ISO 13859:2014

INTERNATIONAL ISO
STANDARD 13859
First edition
2014-02-01
Soil quality — Determination of
polycyclic aromatic hydrocarbons
(PAH) by gas chromatography
(GC) and high performance liquid
chromatography (HPLC)
Qualité du sol — Détermination des hydrocarbures aromatiques
polycycliques (HAP) par chromatographie en phase gazeuse (CPG) et
chromatographie liquide à haute performance (CLHP)
Reference number
©
ISO 2014
© ISO 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 2
3 Terms and definitions . 2
4 Principle . 3
5 Interferences . 3
5.1 Interference with sampling and extraction . 3
5.2 Interference with GC-MS . 4
5.3 Interferences with the HPLC . 4
6 Safety remarks . 4
7 Reagents . 5
7.1 General . 5
7.2 Reagents for extraction . 5
7.3 Reagents for clean-up . 5
7.4 Reagents for chromatographic analysis . 6
7.5 Standards . 7
7.6 Preparation of standard solutions . 8
7.7 Preparation of internal standard solutions . 9
7.8 Preparation of injection standard solution . 9
8 Apparatus . 9
9 Sample storage and preservation .11
9.1 Sample storage .11
9.2 Sample pretreatment .11
10 Procedure.11
10.1 Blank test .11
10.2 Extraction .12
10.3 Concentration or dilution .14
10.4 Clean-up of the extract .15
10.5 Addition of the injection standard .16
10.6 Gas chromatographic analysis (GC) .17
10.7 High performance liquid chromatographic analysis (HPLC) .20
11 Performance characteristics .23
12 Precision .23
13 Test report .23
Annex A (informative) Repeatability and reproducibility data .24
Annex B (informative) Examples of instrumental conditions and chromatograms .27
Bibliography .37
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 3, Chemical
methods and soil characteristics.
iv © ISO 2014 – All rights reserved

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 preceding steps for sampling, pretreatment, 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.
It is to be underlined that the target contamination level of PAH can lie in the range of about 0,01 mg/kg
per individual PAH (agricultural soil and sediment) to about 200 mg/kg and higher (e.g. contaminated
soil at coking plant sites or waste). The use of internal and injection standards is described in order to
have an internal check on execution of the extraction and clean-up procedure. The method is as far as
possible in agreement with the method described for PCBs (see EN 16167).
This International Standard is the result of a desk study “Horizontal International Standard for
determination of PAH in sludge, soil, and biowaste” in the project “Horizontal” and aims at evaluating
the latest developments in assessing PAH in sludge, soil, treated biowaste, and neighbouring fields. After
an evaluation study, in which the ruggedness of the method was studied, a European-wide validation
of the draft standard has taken place. The results of the desk studies as well as the evaluation and
validation studies have been subject to discussions with all parties concerned in CEN.
This International Standard 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 International Standard is applicable and validated
Matrix Materials used for validation
Sludge Municipal sludge
Biowaste Fresh compost
INTERNATIONAL STANDARD ISO 13859:2014(E)
Soil quality — Determination of polycyclic aromatic
hydrocarbons (PAH) by gas chromatography (GC) and high
performance liquid chromatography (HPLC)
WARNING — Persons using this International Standard should be familiar with usual laboratory
practice. This International Standard 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.
IMPORTANT — It is absolutely essential that tests conducted according to this International
Standard be carried out by suitably trained staff.
1 Scope
This International Standard specifies the quantitative determination of 16 PAH (see Table 2) in
sludge, soil, and treated biowaste using GC-MS and HPLC-UV-DAD/FLD covering a wide range of PAH
contamination levels (see also Annex B).
When using fluorescence detection, acenaphthylene cannot be measured.
Table 2 — Polycyclic aromatic hydrocarbons which can be analysed using this International
Standard
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
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.
Typically, a lower limit of application of 0,01 mg/kg (expressed as dry matter) can be ensured for each
individual PAH. This depends on instrument and sample.
