SIST EN 17503:2023

SLOVENSKI STANDARD
01-marec-2023
Nadomešča:
SIST EN 15527:2009
SIST EN 16181:2018
SIST ISO 13877:1999
Tla, blato, obdelani biološki odpadki in odpadki - Določevanje policikličnih
aromatskih ogljikovodikov (PAH) s plinsko kromatografijo (GC) in s tekočinsko
kromatografijo visoke ločljivosti (HPLC)
Soil, sludge, treated biowaste and waste - Determination of polycyclic aromatic
hydrocarbons (PAH) by gas chromatography (GC) and high performance liquid
chromatography (HPLC)
Boden, Schlamm, behandelter Bioabfall und Abfall - Bestimmung von polycyclischen
aromatischen Kohlenwasserstoffen (PAK) mittels Gaschromatographie (GC) und
Hochleistungs-Flüssigkeitschromatographie (HPLC)
Sols, boues, biodéchets traités et déchets - Dosage des hydrocarbures aromatiques
polycycliques (HAP) par chromatographie en phase gazeuse et chromatographie liquide
à haute performance
Ta slovenski standard je istoveten z: EN 17503:2022
ICS:
13.030.01 Odpadki na splošno Wastes in general
13.080.10 Kemijske značilnosti tal Chemical characteristics of
soils
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17503
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2022
EUROPÄISCHE NORM
ICS 13.030.01; 13.080.10 Supersedes EN 15527:2008, EN 16181:2018
English Version
Soil, sludge, treated biowaste and waste - Determination of
polycyclic aromatic hydrocarbons (PAH) by gas
chromatography (GC) and high performance liquid
chromatography (HPLC)
Sols, boues, biodéchets traités et déchets - Dosage des Boden, Schlamm, behandelter Bioabfall und Abfall -
hydrocarbures aromatiques polycycliques (HAP) par Bestimmung von polycyclischen aromatischen
chromatographie en phase gazeuse et Kohlenwasserstoffen (PAK) mittels
chromatographie liquide à haute performance Gaschromatographie (GC) und Hochleistungs-
Flüssigkeitschromatographie (HPLC)
This European Standard was approved by CEN on 3 January 2022.

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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17503:2022 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 8
5 Interferences . 8
6 Safety remarks . 9
7 Reagents . 10
8 Apparatus . 14
9 Sample storage and preservation . 15
10 Procedure . 16
11 Performance characteristics. 29
12 Precision . 29
13 Test report . 30
Annex A (informative) Repeatability and reproducibility data . 31
A.1 Materials used in the interlaboratory comparison study . 31
A.2 Interlaboratory comparison results . 32
Annex B (informative) Examples of instrumental conditions and chromatograms . 39
B.1 Measurement of PAH with GC-MS . 39
B.2 Measurement of PAH with HPLC fluorescence . 45
B.3 Example for measurement conditions of PAH with GC-MS/MS . 51
Bibliography . 53

European foreword
This document (EN 17503:2022) has been prepared by Technical Committee CEN/TC 444
“Environmental characterization of solid matrices”, the secretariat of which is held by NEN.
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 August 2022, and conflicting national standards shall be
withdrawn at the latest by August 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 15527:2008 and EN 16181:2018.
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.
Introduction
Polycyclic aromatic hydrocarbons (PAH) are ubiquitous because they are released in appreciable
quantities every year into the environment through the combustion of organic matters such as coal, fuel
oils, petrol, wood, refuse and plant materials. Since some of these PAH compounds are carcinogenic or
mutagenic, their presence in the environment (air, water, soil, sediment and waste) is regularly
monitored and controlled. At present determination of PAH is carried out in these matrices in most of the
routine laboratories following the prescribed steps specified for sampling, pre-treatment, extraction,
clean-up by measurement of specific PAH by means of gas chromatography in combination with mass
spectrometric detection (GC-MS) or by high performance liquid chromatography (HPLC) in combination
with UV-DAD- or fluorescence detection (HPLC-UV-DAD/FLD). Both the GC-MS and the HPLC methods
are included in this horizontal standard.
This document was developed by the merging of EN 16181:2018, initially elaborated as a CEN Technical
Specification in the European project 'HORIZONTAL' and validated by CEN/TC 400 with the support of
BAM, with EN 15527, published by CEN/TC 292.
Considered the different matrices and possible interfering compounds, this document does not contain
one single possible way of working. Several choices are possible, in particular relating to clean-up.
