CEN/TS 16181:2013

SLOVENSKI STANDARD
oSIST prEN 16181:2011
01-januar-2011
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Sludge, treated biowaste and soil - Determination of polycyclic aromatic hydrocarbons
(PAH) by gas chromatography (GC) and high performance liquid chromatography
(HPLC)
Schlamm, behandelter Bioabfall und Boden - Bestimmung von polycyclischen
aromatischen Kohlenwasserstoffen (PAK) mittels Gaschromatographie (GC) und
Hochleistungs-Flüssigkeitschromatographie (HPLC)
Boue, biodéchet traité et sol - Dosage des hydrocarbures aromatiques polycycliques
(HAP) par chromatographie en phase gazeuse et chromatographie liquide à haute
performance
Ta slovenski standard je istoveten z: prEN 16181
ICS:
13.030.20 7HNRþLRGSDGNL%ODWR Liquid wastes. Sludge
oSIST prEN 16181:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 16181:2011
oSIST prEN 16181:2011
EUROPEAN STANDARD
DRAFT
prEN 16181
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2010
ICS 13.030.20
English Version
Sludge, treated biowaste and soil - Determination of polycyclic
aromatic hydrocarbons (PAH) by gas chromatography (GC) and
high performance liquid chromatography (HPLC)
Boue, bio-déchets traités et sols - Dosage des Schlamm, behandelter Bioabfall und Boden - Bestimmung
hydrocarbures aromatiques polycycliques (HAP) par von polycyclischen aromatischen Kohlenwasserstoffen
chromatographie en phase gazeuse et chromatographie (PAK) mittels Gaschromatographie (GC) und
liquide à haute performance Hochleistungs-Flüssigkeitschromatographie (HPLC)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 400.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16181:2010: E
worldwide for CEN national Members.

oSIST prEN 16181:2011
prEN 16181:2010 (E)
Contents Page
Foreword .4
Introduction .5
1 Scope .6
2 Normative references .7
3 Terms and definitions .7
4 Principle .8
5 Interferences .8
5.1 Interference with sampling and extraction .8
5.2 Interference with GC-MS .9
5.3 Interferences with the HPLC .9
6 Safety remarks .9
7 Reagents . 10
7.1 General . 10
7.2 Reagents for extraction . 10
7.3 Reagents for clean-up . 10
7.3.1 Clean-up using aluminium oxide . 10
7.3.2 Clean-up using Silica gel 60 for column chromatography . 11
7.3.3 Clean-up using Gel permeation chromatography (GPC) . 11
7.3.4 Clean-up using liquid-liquid partition/DMF/cyclohexane . 11
7.4 Reagents for chromatographic analysis . 11
7.4.1 GC-Analysis . 11
7.4.2 HPLC-analysis . 11
7.5 Standards . 12
7.5.1 Reference substances, internal standards . 12
7.5.2 Injection standard . 13
7.6 Preparation of standard solutions . 14
7.6.1 General . 14
7.6.2 Standard solutions for HPLC-Analysis. 14
7.6.3 Standard solutions for GC-MS analysis . 14
7.6.4 Calibration standard solutions . 15
7.7 Preparation of internal standard solutions . 15
7.8 Preparation of injection standard solution . 15
8 Apparatus . 15
8.1 Extraction and clean-up procedures . 15
8.2 Gas chromatograph . 16
8.3 High-performance liquid chromatograph . 16
9 Sample storage and preservation . 17
9.1 Sample storage . 17
9.2 Sample preservation and pretreatment . 17
10 Procedure . 18
10.1 Blank test . 18
10.2 Extraction . 18
10.2.1 General . 18
10.2.2 Extraction procedure 1: samples using acetone/petroleum ether and agitation . 19
10.2.3 Extraction procedure 2: dry samples using Soxhlet. 19
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prEN 16181:2010 (E)
10.2.4 Extraction procedure 3: wet samples acetone/petroleum ether/sodium chloride and
agitation . 20
10.3 Concentration or dilution . 20
10.3.1 General . 20
10.3.2 For HPLC analysis . 20
10.4 Clean-up of the extract . 21
10.4.1 General . 21
10.4.2 Clean-up A – Aluminium oxide . 21
10.4.3 Clean-up B – Silica gel . 22
10.4.4 Clean-up C – Gel permeation chromatography (styrene divinylbenzene resin) . 22
10.4.5 Clean-up D – DMF/cyclohexane partitioning for aliphatic hydrocarbons removal. 22
10.5 Addition of the injection standard . 23
10.6 Gas chromatographic analysis (GC) . 23
10.6.1 Gas chromatographic analysis with mass spectrometric detection . 23
10.6.2 Calibration of the method using an internal standard . 24
10.6.3 Measurement . 25
10.6.4 Identification . 25
10.6.5 Check on method performance . 26
10.6.6 Calculation . 26
10.7 High-performance liquid chromatographic analysis (HPLC) . 27
10.7.1 General . 27
10.7.2 Chromatographic separation . 27
10.7.3 Detection . 27
10.7.4 Calibration . 28
10.7.5 Measurement of samples. 29
10.7.6 Calculation . 29
11 Performance criteria . 30
12 Precision. 30
13 Test report . 30
Annex A (informative) Examples of instrumental conditions and chromatograms . 31
A.1 Measurement of PAH with GC-MS . 31
A.2 Measurement of PAH with HPLC fluorescence . 37
Annex B (informative) Repeatability and reproducibility data . 40
B.1 Materials used in the interlaboratory comparison study . 40
B.2 Interlaboratory comparison results . 41
Bibliography . 43