Sludge, soil, and treated biowaste can differ in properties and also in the expected contamination levels
of PAH and presence of interfering substances. These differences make it impossible to describe one
general procedure. This International Standard contains decision tables based on the properties of
the sample and the extraction and clean-up procedure to be used. Two general lines are followed, an
agitation procedure (shaking) or use of Soxhlet/pressurized liquid extraction.
NOTE Other PAH compounds can also be analysed with this method, provided suitability has been proven.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 5667-15, Water quality — Sampling — Part 15: Guidance on the preservation and handling of sludge
and sediment samples
ISO 8466-1, Water quality — Calibration and evaluation of analytical methods and estimation of performance
characteristics — Part 1: Statistical evaluation of the linear calibration function
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric
method
ISO 14507, Soil quality — Pretreatment of samples for determination of organic contaminants
ISO 18512, Soil quality — Guidance on long and short term storage of soil samples
ISO 22892, Soil quality — Guidelines for the identification of target compounds by gas chromatography and
mass spectrometry
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
critical pair
pair of congeners that will be separated to a predefined degree (e. g. R = 0,5) to ensure chromatographic
separation meets minimum quality criteria
[SOURCE: EN 15308:2008, 3.6]
Note 1 to entry: See Figure 1.
2 © ISO 2014 – All rights reserved

Key
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
4 Principle
After pretreatment to reduce the moisture content and to increase the homogeneity (see 9.2), the test
sample is extracted with a solvent.
The extract is concentrated and interfering compounds are removed by a clean-up method suitable for
the specific matrix. The eluate is concentrated. For HPLC analysis, the concentrated eluate is taken up
in an appropriate less volatile water miscible polar solvent and the non-polar eluate residue is removed.
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 external 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 change 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 can 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 incomplete 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.
Unsymmetrical 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. Triphenylene
cannot be completely separated from benz(a)anthracene and chrysene. In this case it shall be stated in
the report.
5.3 Interferences with the HPLC
Substances that show either fluorescence or quenching and co-elute with the PAH to be determined
can interfere 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-ring and
3-ring PAH. Extracts shall be diluted sufficiently with acetonitrile for the HPLC analysis, otherwise the
detection of naphthalene and 3-ring PAH can be interfered by a broad toluene peak.
Separation between dibenz(a,h)anthracene and indeno(1,2,3-cd)pyrene can be critical. When incomplete
resolution is encountered, peak integration shall be checked and, when necessary, corrected.
Usually perylene is incompletely resolved from 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.
PAH can co-distil with solvent and become deposited outside of stoppered bottles. All containers
containing solutions of PAH in solvent shall therefore always be handled using gloves which are solvent
resistant and preferably disposable.
PAH contamination of vessels can be detected by irradiation with 366 nm UV-light.
Vessels containing PAH solutions should be stored standing in beakers to contain any spillage in the case
of breakage.
Solid PAH are the most dangerous and give rise to a dust hazard due to their crystals becoming
electrostatically charged. These materials shall only be handled where proper facilities are available (e. g.
adequate fume hoods, protective clothing, dust masks). It is strongly advised that standard solutions are
prepared centrally in suitably equipped laboratories or purchased from suppliers specialized in their
preparation.
Solvent solutions containing PAH shall be disposed of in a manner approved for disposal of toxic wastes.
National regulations shall be followed with respect to all hazards associated with this method.
4 © ISO 2014 – All rights reserved

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), C H O.
3 6
7.2.2 Petroleum ether, boiling range 40 °C to 60 °C.
NOTE Hexane-like solvents with a boiling range between 30 °C and 69 °C are allowed.
7.2.3 Toluene, C H .
7 8
7.2.4 Anhydrous sodium sulfate, Na SO .
2 4
The anhydrous sodium sulfate shall be kept carefully sealed.