Quantification with both GC-MS-detection and HPLC-DAD-UV/FLD is possible. Four different extraction
procedures are described and three clean-up procedures. The use of internal and injection standards is
described in order to have an internal check on choice of the extraction and clean-up procedure. The
method is as far as possible in agreement with the method described for polychlorinated biphenyls (PCB)
in EN 17322. It has been tested for ruggedness.
This document is applicable and validated for several types of matrices as indicated in Table 1 (see also
Annex A for the results of the validation).
Table 1 — Matrices for which this document is applicable and validated
Matrix Materials used for validation
Soil Sandy soil
Mix of soil from an industrial area in Brandenburg, Germany and
PAH-free German reference soil
Sludge Mix of municipal waste water treatment plant sludge from the
vicinity of Berlin, Germany
Biowaste Mix of compost from the vicinity of Berlin, Germany
Contaminated soil, building debris, waste wood, roofing tar,
Waste
shredder light fraction
WARNING — Persons using this document should be familiar with usual laboratory practice. This
document does not purport to address all of the safety problems, if any, associated with its use. It is the
responsibility of the user to establish appropriate safety and health practices and to ensure compliance
with any national regulatory conditions.
WARNING — It is absolutely essential that tests conducted according to this document be carried out by
suitably trained staff.
1 Scope
This document specifies different methods for quantitative determination of 16 polycyclic aromatic
hydrocarbons (PAH) (see Table 2) in soil, sludge, treated biowaste, and waste, using GC-MS or HPLC-UV-
DAD/FLD covering a wide range of PAH contamination levels (see Table 2).
NOTE The method can be applied to sediments provided that validity is demonstrated by the user.
When using fluorescence detection, acenaphthylene cannot be measured.
Table 2 —Target analytes of this document
a
Target analyte
CAS-RN
Naphthalene 91–20–3
Acenaphthene 83–32–9
Acenaphthylene 208–96–8
Fluorene 86–73–7
Anthracene 120–12–7
Phenanthrene 85–01–8
Fluoranthene 206–44–0
Pyrene 129–00–0
Benz[a]anthracene 56–55–3
Chrysene 218–01–9
Benzo[b]fluoranthene 205–99–2
Benzo[k]fluoranthene 207–08–9
Benzo[a]pyrene 50–32–8
Indeno[1,2,3-cd]pyrene 193–39–5
Dibenz[a,h]anthracene 53–70–3
Benzo[ghi]perylene 191–24–2
a
CAS-RN Chemical Abstracts Service Registry Number.
The limit of detection depends on the determinants, the equipment used, the quality of chemicals used
for the extraction of the sample and the clean-up of the extract.
Under the conditions specified in this document, the lower limit of application from 10 μg/kg (expressed
as dry matter) for soils, sludge and biowaste to 100 μg/kg (expressed as dry matter) for solid waste can
be achieved. For some specific samples (e.g. bitumen) the limit of 100 μg/kg cannot be reached.
Sludge, waste and treated biowaste can differ in properties as well as in the expected contamination
levels of PAH and presence of interfering substances. These differences make it impossible to describe
one general procedure. This document contains decision tables based on the properties of the sample and
the extraction and clean-up procedure to be used.
The method can be applied to the analysis of other PAH not specified in the scope, provided suitability is
proven by proper in-house validation experiments.
Sampling is not part of this standard. In dependence of the materials, the following standards need to be
considered, e.g. EN 14899, ISO 5667-12 and EN ISO 5667-13.
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 15002, Characterization of waste — Preparation of test portions from the laboratory sample
EN 15934, Sludge, treated biowaste, soil and waste — Calculation of dry matter fraction after
determination of dry residue or water content
EN 16179, Sludge, treated biowaste and soil — Guidance for sample pretreatment
EN ISO 5667-15, Water quality — Sampling — Part 15: Guidance on the preservation and handling of
sludge and sediment samples (ISO 5667-15)
EN ISO 16720, Soil quality — Pretreatment of samples by freeze-drying for subsequent analysis (ISO 16720)
EN ISO 22892, Soil quality — Guidelines for the identification of target compounds by gas chromatography
and mass spectrometry (ISO 22892)
ISO 8466-1, Water quality — Calibration and evaluation of analytical methods — Part 1: Linear calibration
function
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 https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
3.1
critical pair
pair of PAH that are separated to a predefined degree (e.g. R = 0,5) to ensure chromatographic separation
meets minimum quality criteria
EXAMPLE Figure 1 shows an example of a chromatogram of a critical pair.