oSIST prEN 16181:2011
prEN 16181:2010 (E)
Foreword
This document (prEN 16181:2010) has been prepared by Technical Committee CEN/TC 400 “Project
Committee - Horizontal standards in the fields of sludge, biowaste and soil”, the secretariat of which is held by
DIN.
This document is currently submitted to the CEN Enquiry.
This European Standard is part of a modular horizontal approach in which this document belongs to the
analytical step.
The preparation of this document by CEN is based on a mandate by the European Union (Mandate M/330),
which assigned the development of standards on sampling and analytical methods for hygienic and biological
parameters as well as inorganic and organic determinants, aiming to make these standards applicable to
sludge, treated biowaste and soil as far as this is technically feasible.
Until now, test methods determining properties of materials within the environmental area were prepared in
Technical Committees (TCs) working on specific products/matrices (e. g. soil, waste, sludge). However, it is
understood that many steps within individual test procedures may also be used for the analysis of various
other materials. By careful determination of these steps and selection of specific questions within these steps,
elements of the test procedure can be described in a way that can be used for a variety of matrices and
materials with certain specifications. This optimization is in line with the development among end-users of
standards. A majority of routine environmental analyses are carried out by institutions and laboratories
working under a scope that is not limited to one single environmental matrix but covers a wide variety of
matrices. Availability of standards covering more matrices contributes to the optimization of laboratory
procedures and standard maintenance costs, e. g. costs related to accreditation and recognition.
A horizontal modular approach was developed in the project 'Horizontal'. 'Modular' means that a test standard
developed in this approach concerns a specific step in assessing a property and not the whole ”chain of
measurement” (from sampling to analyses). A beneficial feature of this approach is that individual “modules”
can be replaced by improved ones without jeopardizing the standard “chain”.
The results of the desk study as well as the evaluation and validation studies have been subject to
discussions with all parties concerned in the CEN structure during the development by project 'Horizontal'.
The results of these consultations with interested parties in the CEN structure have been presented to and
discussed in CEN/TC 400.
Based on data from interlaboratory studies and consultations with interested parties within CEN member
bodies, it has been concluded that this draft standard prEN XXXX is acceptable for its intended use and is
ready for CEN enquiry.
It is recognized that standardization in the environmental field in most national standardization bodies is
organized in national standardization committees that mirror the vertical structure of technical committees in
the environmental field in CEN. The present CEN enquiry therefore asks for special attention by the NSBs to
assure that the relevant and interested parties are consulted during the CEN enquiry, i. e. to assure that one
single consolidated enquiry reply on this draft standard prEN XXXX can be presented by the NSB that covers
the entire scope of this draft standard.
oSIST prEN 16181:2011
prEN 16181:2010 (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 are carried out in these matrices in most of the routine laboratories following the
preceding steps 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.
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 prEN WI 00400003).
This document is the result of a desk study "Horizontal European 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 European Standard is (applicable and) validated for several types of matrices as indicated below (see
Annex B for the results of the validation):
Table 1 — Matrices for which this European standard is (applicable and) validated
Matrix Validated for
Sludge Municipal sludge
Biowaste Fresh compost
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prEN 16181:2010 (E)
WARNING — Persons using this European Standard should be familiar with normal laboratory
practice. This European 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 European Standard be
carried out by suitably trained staff.
1 Scope
This European Standard specifies the quantitative determination of 16 polycyclic aromatic hydrocarbons
(PAH) (see Table 2) in sludge and treated biowaste using GC/MS and HPLC-UV-DAD/FLD covering a wide
range of PAH contamination levels (see also annex A).
When using fluorescence detection acenaphthylene cannot be measured.
