by the manufacturer.
As the accuracy of the calibration depends on the air resistance and characteristic curves of the individual
trap, vacuum pump and flow meter used [10], the validity has to be certified accordingly, i.e. specifically
for the trap/vacuum pump/flow meter combination delivered by the manufacturer.
The flow rate shall be checked at every change of the impaction support.
Key
1 lid
2 start reference pointer
3 lock nut
4 orifice position
5 trapping surface
Figure 3 — The Hirst volumetric trap showing 7-day lid assembly with drum
5.1.1.2 Orifice (inlet) (Figure 2)
The orifice shall have the following dimensions (with associated tolerances):
— rectangular opening: 14 mm (±0,1 mm) × 2 mm (±0,1 mm);
— specific orifice length: from 19 mm to 25 mm;
— distance D from the inside orifice to the drum without the tape: 0,70 mm (±0,1 mm).
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 distance D between orifice and drum shall be 0,70 mm (±0,1 mm) (see Figure 4 – distance D = A-B).
The distance D allows efficient particle impaction for pollen grains and fungal spores. It shall be controlled
[11] [12] [13].
The orifice should be directed into the air-stream using a wind vane.
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
D 0,7 mm (±0,1 mm)
Figure 4 — Pollen trap (Head of Hirst system)
5.1.2 Sampling support
5.1.2.1 General
Two possibilities are widely used depending on the requested sampling period as a sampling support:
— A glass slide for microscopy (76 mm × 26 mm) on which a transparent tape is fixed
(48 mm × 19 mm) reagents (see Figure B.1 in Annex B) for one
coated with specific day of sampling.
— A drum (110 mm to 112 mm in diameter) on which a transparent coated flexible tape is attached for
seven days of sampling (see Figure B.2 in Annex B). The length of this tape ranges from 345 mm to
350 mm (±0,5 mm) depending on the size of the drum.
The sampling support is driven by a clockwork with a scrolling speed ranging from 2 mm/h to 14 mm/h
(±0,01 mm/h) depending on the sampling period.
The sampling support shall scroll regularly in front of the back outlet of the orifice. Sampling shall always
be continuous and stable and not be stopped during the requested sampling period.
5.1.2.2 Transparent tape
The transparent tape is coated with an adhesive in order to fix the particles.
The following requirements shall be fulfilled:
— The transparent tape shall be not hygroscopic.
— 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 transparent to allow the passing of microscopic light.
— The length shall be adapted to the support used (see requirements of use by the trap manufacturer).
5.1.2.3 Reagents
Two transparent coating products (solubilized in specific solvents or not) are useable: Vaseline
(petroleum jelly) or silicone.
NOTE See the Material Safety Data Sheet (MSDS) for the products used for special instructions before use (see
Annex C).
5.1.2.3.1 Vaseline (petroleum jelly)
The main characteristics of Vaseline (petroleum jelly) (n° CAS: [8009 03 8]), see Annex C, are the
following:
— purity > 99 %;
— odorless;
— colourless;
— viscous liquid;
— melting point: 38 °C to 60 °C.
Solubilize the Vaseline (petroleum jelly) with toluene (n° CAS [108 88 3], see Annex C). It is also possible
to use insolubilized Vaseline (petroleum jelly) if there is a guarantee to have a regular thickness along the
strip.
The purity of toluene shall be > 99 %.
5.1.2.3.2 Silicone
The main characteristics of silicone fluid 1,000,000 (one million) cSt (Polydimethylsiloxane Fluid, CAS
No 63148-62-9), see Annex C, are the following:
— purity > 99 %;
— odorless;
— colourless to white;
— pasty;
— high viscosity stable from –20 °C to +150 °C;
— flammable over 400 °C;
— non-biodegradable.
Solubilize the silicone fluid 1,000,000 (one million) cSt at 3 % 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.
5.1.3 Installation conditions
For pollen grains and fungal spores 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 near stationary or moving sources of biological or non-biological
particles.