7.2.5 Distilled water, or water of equivalent quality, H O.
7.2.6 Sodium chloride, NaCl, anhydrous.
7.3 Reagents for clean-up
7.3.1 Clean-up using aluminium oxide
7.3.1.1 Aluminium oxide, Al O , basic or neutral, specific surface 200 m /g, activity Super I according
2 3
to Brockmann.
NOTE 1 Hexane-like solvents with a boiling range between 30 °C and 69 °C are allowed.
NOTE 2 Brockman Activity Scale is a measure of the percentage of water added to the adsorbent based upon
[14] [15]
weight/weight relationships between water and the adsorbent. Grade I corresponds to 0 % water added.
7.3.1.2 Deactivated aluminium oxide, deactivated with approximately 10 % 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.
NOTE The activity depends on the water content. It can be necessary to adjust the water content.
7.3.2 Clean-up 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, heated for at least 3 h at 450 °C, cooled down in a desiccator, and stored containing magnesium
perchlorate or a suitable drying agent. Before use, heat at least for 5 h at 130 °C in a drying oven. Allow
cooling in a desiccator and add 10 % water (mass fraction) in a flask. Shake for 5 min intensively until
all lumps have disappeared, and then for 2 h in a shaking device (8.1.2). Store the deactivated silica gel
in the absence of air; use it for maximum of two weeks.
Silica gel 60 is stable for at most two weeks.
7.3.3 Clean-up 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
7.3.3.4 Spherical, porous styrene divinylbenzene resin. ®
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)
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 through the column at a flow
−1
rate of 5 ml · min and push in the plungers to obtain a filling level of approximately 33 cm.
7.3.4 Clean-up using liquid-liquid partition/DMF/cyclohexane
7.3.4.1 Dimethylformamide (DMF), C H NO.
3 7
7.3.4.2 Dimethylformamide:water, 9:1.
7.4 Reagents for chromatographic analysis
7.4.1 GC-analysis
Carrier gas for GC-MS: helium or hydrogen of high purity and in accordance with the manufacturer’s
specifications.
7.4.2 HPLC analysis
7.4.2.1 Mobile phase.
7.4.2.2 Acetonitrile, CH CN or methanol, CH OH, HPLC purity grade.
3 3
7.4.2.3 Ultra-pure water, HPLC purity grade.
7.4.2.4 Helium, He, of suitable purity for degasification of solvents.
1) Bio-Beads© 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 ISO of this product. Equivalent
products may be used if they can be shown to lead to the same results.
6 © ISO 2014 – All rights reserved

7.5 Standards
7.5.1 Reference substances, internal standards
Choose the internal standards substances whose physical and chemical properties (such as extraction
behaviour, retention time) are similar to those of the compounds to be analysed. A minimum of five
labelled internal standards shall be used as internal standards for the GC-MS method for evaluation of
results. Verify the stability of the internal standards regularly. Table 3 contains native and deuterated
PAH to be used for calibration of specific analytes.
Table 3 — Native PAH and deuterated PAH
PAH reference substances Internal standard substances deuterated PAH
Naphthalene (CAS-RN 91-20-3) Naphthalene-d8
Acenaphthene (CAS-RN 83-32-9) Acenaphthene-d10
Acenaphthylene (CAS-RN 208-96-8) Acenaphthylene-d8
Fluorene (CAS-RN 86-73-7) Fluorene-d10
Anthracene (CAS-RN 120-12-7) Anthracene-d10
Phenanthrene (CAS-RN 85-01-8) Phenanthrene-d10
Fluoranthene (CAS-RN 206-44-0) Fluoranthene-d10
Pyrene  (CAS-RN 129-00-0) Pyrene-d10
Benz(a)anthracene (CAS-RN 56-55-3) Benz(a)anthracene-d12
Chrysene (CAS-RN 218-01-9) Chrysene-d12
Benzo(b)fluoranthene (CAS-RN 205-99-2) Benzo(b)fluoranthene-d12
Benzo(k)fluoranthene (CAS-RN 207-08-9) Benzo(k)fluoranthene-d12
Benzo(a)pyrene (CAS-RN 50-32-8) Benzo(a)pyrene-d12
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
Dibenz(a,h)anthracene (CAS-RN 53-70-3) Dibenz(a,h)anthracene-d14
Benzo(ghi)perylene (CAS-RN 191-24-2) Benzo(ghi)perylene-d12
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 standard.