Table 2 — Polycyclic aromatic hydrocarbons
which can be analysed using this European 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[ah]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) may be ensured for each
individual PAH. This depends on instrument and sample.
oSIST prEN 16181:2011
prEN 16181:2010 (E)
Sludge and treated biowaste may differ in properties and also in the expected contamination levels of PAHs
and presence of interfering substances. These differences make it impossible to describe one general
procedure. This European 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/pressurised liquid extraction.
NOTE Other PAH compounds can also be analysed with this method, provided suitability has been proven.
2 Normative references
The following referenced documents are indispensable for the application 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.
prEN WI00400022, Sludge, treated biowaste and soil — Guidance for sample pretreatment
EN 15308, Characterization of waste — Determination of selected polychlorinated biphenyls (PCB) in solid
waste by using capillary gas chromatography with electron capture or mass spectrometric detection
prEN 15934, Sludge, treated biowaste, soil and waste — Calculation of dry matter by determination of dry
residue or water content
EN ISO 5667-15, Water quality — Sampling — Part 15: Guidance on the preservation and handling of sludge
and sediment samples
EN ISO 16720, Soil quality — Pretreatment of samples by freeze-drying for subsequent analysis
ISO 8466-1, Water quality — Calibration and evaluation of analytical methods and estimation of performance
characteristics — Part 1: Statistical evaluation of the linear calibration curve
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 European Standard, the following terms and definitions apply.
3.1
critical pair
pair of congeners that must be separated to a predefined degree (e. g. R = 0,5) to ensure chromatographic
separation meets minimum quality criteria
[EN 15308:2008]
NOTE see Figure 1.
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prEN 16181:2010 (E)
Key
∆t is the difference in retention times of the two peaks a and b in seconds (s)
Y is the peak width at the base of peak a in seconds (s)
a
Y is the 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.
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prEN 16181:2010 (E)
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 may interfere with the determination. These interferences may
lead to incompletely resolved signals and may, 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 being broader than the corresponding peaks of the reference substance suggest interferences.
Chromatographic separation between dibenzo[ah]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, when necessary, corrected.
Sufficient resolution (e. g. 0,8) between the peaks of benzo[b]fluoranthen and benzo[k]fluoranthen as well as
of benzo[a]pyrene and benzo[e]pyrene shall be set as quality criteria for the capillary column.
Benzo[b]fluoranthen and benzo[j]fluoranthen cannot be separated. Triphenylene may not be completely
separated from benzo[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 PAHs to be determined may
interfere the determination. These interferences may lead to incompletely resolved signals and may,
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 being broader than the corresponding
peaks of the reference substance suggest interferences. This problem may 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 (hexane, acetone,
dichloromethane) may 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 by a broad toluene
peak.
Separation between dibenzo[ah]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 of solid materials,
solvent extracts and solutions of standard PAH.
PAH may 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 may 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 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 are purchased from suppliers specialised in their preparation.
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prEN 16181:2010 (E)
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 shall be followed with respect to all hazards associated with this method.
7 Reagents
7.1 General
All reagents shall be of recognised 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, C H O.
2 4
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 . The anhydrous sodium sulfate shall be kept carefully sealed.
2 4
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
2 3
1)
Basic or neutral, specific surface 200 m /g, activity Super I according to Brockmann .
7.3.1.2 Deactivated aluminium oxide
Deactivated with 10 % water.
Add 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.