5.2 Operating procedure
5.2.1 Preparation of the coating medium
Prepare the coating medium as follows [12] [13]:
— Vaseline (petroleum jelly (18 g (±1 g)) and toluene (1 l) (purity > 99 %);
— under the hood, in a laboratory glass device, 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;
— spread the solution on the tape, e.g. with a brush , and let it dry under the hood for at least 1h.
The brush should be a pure Ox-hair brush N°14, i.e. ref 922 N°14 OMEGA, made in Italy or comparable
products.
Or use:
— Pure Vaseline (petroleum jelly).

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.
Or use:
— Silicone fluid 1,000,000 (one million) cSt (30 g (±1 g)) and 1 l of a specific solvent;
— under the hood, add silicone to the solvent (e.g. cyclohexane) 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.
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 5). Conservation at
ambient temperatures shall not exceed 12 months for silicone and one month for Vaseline (petroleum
jelly).
Figure 5 — 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;
— remove the drum, clean the orifice (inlet), clean the seal and check its condition;
— wind the clock;
— put in the new drum;
— control the flow rate;
— 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
— optical microscope;
— magnetic stirrer;
— hot plate;
— bench;
— tools to use (tweezers, scalpel);
— winding support;
— extractor hood;
— cutting rule;
— brush;
— glass slide;
— cover glass;
— ethyl alcohol (70 %);
— reagents for coating medium;
— reagents for mounting medium;
— transparent tape;
— double-sided adhesive;
— drum (with its box).
6.2 Operating procedure
6.2.1 Support
6.2.1.1 Microscope glass slide
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 fixation of the preparation and staining are the following:
The appropriate fixation agents are glycerine/gelatin, simple glycerine, specific mixtures containing
2 3
Polyvinyl alcohol (e.g. Gelvatol /Mowiol ) or other ready to use products.
For pollen, staining agents shall be fuchsin (N° CAS [632 99 5], see Annex C) or safranine (N° CAS [477-
74 6], see Annex C).
6.3 Methodology for counting
6.3.1 Glass slide preparation for microscopy analysis for drum tape
The preparation of the slides for the microscopic analysis shall be performed at room temperature as
soon as possible and in less than 14 days after removing the drum from the trap (to keep the migration
of pollen grains as low as possible).
Operating procedure:
— If necessary, put the mounting medium on the hot plate.
— Clean bench, hot plate and utensils before use (tweezers, scalpel).
— Take one slide per day (seven to eight if weekly monitoring) and label it (location, date and initials
of the operator) onto one side of the glass.

Gelvatol is an example 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 to this product.
Mowiol is an example 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 to this product.
— Degrease the glass slide with ethyl alcohol (70 %) if necessary.
— Fix the drum on the winding support (see Figure 6).
— Cut the tape at the junction point (double-sided adhesive).
— Take the tape with the tweezers (do not touch the tape with your fingers) and place the tape on the
cutting rule (see Figure 7).
— Match the beginning or the end of the exposure period and place it taking into account the time.
— Cut the tape into daily portions (see Figure 7). Put three drops or a line of liquid mounting medium
(with or without colour) on the glass slide (see Figure 8 and Figure 9).
— Using the tweezers, gently deposit the first tape section in the centre of the glass slide on the
mounting medium. The beginning of the recording shall be on the label side.
— Place three drops or a line of coloured mounting medium on this tape or on the cover glass.
— Place slowly cover glass on the preparation, avoiding bubbles and movement of particles.
— In case of staining medium containing gelatin, leave the glass slide on the hot plate from 50 °C
to 60 °C to remove air bubbles.
—
Leave the slide to cool at room temperature for at least 15 min.
— Use the same procedure for the other daily sections of tapes.

Figure 6 — Drum on the winding support
Dimensions in millimetres
Key
1 cutting rule
2 tape
Figure 7 — Transparent tape on the cutting rule INSERT: Cutting rule (white) – Tape (grey part)
Dimensions in millimetres
Figure 8 — Glass slide preparation – Method 1
Dimensions in millimetres
Figure 9 — Glass slide preparation – Method 2
6.3.2 Optical microscopy
The slides are examined under an optical microscope at variable magnification [1] [16] [17].