NOTE 2 Certified solutions of PAH, and single solid PAH substances with certified purity are available from
a limited number of suppliers e. g. Institute for Reference Materials and Measurements (IRMM) B-2440 Geel,
Belgium; National Institute of Science and Technology; Office of Standard Ref. Data, Washington DC 20 234 U.S.A;
or from other commercial providers.
When highly contaminated samples are analysed, an aliquot of the extract is often used for a further
clean-up.
7.5.2 Injection standard
7.5.2.1 GC-MS
A deuterated PAH such as 1-Methylnaphthalene-d10, Triphenylene-d12, and Perylene-d12 shall be added
to the final extract before GC-MS injection to check the recovery of the deuterated internal standards.
7.5.2.2 HPLC
For this method, a recovery control shall be made by addition, and which is not interfering with the
target analytes, of a suitable native PAH not mentioned in the scope, e. g. 6-methylchrysene, to the
sample before extraction. The recovery range for this control should be between 70 % and 110 %.
7.6 Preparation of standard solutions
7.6.1 General
Commercially available, preferably certified standard solutions should be used due to the dangerous
nature of the substances. Avoid skin contact.
7.6.2 Standard solutions for HPLC analysis
7.6.2.1 Single substance stock solutions
Prepare solutions of the single substances (see Table 3) in acetonitrile (7.4.2.2) to achieve a mass
concentration of 10 µg/ml. These solutions are used for confirmation and identification of single PAH in
the chromatogram.
7.6.2.2 Multiple substance stock solution
Prepare solutions of the reference substances (see Table 3) in acetonitrile (7.4.2.2) to achieve a mass
concentration of the respective individual substance of 10 µg/ml.
Solutions according to 7.6.2.1 and 7.6.2.2 are stable for at least one year when stored in the dark at room
temperature and protected from evaporation.
7.6.2.3 Calibration solutions
Prepare at least five calibration solutions by appropriate dilution of the stock solution (7.6.2.1 or
7.6.2.2), using acetonitrile (7.4.2.2) or methanol (7.4.2.2) as solvent. The choice of solvent depends on
the composition of the mobile phase.
Transfer 50 µl of the stock solution into a graduated 5-ml flask and fill up to the mark with acetonitrile.
1 µl of this reference solution contains 100 pg of the respective individual substances.
Check the stability of the reference solutions regularly.
Check the mass concentration of the PAH in the stock solution by comparison with an independent,
preferably certified, standard solution.
7.6.3 Standard solutions for GC-MS analysis
7.6.3.1 Single substance stock solution
Prepare solutions of the single substances of native and deuterated PAH (see Table 3) in toluene (7.2.3)
or cyclohexane (7.3.3.3) to achieve a mass concentration of 10 µg/ml. These solutions are used for
confirmation and identification of single PAH in the chromatogram.
The single substance stock solutions shall be stored in a dark place at about –15 °C to –18 °C. Store the
diluted standard solutions at (5 ± 3) °C protected from light and evaporation. The solutions are stable
for about one year.
8 © ISO 2014 – All rights reserved

7.6.3.2 Multiple substance stock solution of native PAH
Dilute the solution of the reference substances, i. e. native PAH (see Table 3) in toluene (7.2.3) or
cyclohexane (7.3.3.3), to achieve a mass concentration of the respective individual substance, i. e.
10 µg/ml.
7.6.3.3 Multiple substance stock solution of deuterated or labelled PAH (internal standard)
Multiple deuterated or labelled PAH standards for use as internal standard, also available as mixtures
in suitable solvent, can be diluted to the same mass concentration, i. e. 10 µg/ml for each individual
deuterated standard.