1) Brockman Activity Scale : a measure of the percentage of water added to the adsorbent based upon weight/weight
relationships between water and the adsorbent. Grade I corresponds to 0 % water added,
H. Brockmann and H. Schrodder; Ber. Deut. Chem. Ges. 74, 73 (1941)
J. W. Hampson, W. F. Bleam, Thermochimica Acta 288 (1996) 179 189
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prEN 16181:2010 (E)
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. Then allow
cooling in a desiccator and adding 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 2 weeks.
7.3.3 Clean-up using Gel permeation chromatography (GPC)


2)
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 for 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,
-1
pump approximately 2 l of elution mixture 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.
7.3.4 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 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
2)
Bio-Beads is an example of a suitable product available commercially. This information is given for the convenience of

this European Standard and does not constitute an endorsement by CEN of this product.

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prEN 16181:2010 (E)
7.4.2.3 Ultra-pure water, HPLC purity grade.
7.4.2.4 Helium, He, of suitable purity for degasification of solvents.
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.
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prEN 16181:2010 (E)
Table 3 — Native PAH and deuterated PAH
PAH reference substances Internal standard substances deuterated PAHs
Naphthalene (CAS-RN 91-20-3) Naphthalene-D
Acenaphthene (CAS-RN 83-32-9) Acenaphthene-D
Acenaphthylene-D
Acenaphthylene (CAS-RN 208-96-8)
Fluorene (CAS-RN 86-73-7) Fluorene-D
Anthracene (CAS-RN 120-12-7) Anthracene-D
Phenanthrene-D
Phenanthrene (CAS-RN 85-01-8)
Fluoranthene (CAS-RN 206-44-0) Fluoranthene-D
Pyrene (CAS-RN 129-00-0) Pyrene-D
Benz[a]anthracene-D
Benz[a]anthracene (CAS-RN 56-55-3)
Chrysene (CAS-RN 218-01-9) Chrysene-D
Benzo[b]fluoranthene (CAS-RN 205-99-2) Benzo[b]fluoranthene-D
Benzo[k]fluoranthene-D
Benzo[k]fluoranthene (CAS-RN 207-08-9)
Benzo[a]pyrene (CAS-RN 50-32-8) Benzo[a]pyrene-D
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-D
Dibenz[ah]anthracene (CAS-RN 53-70-3) Dibenz[ah]anthracene- D
Benzo[ghi]perylene-D
Benzo[ghi]perylene (CAS-RN 191-24-2)
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 D.C. 20 234 U.S.A or from other
commercial providers.
When highly contaminated samples are analysed, often an aliquot of the extract is used for a further clean-up.
7.5.2 Injection standard
7.5.2.1 GC-MS
A deuterated PAH such as 1-Methylnaphthalene-D , Triphenylene-D and Perylene-D shall be added to
10 12 12
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 %.
oSIST prEN 16181:2011
prEN 16181:2010 (E)
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 PAHs 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 1 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 PAHs 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 about 4 °C protected from light and evaporation. The solutions are stable for
about one year.
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.
oSIST prEN 16181:2011
prEN 16181:2010 (E)
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 2 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
before use.
8.1.1 Sample bottles. Glass or stainless steel or aluminium bottles with glass stopper or screw top and
polytetrafluoroethylene (PTFE) seal. Size in agreement with the amount of sample taken.
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 to 300 strokes per min).
8.1.3 Water bath, adjustable up to 100 °C.
8.1.4 Separating funnels of appropriate volume.
oSIST prEN 16181:2011
prEN 16181:2010 (E)
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, e. g. 27 mm × 100 mm, vertical condensers, e. g. 300 mm, heating device.
8.1.7 Concentrator, Kuderna Danish type.
NOTE Other evaporators, e. g. a rotary evaporator
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