∆ t
R 2 ×
YY +
ab
(1)
where:
R peak separation
Δt difference in retention times of the two peaks a and b in seconds (s)
Y peak width at the base of peak a in seconds (s)
a
Y peak width at the base of peak b in seconds (s)
b
Figure 1 — Example of a chromatogram of a critical pair
3.2
internal standard
compound added in a known amount to the sample from the beginning of the protocol and enabling
analytical coverage throughout the procedure, and that is used to correct for losses during sample
preparation and analysis by accounting for all-system matrix effects (recoveries, ionization effect,
variability of the detector response of the instrument for example)
Note 1 to entry: isotopically labelled mostly deuterated PAH or native PAH unlikely to be present in the sample
[SOURCE: EN ISO 21253-2:2019, 3.10]
3.3
injection standard
standard mixture added to a sample before injection into the GC-MS apparatus, to monitor variability of
instrument response and to calculate internal standard recovery
[SOURCE: ISO 28540:2011, 3.4]
=
3.4
extraction standard
PAH that is unlikely to be present in samples added to the sample prior to extraction, used for checking
the extraction efficiency and not used for quantification purposes
3.5
sediment
solid material, both mineral and organic, deposited in the bottom of a water body
[SOURCE: ISO 5667-12:2017, 3.5]
4 Principle
Due to the multi matrix character of this document, different procedures for different steps (modules)
are allowed. Which modules should be used depends on the sample. A recommendation is given in this
document. Performance criteria are described and it is the responsibility of the laboratories applying this
document to show that these criteria are met. Using of spiking internal standards allows an overall check
on the efficiency of a specific combination of modules for a specific sample. But the use of internal
standards does not necessarily give the information regarding the extensive extraction efficiency of the
native PAH bonded to the matrix.
After pre-treatment, the sample is extracted with a suitable solvent.
The extract is concentrated by evaporation. If necessary, interfering compounds are removed by a clean-
up method suitable for the specific matrix, after the concentration step.
If a solvent exchange for HPLC analysis is necessary, the concentrated extract is taken up in an
appropriate less volatile water miscible polar solvent and the non-polar extract residue is removed under
a gentle flux of inert gas.
The extract is analysed by GC-MS using a capillary column with a stationary phase of low polarity or by
HPLC-UV-DAD/FLD with an appropriate reversed phase column.
PAH are identified and quantified with GC-MS by comparison of relative retention times and relative peak
heights (or peak areas) with respect to internal standards added, and with HPLC by using the
corresponding variables of the extraction standard solutions. The efficiency of the procedure depends on
the composition of the matrix that is investigated.
5 Interferences
5.1 Interference with sampling and extraction
Use sampling containers of materials (preferably of steel, aluminium or glass) that do not affect the
sample during the contact time. Avoid plastics and other organic materials during sampling, sample
storage or extraction. Keep the samples from direct sunlight and prolonged exposure to light.
During storage of the samples, losses of PAH may occur due to adsorption on the walls of the containers.
The extent of the losses depends on the storage time.
5.2 Interference with GC-MS
Substances that co-elute with the target PAH can interfere with the determination. These interferences
can lead to incompletely resolved signals and can, depending on their magnitude, affect accuracy and
precision of the analytical results. Peak overlap does not allow an interpretation of the result.
Asymmetrical peaks and peaks broader than the corresponding peaks of the reference substance suggest
interferences.
Chromatographic separation between dibenz[a,h]anthracene and indeno[1,2,3-cd]pyrene are mostly
critical. Due to their molecular mass differences, quantification can be made by mass selective detection.
When incomplete resolution is encountered, peak integration shall be checked and, if necessary,
corrected.
Sufficient resolution (e.g. 0,8) between the peaks of benzo[b]fluoranthene and benzo[k]fluoranthene as
well as of benzo[a]pyrene and benzo[e]pyrene shall be set as quality criteria for the capillary column.
Benzo[b]fluoranthene and benzo[j]fluoranthene cannot be separated. It can happen that the signal of
triphenylene is not completely separated from the signals of benz[a]anthracene and chrysene. In this case
it shall be stated in the report.
5.3 Interference with the HPLC
Substances that show either fluorescence or quenching and co-elute with the PAHs to be determined can
interfere with the determination. These interferences can lead to incompletely resolved signals and can,
depending on their magnitude, affect accuracy and precision of the analytical results. Peak overlap does
not allow an interpretation of the result. Asymmetrical peaks and peaks being broader than the
corresponding peaks of the reference substance suggest interferences. This problem can arise for
naphthalene and phenanthrene depending on the selectivity of the phases used.