7.6.4 Calibration standard solutions
Prepare a series of calibration standard solutions (at least five) over a suitable range by transferring
different volumes of the multiple substance stock solution of native PAH standards (7.6.3.2) and
a constant volume of the internal standard solution (7.6.3.3) into a volumetric flask and fill up with
cyclohexane (7.3.3.3).
7.7 Preparation of internal standard solutions
Multiple substance stock solution of deuterated or labelled PAH (7.6.3.3) can be used for spiking of
the sample before extraction. The use of the spiked internal standards shall be adjusted so that their
concentration in the final extract for GC-MS determination is the same as in the calibration solutions
(e. g. 100 pg/µl). For HPLC analysis, where external calibration is applied, a PAH which is not interfering
with the target PAH, e. g. 6-methylchrysene, is added to the sample before extraction to check for the
recovery of this substance throughout the whole procedure.
7.8 Preparation of injection standard solution
This is needed to check the recovery of the deuterated internal standards.
A single substance stock solution (7.6.3.1), e. g. deuterated benzo(e)pyrene, which is not interfering with
the target analyte, can be used.
NOTE A deuterated or a C -labelled PAH not mentioned in Table 3 is added before injection into the GC-
system to monitor variability of the instrument response. The recovery of the internal standards throughout the
whole method can be calculated by the related response of the internal standard to the injection standard. Add
such an amount to give a peak with measurable peak area or peak surface in the chromatogram (at least 10 times
the detection limit).
8 Apparatus
8.1 Extraction and clean-up procedures, usual laboratory glassware.
All glassware and material that comes into contact with the sample or extract shall be thoroughly
cleaned.
8.1.1 Sample bottles, made of glass, stainless steel, or aluminium, with glass stopper or screw top and
polytetrafluoroethylene (PTFE) seal of appropriate volume.
NOTE Glass is not appropriate for sludge samples.
WARNING — For safety reasons, biologically active sludge samples shall not be stored in a sealed
container.
8.1.2 Shaking device, with horizontal movement (200 strokes to 300 strokes per minute).
8.1.3 Water bath, adjustable up to 100 °C.
8.1.4 Separating funnels, of appropriate volume.
8.1.5 Conical flasks, of appropriate volume.
8.1.6 Soxhlet extraction apparatus, consisting of round bottom flask, e. g. 100 ml Soxhlet extractors
and Soxhlet thimbles, 27 mm × 100 mm vertical condensers, 300 mm heating device.
8.1.7 Concentrator, Kuderna Danish type.
NOTE Other evaporators, e. g. a rotary evaporator, can be used if found to be equally suitable.
8.1.8 Boiling chips, glass or porcelain beads.
8.1.9 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.
8.1.10 Calibrated test tubes, with a nominal capacity of 10 ml to 15 ml and ground glass stopper.
8.1.11 Chromatography tubes, chromatography column of glass, with 5 mm to 10 mm inside diameter
length, e. g. 600 mm.
8.2 Gas chromatograph, equipped with a non-discriminating injection system, capillary column, and
a mass spectrometric detector (GC-MS).
8.2.1 Capillary columns, each comprising a 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 below. In general, column length should be at least
30 m,with internal diameter of 0,25 mm and film thickness of 0,2 µm.
Sufficient resolution (0,8) between the chromatographic peaks of critical pairs as 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.
8.3 High performance liquid chromatograph, HPLC system, equipped according to requirements
with ultraviolet (UV) and a fluorescence detection (FLD) system and a data evaluation system.
This includes the following:
— 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, preferably equipped with monochromator(s);
— UV detector (with variable wavelength) or diode array.
8.3.1 Analytical separation column, a reversed phase HPLC column meeting the separation
requirements described in Annex B.
10 © ISO 2014 – All rights reserved

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 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 ISO 18512.
