SIST-TS CEN/TS 16182:2012

SLOVENSKI STANDARD
SIST-TS CEN/TS 16182:2012
01-junij-2012
%ODWRREGHODQLELRORãNLRGSDGNLLQWOD'RORþHYDQMHQRQLOIHQRORY13WHU
QRQLOIHQROPRQRHWRNVLODWRYLQGLHWRNVLODWRY]XSRUDERSOLQVNHNURPDWRJUDILMH]
PDVQRVHOHNWLYQRGHWHNFLMR*&06
Sludge treated biowaste and soil - Determination of nonylphenols (NP) and nonylphenol-
mono- and diethoxylates using gas chromatography with mass selective detection (GC-
MS)
Schlamm, behandelter Bioabfall und Boden - Bestimmung von Nonylphenolen (NP) und
Nonylphenol-Mono- und Diethoxylaten mittels Gaschromatographie mit massenselektiver
Detektion (GC-MS)
Boues, bio-déchets traités et sols - Détermination des nonylphénols et nonylphénol-
mono- et di-éthoxylates par chromatographie en phase gazeuse avec détection sélective
de masse (GC-MS)
Ta slovenski standard je istoveten z: CEN/TS 16182:2012
ICS:
13.030.20 7HNRþLRGSDGNL%ODWR Liquid wastes. Sludge
SIST-TS CEN/TS 16182:2012 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------

SIST-TS CEN/TS 16182:2012

---------------------- Page: 2 ----------------------

SIST-TS CEN/TS 16182:2012


TECHNICAL SPECIFICATION
CEN/TS 16182

SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION
February 2012
ICS 13.030.01
English Version
Sludge treated biowaste and soil - Determination of
nonylphenols (NP) and nonylphenol-mono- and diethoxylates
using gas chromatography with mass selective detection (GC-
MS)
Boues, biodéchets traités et sols - Détermination des Schlamm, behandelter Bioabfall und Boden - Bestimmung
nonylphénols et nonylphénol-mono- et di-éthoxylates par von Nonylphenolen (NP) und Nonylphenol-Mono- und
chromatographie en phase gazeuse avec détection Diethoxylaten mittels Gaschromatographie mit
sélective de masse (GC-MS) massenselektiver Detektion (GC-MS)
This Technical Specification (CEN/TS) was approved by CEN on 24 April 2011 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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, Turkey and United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16182:2012: E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Principle .5
4 Interferences .5
4.1 Interferences from sampling .5
4.2 Interferences by GC-MS .6
5 Reagents .6
6 Apparatus .8
7 Sample storage and sample pretreatment .9
7.1 Sample storage .9
7.2 Sample pretreatment .9
8 Procedure .9
8.1 Extraction .9
8.1.1 General .9
8.1.2 Extraction of wet sludge samples . 10
8.1.3 Extraction of freeze-dried sludge samples . 10
8.1.4 Extraction of soil and treated biowaste samples . 10
8.1.5 Extraction of freeze-dried soil and treated biowaste samples . 11
8.2 Concentration (optional) . 11
8.3 Clean-up (optional) . 12
8.4 Derivatization . 12
8.5 Blank test . 12
8.6 GC-MS analysis . 13
8.7 Calibration . 13
8.7.1 General . 13
8.7.2 Initial calibration . 13
8.7.3 Verification of calibration . 14
8.8 Analysis of samples and identification . 14
9 Calculation and expression of results . 14
9.1 General . 14
9.2 Calibration . 15
9.3 Calculation . 15
10 Precision . 16
11 Test report . 16
Annex A (informative) Repeatability and reproducibility data . 17
A.1 Materials used in the interlaboratory comparison study . 17
A.2 Interlaboratory comparison results . 18
Annex B (informative) Example of chromatographic conditions and example of a chromatogram . 19
Bibliography . 21


2

---------------------- Page: 4 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
Foreword
This document (CEN/TS 16182:2012) 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.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
The preparation of this document by CEN is based on a mandate by the European Commission (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.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: 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, Turkey and the United Kingdom.

3

---------------------- Page: 5 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
Introduction
Nonylphenols (NP) are mainly found in the environment as degradation products of nonylphenol
polyethoxylates (NPEO). NPEO have many applications as non-ionic detergents in washing and cleaning
agents.
After use NPEO are degraded by de-ethoxylation, resulting in polyethoxylates with fewer ethoxy-groups.
Nonylphenol-diethoxylates (NP2EO), nonylphenol-monoethoxylates (NP1EO) and nonylphenols (NP) are the
last three products in the degradation chain. Due to their significant presence in sewage sludge, all three
components are included in this Technical Specification.
This Technical Specification is applicable for several types of matrices and validated for municipal sewage
sludge (see also Annex A for the results of the validation).
WARNING — Persons using this Technical Specification should be familiar with usual laboratory
practice. This Technical Specification 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 Technical
Specification be carried out by suitably trained staff.
4