Incomplete removal of the solvents used for sample extraction and clean-up can lead to poor
reproducibility of the retention times and wider peaks or double peaks especially for the 2- and 3-ring
PAHs. Extracts shall be diluted sufficiently with acetonitrile for the HPLC-analysis, otherwise the
detection of naphthalene and 3-ring PAH can be interfered with by a broad toluene peak.
Usually the signal of perylene is incompletely resolved from the signal of benzo[b]fluoranthene, but by
choosing a selective wavelength, the perylene peak can be suppressed.
6 Safety remarks
Certain PAH are highly carcinogenic and shall be handled with extreme care. Avoid contact with solid
materials, solvent extracts and solutions of standard PAH. It is strongly advised that standard solutions
are prepared centrally in suitably equipped laboratories or are purchased from suppliers specialized in
their preparation.
PAH contamination of vessels may be detected by irradiation with 366 nm UV-light.
Solvent solutions containing PAH shall be disposed of in a manner approved for disposal of toxic wastes.
For the handling of hexane precautions shall be taken because of its neurotoxic properties.
National regulations should be followed with respect to all hazards associated with this method.
7 Reagents
7.1 General
All reagents shall be of recognized analytical grade. The purity of the reagents used shall be checked by
running a blank test as described in 10.1. The blank shall be less than 50 % of the lowest reporting limit.
7.2 Reagents for extraction
7.2.1 Acetone, (2-propanone), (CH ) CO.
3 2
7.2.2 Toluene, C H .
7 8
7.2.3 Petroleum ether, boiling range 40 °C to 60 °C.
Hexane-like solvents with a boiling range between 40 °C and 98 °C are allowed.
7.2.4 Sodium sulfate, Na SO .
2 4
The anhydrous sodium sulfate shall be kept carefully sealed.
O.
7.2.5 Distilled water or water of equivalent quality, H
7.2.6 Sodium chloride, NaCl.
7.2.7 Keeper substance (High boiling compound, e.g. octane, nonane).
7.3 Reagents for clean-up
7.3.1 Clean-up A using aluminium oxide
7.3.1.1 Aluminium oxide, Al O
2 3.
Basic or neutral, specific surface 200 m /g, activity Super I [9].
7.3.1.2 Deactivated aluminium oxide.
Deactivated with approximately 100 g/kg water.
Add approximately 10 g of water (7.2.5) to 90 g of aluminium oxide (7.3.1.1). Shake until all lumps have
disappeared. Allow the aluminium oxide to condition before use for some 16 h, sealed from the air, use it
for maximum of two weeks.
The activity depends on the water content. It can be necessary to adjust the water content.
NOTE Commercially available aluminium oxides with 100 g/kg mass fraction water can also be used.
7.3.2 Clean-up B using silica gel 60 for column chromatography
7.3.2.1 Silica gel 60, particle size 63 µm to 200 µm.
7.3.2.2 Silica gel 60, water content (mass fraction) w(H O) = 10 %.
Silica gel 60 (7.3.2.1), heated for at least 3 h at 450 °C, cooled down and stored in a desiccator containing
magnesium perchlorate or a suitable drying agent. Before use, heat for at least for 5 h at 130 °C in a drying
oven. Then allow cooling in a desiccator. Put the silica gel in a stopperad flask and add 10 % water (7.2.5)
(mass fraction), shake for 5 min intensively until all lumps have disappeared and then for 2 h in a shaking
device (8.1.3). Store the deactivated silica gel in the absence of air, use it for a maximum of two weeks.
7.3.3 Clean-up C using gel permeation chromatography (GPC)
1)
7.3.3.1 Bio-Beads® S-X3 .
7.3.3.2 Ethyl acetate, C H O .
4 8 2
7.3.3.3 Cyclohexane, C H .
6 12 ®
Preparation of GPC, for example: Put 50 g Bio-Beads S-X3 (7.3.3.1) into a 500 ml Erlenmeyer flask and
add 300 ml elution mixture made up of cyclohexane (7.3.3.3) and ethyl acetate (7.3.3.2) 1: 1 (volume
fraction) in order to allow the beads to swell. After swirling for a short time until no lumps are left,
maintain the flask closed for 24 h. Drain the slurry into the chromatography tube for GPC. After
approximately three days, push in the plungers of the column so that a filling level of
approximately 35 cm is obtained. To further compress the gel, pump approximately 2 l of elution mixture
−1
through the column at a flow rate of 5 ml · min and push in the plungers to obtain a filling level of
approximately 33 cm.