The magnification shall be sufficient to distinguish the fine structure of pollen and fungal spore.
NOTE Lower magnification might increase the error of identification but a higher magnification requires a
greater reading area in consideration, for instance using the process of dynamic counting (see 6.3.4).
6.3.3 Identification
The identification shall be made by a specifically trained operator [16] [17].
The identification takes place according to a detailed key of determination including all relevant taxa. The
classification is achieved thanks to a key of determination which is based on size, shape, number and
types of apertures on the surface, ornamentation of exine, etc.
NOTE The most widely used key of determination is the one provided by RNSA (see Annex D).
More details and information about this key of determination are given in Annex D.
6.3.4 Counting method
Horizontal or vertical parallel sweeps can be used for reading slides. Due to the area of slides examined
depending on the size of the microscope’s field of view and amount of magnification, a minimum area of
the slide recommended for counting shall be defined rather than the number of transept (horizontal or
vertical) used. A minimum surface of 10 % for pollen analysis is required [17], or the use of dynamic
counting [20].
NOTE Horizontal sweeps, i.e. sweeps running parallel to the time axis are required, following the same drum
rotation continuously, to provide better results.
In order to optimize efficiency and precision whilst taking into consideration increasing counting error
by low or high counts, dynamic counting is possible:
— For efficiency when individual pollen types occur with high concentration, counting of that type may
be stopped, if pollen counts exceed the number of 50 throughout the whole of the first sweep.
— To improve precision in case of low total pollen concentrations (e.g. in the beginning or the end of
the season), the counting area may be successively expanded by analysing more sweeps until 50 total
pollen counts (not related to individual pollen type) are reached or until 50 % of the deposition area
has been analysed.
In the examined area, the number of each pollen grain and fungal spore identified is counted; this
provides information on the pollen and fungal spore count throughout the day.
6.3.5 Data recording
The data recording may be done by manual recording or semi-automatic recording (computer-based
software e.g. EAN poll or Pollrec, voice recognition and incrementing in a database).
The minimum information that is collected is:
— location;
— year;
— month;
— day;
— hour;
— counter’s name;
— number and type of counted pollen grains and fungal spores;
— conversion factor.
6.3.6 Conversion factor
Pollen or fungal spore counts should be expressed as the daily average pollen or fungal spores
concentration (number of particles per cubic meter of air). For this purpose, the number of pollen or
fungal spores counted is multiplied by a factor that takes into account the volume of air sampled
(10 l/min), the sampling area and the size of the microscope’s field of view used.
NOTE It is also possible to express the results as bi-hourly data/m of air, in accordance with the needs.
Calculate the conversion factor (CF) for the unit of time chosen by using Formulae (1) and (2).
CF = S total sample / S analysed × (1/ V) = (L × l) / (L × d × N) × (1/ V) = l / (d × N) × (1/V) (1)
Pollen or fungal spore concentration = n × CF (2)
where
S 2
is the surface (total sample or analysed) (mm )
V 3
is the volume of sucked air (V = D × t) (m )
D 3
is the flow rate of the equipment (l/min converted in m / unit of time)
t is the duration of the sampling period (min)
L is the length of the line (mm)
l is the width of the line (mm)
d is the used diameter of the microscope's field of vision (mm)
N is the number of sweeps
n is the number of pollen or fungal spores counted, the analysed area of the microscope slide
The first step to calculate the conversion factor is to measure the microscope’s field of vision at the
magnification which is used.
Example for daily concentration [2]:
If the diameter of the field of view is 0,45 mm:
Air sampling rate: 10 l/min = 600 l/h = 14 400 l/day = 14,4 m /day
Length of the tape (or line): 48 mm
Width of impaction area on the tape: 14 mm
Area of one horizontal sweep = 48 mm × 0,45 mm = 21,6 mm
2 2
Su
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