---------------------- Page: 6 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
1 Scope
This Technical Specification specifies a method for the determination of nonylphenols (NP), nonylphenol-
monoethoxylates (NP1EO) and nonylphenol-diethoxylates (NP2EO) in sludge, treated biowaste and soil using
GC-MS.
For sludge a limit of detection of 0,1 mg/kg and for soil and treated biowaste 0,02 mg/kg (expressed as dry
matter) may be achieved.
Lower limits of detection may be achieved by concentrating the extract by solvent evaporation.
NOTE 4-tert-octylphenol can also be analysed with this method.
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.
EN 15934, Sludge, treated biowaste, soil and waste — Calculation of dry matter fraction after determination of
dry residue or water content
EN 16179, Sludge, treated biowaste and soil — Guidance for sample pretreatment
EN ISO 16720, Soil quality — Pretreatment of samples by freeze-drying for subsequent analysis (ISO 16720)
EN ISO 22892, Soil quality — Guidelines for the identification of target compounds by gas chromatography
and mass spectrometry (ISO 22892)
ISO 8466-1, Water quality — Calibration and evaluation of analytical methods and estimation of performance
characteristics — Part 1: Statistical evaluation of the linear calibration function
3 Principle
After pretreatment, the test sample is extracted by shaking with a mixture of acetone and petroleum ether
(1:1). If necessary, interfering compounds are removed from the extract by a clean-up on a suitable column.
The extract is treated with N-methyl-N-(trimethylsilyl)-trifluoracetamide (MSTFA) reagent for the derivatization
(silylation) of the analytes, and subsequently analysed by gas chromatography and mass selective detection
(GC-MS).
Nonylphenols and nonylphenol-mono- and diethoxylates are identified from the GC fingerprint, the relative
retention times and the relative intensities of two diagnostic ions. The quantification is based on an internal
13 13
standard procedure. The internal standards ( C-labelled 4-n-NP and C-labelled 4-n-NP2EO) are taken
through the whole analytical procedure.
4 Interferences
4.1 Interferences from sampling
Use sampling containers of materials (preferably glass or steel) that do not significantly affect the sample
during the contact through sampling and storage. Plastic containers may be used if it has been proven that
they do not significantly affect the sample.
5

---------------------- Page: 7 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
4.2 Interferences by GC-MS
Substances that co-elute with NP, NP1EO or NP2EO and give the same ion(s) may interfere with the
determination. This may have a large influence on the result, since all three analytes are determined from the
sum of a cluster of five to nine chromatographic peaks. It is essential that the interfering peaks are not
included in the calculations. A peak is excluded if the retention times are not the same as expected from the
calibration standard and if the relative peak areas from the two diagnostic ions differ more than 30 % from the
same peak in the calibration standard. Interfering peaks may usually be spotted by comparing the fingerprints
of the sample with the fingerprints of the calibration standard, although the isomer distribution in the
environmental samples may differ from the distribution in the calibration standard.
5 Reagents
5.1 General
All reagents shall be of recognised analytical grade.
The purity of the reagents used shall be checked by running a blank determination as described in 8.5.
5.2 Acetone, C H O.
3 6
5.3 Hexane-like solvent
Any aliphatic hydrocarbon solvent with a boiling point or boiling range between 34 °C and 100 °C may be
applied.
5.4 Anhydrous sodium sulphate, Na SO , powdered.
2 4
Heated for at least 6 h to (550 ± 20) °C, cooled to about 200 °C in the furnace and then to ambient
temperature in a desiccator containing magnesium perchlorate or a suitable alternative. The anhydrous
sodium sulphate shall be kept carefully sealed.
1)
5.5 N-methyl-N-(trimethylsilyl)-trifluoracetamide, C H F NOSi, (MSTFA), CAS-RN 24589-78-4, for
6 12 3
derivatization.
5.6 Isooctane, C H , boiling point 99 °C.
8 17
5.7 Derivatization solution, 5 % MSTFA (5.5) in isooctane (volume fraction).
Dissolve e. g. 1 ml of MSTFA (5.5) in isooctane (5.6) in a 20 ml volumetric flask and make up to volume with
isooctane (5.6).
Store the derivatization solution in a dark place at a temperature of (4 ± 3) °C. The solution is stable for at
least two months.
5.8 Operating gas for gas chromatography with MS-detector
Helium of sufficient purity and in accordance with the manufacturer's specification.
5.9 Nitrogen, N , for solvent evaporation.
2
Nitrogen of sufficient purity.