7.4 Reagents for chromatographic analysis
7.4.1 GC-Analysis
7.4.1.1 Carrier gas for GC-MS.
Operating gases of high purity and in accordance with the manufacturer’s specifications.
7.4.2 HPLC-analysis
7.4.2.1 Acetonitrile (CH CN) or methanol (CH OH), HPLC purity grade.
3 3
7.4.2.2 Ultra-pure water, HPLC purity grade.
7.4.2.3 Helium, He, of suitable purity for degasification of solvents.
7.5 Standards
7.5.1 General
Choose the internal and/or extraction standards whose physical and chemical properties (such as
extraction behaviour, retention time) are similar to those of the compounds to be analysed. For GC-MS a
minimum of four deuterated internal standards shall be used as internal standards for evaluation of
results.
7.5.2 Calibration substances and internal standards
Table 3 contains native and deuterated PAH to be used for calibration. Verify the stability of the internal
standards regularly.
1)
Bio-Beads® is an example of a suitable product available commercially. This information is given for
convenience of users of this European Standard and does not constitute an endorsement by CEN of this product.
Equivalent products can be used if they can be shown to lead to the same results.
Table 3 — Native PAH and deuterated PAH
PAH reference substances Internal standard substances (deuterated PAHs)
Naphthalene (CAS-RN 91-20-3) Naphthalene-d8 (CAS-RN 1146-65-2)
Acenaphthene (CAS-RN 83-32-9) Acenaphthene-d10 (CAS-RN 15067-26-2)
Acenaphthylene (CAS-RN 208-96-8) Acenaphthylene-d8 (CAS-RN 93951-97-4)
Fluorene (CAS-RN 86-73-7) Fluorene-d10 (CAS-RN 81103-79-9)
Anthracene (CAS-RN 120-12-7) Anthracene-d10 (CAS-RN 1719-06-8)
Phenanthrene (CAS-RN 85-01-8) Phenanthrene-d10 (CAS-RN 1517-22-2)
Fluoranthene (CAS-RN 206-44-0) Fluoranthene-d10 (CAS-RN 93951-69-0)
Pyrene (CAS-RN 129-00-0) Pyrene-d10 (CAS-RN 1718- 52–1)
Benz[a]anthracene (CAS-RN 56-55-3) Benz[a]anthracene-d12 (CAS-RN 1718-53-2)
Chrysene (CAS-RN 218-01-9) Chrysene-d12 (CAS-RN 1719-03-5)
Benzo[b]fluoranthene (CAS-RN 205-99-2) Benzo[b]fluoranthene-d12 (CAS-RN 93951-98-5)
Benzo[k]fluoranthene (CAS-RN 207-08-9) Benzo[k]fluoranthene-d12 (CAS-RN 93952-01-3)
a
(CAS-RN 205-82-3)
Benzo[j]fluoranthene
Benzo[a]pyrene (CAS-RN 50-32-8) Benzo[a]pyrene-d12 (CAS-RN 63466-71-7)
a
Benzo[e]pyrene (CAS-RN 192-97-2)
Indeno[1,2,3-cd]pyrene (CAS-RN 193-39-5) Indeno[1,2,3-cd]pyrene-d12 (CAS-RN 203578-33-0)
Dibenz[a,h]anthracene (CAS-RN 53-70-3) Dibenz[a,h]anthracene -d14 (CAS-RN 13250-98-1)
Benzo[ghi]perylene (CAS-RN 191-24-2) Benzo[ghi]perylene-d12 (CAS-RN 93951-66-7)
a
Not part of 16 target analytes, but applicable for resolution check for the separation with benzo[a]pyrene for

GC measurements.
NOTE 1 C -labelled PAH standards can also be used as internal standards.
NOTE 2 Certified solutions of PAH, and single solid PAH substances with certified purity are available from a
limited number of suppliers.
When highly contaminated samples are analysed, an aliquot of the extract is often used for further clean-
up. This makes the costs of analyses caused by the use of deuterated standard very high. In these cases,
it is allowed to add the internal standard in two steps. Step 1 addition of unlabelled internal standards to
the sample. Step 2 addition of deuterated compounds to the aliquot of the extract used for clean-up.
For HPLC 6-methylchrysene, 1-methylnaphthalene or other alkylated PAH which are not present in the
sample and which are sufficient separated from the target PAH can be used as internal or extraction
standard.
7.5.3 Injection standard
If required for GC-MS a deuterated PAH such as 1-methylnaphthalene-d10, triphenylene-d12 and
perylene-d12 can be added to the final extract before GC-MS injection to verify the recovery of the
deuterated internal standards.