9.2 Sample pretreatment
Pretreat samples according to ISO 14507, if not otherwise specified, and considering the specific drying
procedures as specified in Table 4 to obtain a test sample.
Pretreatment is necessary to reduce the moisture content to enable extraction of the PAH and to increase
the homogeneity.
Complete drying of the sample is essential if Soxhlet is used for extraction and to increase the homogeneity.
Complete drying is also recommended if the sample shall be stored for a long period.
If necessary, increase the amount of solid matter of the sample by centrifugation and/or filtration (see
Notes 1 and 2).
NOTE 1 Centrifugation is possible for several samples, but not practicable with material having approximately
the same density of water.
NOTE 2 Filtration is possible, but the handling of some samples can cause problems due to blockages, too high
water content, or extraction of target compound with filter paper.
Table 4 — Drying techniques for samples of different matrices for subsequent analyses of PAH
Drying technique
Matrix
Freeze drying
Na SO No drying
2 4
(ISO 16720)
a b
Sludge x x
Biowaste
(compost, mixed x x x
waste)
Soil
x x x
(e. g. sand, clay)
a
Loss of volatile PAH is possible.
b
Na SO can be used for the preservation of hygroscopic dried sludge.
2 4
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 pretreatment, 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 more crucial than the procedure and the extraction
devices itself for the extraction of PAH from the matrices. Since some of the target PAH are relatively
insoluble in the usual non-polar solvents, such as petroleum ether and other hydrocarbons, the choice of
the solvents shall be made in accordance with the expected contamination level.
Extraction procedure 1 (see 10.2.2) is recommended if it is important to break up aggregates in the
sample to reach the PAH. 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 (see 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. ultrasonic extraction, microwave, or pressurized extraction can be
used provided
— the laboratory can show that the extraction efficiency is equivalent to one of the extraction
procedures 1 (see 10.2.2) or 2 (see 10.2.3) as described in this International Standard, 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 Clause 11.
NOTE For application of this International Standard for some types of waste, addition of acetone with Soxhlet
extraction has been shown to be effective.
Extraction procedures described in this International Standard 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 soil; 2 g to 10 g of sewage sludge, 5 g to 20 g of compost,
or 2 g to 20 g of (bio)waste. 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 status Matrix Extraction Extraction Extraction Remark
of the test solvent technique procedure
sample
Dry Soil-like material, Acetone/ Agitation Extraction Also applicable for field
sludge, biowaste, petroleum procedure 1 moist samples with dry
compost ether (see 10.2.2) matter content > 75 %
Highly contami- Toluene Soxhlet, pres- Extraction
nated soil, sludge, surized liquid procedure 2
biowaste, compost extraction (see 10.2.3)
Wet Soil-like material, Acetone/ Agitation Extraction Limitations for the
biowaste compost, petroleum procedure 3 amount of water in the
sludge ether (see 10.2.4) sample are given
NaCl
10.2.2 Extraction procedure 1: acetone/petroleum ether and agitation
For GC-MS analysis with internal calibration, add a definite volume of the internal standard solution.
Add 50 ml of acetone (7.2.1) to the test sample and extract by shaking thoroughly with the shaking
device (8.1.2) for 30 min to break up aggregates. Add 50 ml of petroleum ether (7.2.2) and shake again
12 © ISO 2014 – All rights reserved

thoroughly at least for 12 h. After the solids have been settled, decant the supernatant. Wash the solid
phase with 50 ml of petroleum ether (7.2.2) and decant again. Collect the extracts in a separating funnel
(8.1.4) and remove the acetone by shaking twice with 400 ml of water. Dry the extract over anhydrous
sodium sulfate (7.2.4). Rinse the sodium sulfate with petroleum ether (7.2.2) and add the rinsing to the
extract.
NOTE 1 Tap water has shown to be applicable for removal of the acetone because target compounds are not
present.
If the sample contains water up to 25 %, the same procedure can be used. If the water content of
the sample is greater than 25 %, this procedure is less effective and the amount of acetone shall be
increased. The
...