Soil quality — Determination of polycyclic aromatic hydrocarbons (PAH) by gas chromatography (GC) and high performance liquid chromatography (HPLC)

ISO 13859:2014 specifies the quantitative determination of 16 PAH in sludge, soil, and treated biowaste using GC-MS and HPLC-UV-DAD/FLD covering a wide range of PAH contamination levels. When using fluorescence detection, acenaphthylene cannot be measured. 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. 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. ISO 13859:2014 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.

Qualité du sol — Détermination des hydrocarbures aromatiques polycycliques (HAP) par chromatographie en phase gazeuse (CPG) et chromatographie liquide à haute performance (CLHP)

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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 13859:2014(E)
©
ISO 2014

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ISO 13859:2014(E)

COPYRIGHT PROTECTED DOCUMENT
© 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
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Published in Switzerland
ii © ISO 2014 – All rights reserved

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ISO 13859:2014(E)

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
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ISO 13859:2014(E)

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.
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ISO 13859:2014(E)

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
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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.
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ISO 13859:2014(E)

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.
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ISO 13859:2014(E)

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.
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ISO 13859:2014(E)

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

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ISO 13859:2014(E)

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.
2
7.2.6 Sodium chloride, NaCl, anhydrous.
7.3 Reagents for clean-up
7.3.1 Clean-up using aluminium oxide
2
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 %.
2
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
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ISO 13859:2014(E)

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.
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ISO 13859:2014(E)

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.
13
NOTE 1 C -labelled PAH standards can also be used as internal standard.
12
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-
...

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