7.6 Preparation of standard solutions
7.6.1 General
The procedure for the preparation of standard solutions for GC-MS and HPLC is the same only different
solvents are used.
Because of the dangerous nature of the substances to be used, commercially available – preferably
certified – standard solutions or mixed standard solutions are preferred. It is very strongly recommended
to avoid skin contact.
The working standard solutions shall be in the same solvent as the extract.
Store the primary and diluted standard solutions in a dark place at a temperature of (5 ± 3) °C. The
solutions are stable for at least one year, provided that evaporation of solvent is negligible.
PAH to analyse present in mixed standard solutions should be separated by the chromatographic
columns used (see 3.1, 5.2, 5.3).
7.6.2 Preparation of calibration standard solutions for GC-MS
Prepare individual concentrated primary standard solutions of about 0,4 mg/ml in hexane-like solvent
(7.2.3) by weighing approximately 10 mg of each of the calibration standards (7.5.2 Table 3 left column)
to the nearest 0,1 mg and dissolving them in 25 ml of hexane-like solvent.
Combine small quantities (2 ml to 10 ml) of these individual primary standard solutions into a mixed
standard solution of PAH.
7.6.3 Preparation of internal standard solution for GC-MS
Prepare a concentrated primary internal standard solution, containing at least four different components
(7.5.2 Table 3 right column), of about 0,4 mg/ml in one of the solvents used for extraction (Table 4
specifies the adequate solvents) by weighing approximately 10 mg of each of the chosen internal
standards to the nearest 0,1 mg and dissolving them in 25 ml of extraction solvent or hexane-like solvent.
Prepare from this a secondary internal standard solution with such a concentration that the added
amount gives a peak with measurable peak area or peak height in the chromatogram (at least 10 times
the detection limit).
If the two-step procedure for GC-MS is used, make two different internal standard solutions, one
containing the non-labelled compounds. At least two unlabelled compounds shall be used in the first
internal standard solution and at least four deuterated compounds in the second solution.
7.6.4 Preparation of injection standard solution for GC-MS
Prepare a concentrated primary injection standard solution, containing at least one compound (7.5.3), of
about 0,4 mg/ml in an appropriate solvent as the one used for the extract, by weighing approximately
10 mg of the chosen injection standards to the nearest 0,1 mg and dissolving it in 25 ml. Prepare from
this a secondary injection standard solution with such a concentration that the added amount gives a
peak with measurable peak area or peak height in the chromatogram (at least 10 times the detection
limit).
7.6.5 Preparation of calibration standard solutions for HPLC
Prepare individual concentrated primary standard solutions of about 0,4 mg/ml in acetonitrile (7.4.2.1)
by weighing approximately 10 mg of each of the calibration standards (7.5.2 Table 3 left column) to the
nearest 0,1 mg and dissolving them in 25 ml of acetonitrile.
Combine small quantities (2 ml to 10 ml) of these individual primary standard solutions into a mixed
standard solution of PAH.
7.6.6 Preparation of extraction standard solution for HPLC
Prepare a concentrated primary extraction standard solution, containing one or more alkylated PAH e.g.
6-methylchrysene, 1-methylnaphthalene of about 0,4 mg/ml in acetonitrile (7.4.2.1) by weighing
approximately 10 mg of each of the chosen standards to the nearest 0,1 mg and dissolving them in 25 ml
of acetonitrile. Prepare from this a secondary extraction standard solution with such a concentration that
the added amount gives a peak with measurable peak area or peak height in the chromatogram (at least
10 times the detection limit).
8 Apparatus
8.1 Extraction and clean-up procedures
8.1.1 General
Usual laboratory glassware.
All glassware and material that comes into contact with the sample or extract shall be thoroughly cleaned.
8.1.2 Sample bottles, made of glass, stainless steel, aluminium, perfluoroalkoxy or fluorinated
ethylene propylene with glass stopper or screw top and polytetrafluoroethylene (PTFE) seal of
appropriate volume.
WARNING — For safety reasons, biologically active sludge samples shall not be stored in a sealed
container.
8.1.3 Shaking device, with horizontal movement (200 strokes to 300 strokes per min) or an end-over-
end shaking device.
8.1.4 Water bath, adjustable up to 100 °C.
8.1.5 Separating funnels of appropriate volume.
8.1.6 Conical flasks of appropriate volume.
8.1.7 Soxhlet extraction apparatus, consisting of round bottom flask, e.g. 100 ml, Soxhlet extractors
and Soxhlet thimbles, e.g. 27 mm × 100 mm, vertical condensers, e.g. 300 mm, heating device.
8.1.8 Concentrator, Kuderna Danish type.
Other evaporators, e.g. a rotary evaporator, may be used if found to be equally suitable.
8.1.9 Boiling chips, glass or porcelain beads.
8.1.10 Quartz wool or silanized glass wool.
WARNING — Working with quartz wool imposes a risk to health through the release of fine quartz
particles. Inhalation of these should be prevented by using a fume cupboard and wearing a dust mask.
8.1.11 Calibrated test tubes, with a nominal capacity of 10 ml to 15 ml and ground glass stopper.
8.1.12 Chromatography tubes (Chromatography column of glass, 5 mm to 10 mm inside diameter,
length e.g. 600 mm).
8.1.13 Sonication apparatus.
8.1.14 Pressurized Liquid Extraction equipment (PLE), combining elevated temperature and
pressure with liquid solvents to achieve extraction of the analytes from the solid matrix.
8.1.15 Gel permeation chromatograph, kind of liquid chromatograph which separates molecules on
the basis of their size by using columns packed with very small, porous particles.
8.2 Gas chromatograph
8.2.1 General
Gas chromatograph equipped with a non-discriminating injection system, capillary column and a mass
spectrometric detector (GC-MS).
8.2.2 Capillary columns
Low to medium capillary columns such as 5 % phenyl-methyl silicone stationary phase coated onto fused
silica capillary column or an equivalent chemically bonded phase column.
Their dimensions should be sufficient to separate the critical pairs mentioned in 5.2. In general column
length is typically 25 m to 60 m, internal diameter 0,18 mm to 0,325 mm and film thickness 0,1 µm to
0,5 µm.
8.3 High-performance liquid chromatograph
8.3.1 General
A HPLC system equipped according to requirements with ultraviolet (UV) and a fluorescence detection
(FLD) system and a data evaluation system, including:
— degassing assembly, e.g. for degassing with vacuum or helium;
— analytical pumps, capable of binary gradient elution;
— column thermostat, capable of maintaining the temperature constant to within ± 0,5 °C;
— fluorescence detector capable of programming at least six pairs of wavelengths, including
damping/amplification, equipped with monochromator(s);
— UV detector (with variable wavelength) or diode array.
8.3.2 Analytical separation column
A reversed phase HPLC column meeting the separation requirements described in Annex B.
9 Sample storage and preservation
9.1 Sample storage
The samples shall be analysed as soon as possible after sampling. This applies in particular to the
examination of microbiologically active solids.
If necessary, sludge samples shall be stored according to EN ISO 5667-15.
Dried samples can be stored at room temperature in a dark place up to one month. Soil samples shall be
stored according to e.g. ISO 18512.
During storage of the samples, losses of PAH can occur due to adsorption to the walls of the containers.
The extent of the losses depends on the storage time.
9.2 Sample pre-treatment
To obtain a test sample, suitable pre-treatment of samples shall be performed according to EN 16179
(sludge, biowaste and soil) or EN 15002 (waste), if not otherwise specified, considering the specific
drying procedures as specified in Table 4.
Complete drying of the sample is essential if Soxhlet or PLE is used for extraction. Complete drying is also
recommended if the sample shall be stored for a long period.
Table 4 — Drying techniques for samples of different matrices for subsequent analyses of PAH
Matrix  Drying technique
Freeze drying Na SO Drying No drying
2 4
(EN ISO 16720)
T < 40°C
a b a
Sludge
x x x
a a
Biowaste x x
x x
(compost, mixed
waste)
a a
Soil x x
x x
Sediment
a a
Waste x x
x x
a
Loss of naphthalene is possible.
b
Na SO can be used for the preservation of hygroscopic dried sludge.
2 4
To achieve a homogeneous and representative test portion, one or more particle size reduction steps can
be needed. The choice of the technique depends on the nature of the sample and on the particle size
needed. Typically, particle size reduction is a multi-step operation that implies the use of a sequence of
different techniques like crushing, cutting or grinding.
Grinding of samples which have a plastic or paste-like consistency requires embrittlement with liquid
nitrogen and particle size reduction to less than 0,5 mm, e.g. by using an ultra-centrifugal mill.
10 Procedure
10.1 Blank test
Perform a blank test following the applied procedure (selected extraction and clean-up procedure) using
the same amount of reagents that are used for the pre-treatment, extraction, clean-up and analysis of a
sample. Analyse the blank immediately prior to analysis of the samples to demonstrate sufficient freedom
from contamination. The blank shall be less than 50 % of the lowest reporting limit.
10.2 Extraction
10.2.1 General
Depending on the test sample (matrix and moisture content), choose a suitable extraction procedure
(see Table 5). The choice of the extraction solvent is considered more crucial than the procedure and the
extraction devices itself for the extraction of PAH from the matrices. Since some of the target PAHs are
relatively insoluble in the usual non-polar solvents such as hexane-like solvent, ether and other
hydrocarbons, the choice of the solvents shall be made in accordance with the expected contamination
level.
Extraction procedure 1 (10.2.2) is recommended if it is important to break up aggregates in the sample
to reach the PAHs. This is especially important with soil samples containing clay particles. With wet
samples these procedures shall be applied in order to remove the water.
If dissolving of the PAHs is the most important step (waste and highly contaminated soil and organic
matter rich materials) and the sample is dry, extraction procedure 2 (10.2.3) using Soxhlet is
recommended. For sludges, it has been shown that Soxhlet or pressurized liquid extraction is applicable.
However, a general rule cannot be given, because samples can contain all: aggregates, organic matter and
(plastic) waste.
Other extraction procedures, e.g. microwave may be used provided:
— the laboratory can show that the extraction efficiency is equivalent to one of the extraction
procedures 1, 2 or 3 as described in this document, or
— the sample requires another approach as shown by the laboratory and the results of the procedure
are in agreement with the performance criteria described in 10.6.6 and 11.
NOTE For application of this document for some types of waste, addition of acetone with Soxhlet extraction
has been shown to be effective.
Extraction procedures described in this document are able to extract up to 20 g of dry sample. If the test
sample has a low density (i.e. some matrices) or the sample is homogeneous, depending on the expected
PAH content and on the homogeneity of the sample, less sample can be used. In general, the following
amounts can be used: 10 g to 20 g of sewage sludge, 10 g to 25 g waste, 5 g to 20 g of compost or 2 g to
20 g of soil or sediment. The amount of sample shall be weighed with an accuracy of at least 1 %.
Table 5 — Extraction procedure to be used for different matrices
Moisture Matrix Extraction Extraction Extraction procedure Remark
status of the solvent technique
test sample
Dry Soil, sediment, Acetone/ Agitation, end- Extraction procedure 1
sludge, biowaste, petroleum over-end (see 10.2.2)

waste ether or shaking,
hexane-like sonication
solvent
Plastic-rich waste petroleum Agitation, end- Extraction procedure 1
ether or over-end (see 10.2.2)

hexane-like shaking,
solvent sonication
Highly Toluene, Soxhlet, Extraction procedure 2
contaminated soil, Petroleum Pressurized (see 10.2.3), Extraction
sediment, sludge, ether or Liquid procedure 3
biowaste, compost hexane-like Extraction (see 10.2.4)
solvent
Wet Soil, sediment, Acetone/ Agitation, end- Extraction procedure 1 Also applicable for
sludge, petroleum over-end (see 10.2.2) field moist
biowaste, waste ether or shaking, samples with dry
hexane-like matter
sonication
solvent content > 75 %
Soil, sediment, Acetone/ Agitation Extraction procedure 4
sludge, biowaste petroleum (see 10.2.5)
ether or
hexane-like
solvent /
NaCl
10.2.2 Extraction procedure 1: acetone/hexane-like solvent and agitation or sonication
Place the sample in a bottle (8.1.2). Add a definite volume of the secondary internal standard solution
(7.6.3 or 7.6.6). Add 50 ml of acetone (7.2.1) to the test sample and extract by shaking thoroughly with
the shaking device (8.1.3) or sonication for 30 min to break up aggregates. Then add 50 ml of petroleum
ether or hexane-like solvent (7.2.3) and shake again or sonicate thoroughly at least for 1h. Use a
horizontal shaking device or end-over-end shaking (8.1.3) and have the solvent movement in the sample
bottle as long as possible (horizontal position). After the solids have been settled decant the supernatant.
Wash the solid phase with 50 ml of petroleum ether or hexane-like solvent (7.2.3) and decant again.
Collect the extracts in a separating funnel (8.1.5) and remove the acetone by shaking twice with 400 ml
of water (7.2.5). Dry the extract over sodium sulfate (7.2.4). Rinse the sodium sulfate with petroleum
ether or hexane-like solvent (7.2.3) and add the rinsing to the extract.
NOTE 1 Tap water has sh
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