1) CAS-RN Chemical Abstracts Service Registry Number.
6

---------------------- Page: 8 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
5.10 Standards for calibration
The following standard substances shall be used:
 4-Nonylphenols (NP), mixture of isomers, CAS-RN 84852-15-3;
 4-Nonylphenol monoethoxylates (NP1EO), mixture of isomers, CAS-RN 26027-38-3;
 4-Nonylphenol diethoxylates (NP2EO), mixture of isomers, CAS-RN 20427-84-3.
The two nonylphenol ethoxylates may contain small amounts of other ethoxylates. It is important to check the
purity of all the standards used for calibration.
The standards may be taken from pure compounds or from solutions with a guaranteed concentration.
The standards shall be stored in a freezer at a temperature of (–18 ± 3) °C.
NOTE 1 If 4-tert-octylphenol is included: 4-(1,1,3,3-tetramethylbutyl)phenol, CAS-RN 140-66-9.
NOTE 2 For NP, NP1EO and NP2EO conflicting information about CAS-RN may be found.
5.11 Internal standards
The following internal standard substances may be used:
13 13
 C-labelled 4-n-nonylphenol (4-n-NP), C H -[ C ]H -OH;
9 19 6 4
13
 C-labelled 4-n-nonylphenol-diethoxylate (4-n-NP2EO).
The internal standards shall be stored in a freezer at a temperature of (–18 ± 3) °C.
D -labelled 4-n-nonylphenol or 4-n-nonylphenol (non-labelled) may be used as an alternative internal standard
4
13
to C-labelled 4-n-nonylphenol. 4-n-nonylphenol-diethoxylate (non-labelled) may be used as an alternative
13
internal standard to C-labelled 4-n-nonylphenol-diethoxylate. Non-labelled compounds may only be used if it
is shown that they are not present in the sample.
For ion trap MS deuterated internal standards shall not be used.
5.12 Internal standard solution
Prepare an internal standard solution with the two internal standards in isooctane (5.6). The concentrations
are 20 mg/l for 4-n-NP and 100 mg/l for 4-n-NP2EO.
It is essential that the same internal standard solution is used for calibration standard solutions and for
samples, blank and internal quality control samples.
Store the internal standard solution in a dark place at a temperature of (4 ± 3) °C. The solution is stable for at
least two years, provided that evaporation of solvent is negligible.
5.13 Stock solutions
Prepare individual stock solutions of about 100 mg/l in isooctane (5.6), either from solid standard substances
or from solutions with a certified concentration.
Store the stock solutions in a dark place at a temperature of (4 ± 3) °C. The solutions are stable for at least
2 years, provided that evaporation of solvent is negligible.
7

---------------------- Page: 9 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
5.14 Calibration standard solutions
A mixed calibration standard solution is prepared from the stock solutions (5.13) by diluting the stock solutions
with isooctane (5.6). Internal standard solution (5.12) is added to a concentration of 0,2 mg/l for 4-n-NP and
1,0 mg/l for 4-n-NP2EO. The calibration standards are made to concentrations from 0,01 mg/l to 5 mg/l.
Store the calibration standard solutions in a dark place at a temperature of (4 ± 3) °C. The solutions are stable
for at least two weeks, provided that evaporation of solvent is negligible.
6 Apparatus
6.1 General
All equipment that comes into contact with the sample or extract shall be free from nonylphenols and
nonylphenol ethoxylates. Glassware may be cleaned by heating, at least for 2 h at 450 °C.
6.2 Usual laboratory glassware
6.2.1 Screw cap glass flask with polytetrafluoroethylene (PTFE) seal; volume 100 ml and 250 ml.
6.2.2 Round-bottom flasks, volume 100 ml and 250 ml.
6.2.3 Test tubes and vials
6.3 Shaking device
Reciprocating shaker, with horizontal movement (up to at least 250 strokes per minute).
6.4 Evaporator
2)
Rotary evaporator. Other device like turbo evaporator or Kuderna Danish may be used.
6.5 Clean-up column
Silica column. Commercial columns or freshly prepared columns may be used.
3)
Alternative materials like aluminium oxide or Florisil may be used, provided that a sufficient recovery of the
analytes has been proven.
6.6 Freeze drying apparatus
6.7 Gas chromatograph with mass selective detector equipped with a capillary column: 5 % phenyl-
methyl silicone stationary phase coated onto fused silica or an equivalent chemically bonded phase.
The dimensions should be sufficient to separate the nonylphenols as described below. In general column
length should be 25 m to 50 m. An example of a column is given in Annex B.

2) Kuderna Danish is an example of a suitable product available commercially. This information is given for the
convenience of users of this Technical Specification and does not constitute an endorsement by CEN of this product.

3) Florisil is a trade name for a prepared diatomaceous substance, mainly consisting of anhydrous magnesium silicate.
This information is given for the convenience of users of this Technical Specification and does not constitute an
endorsement by CEN of this product.
8

---------------------- Page: 10 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
The first two peaks in the SIM chromatogram of the nonylphenols are selected as critical pairs for the quality
criteria for the chromatographic system. The resolution shall be sufficiently high, so that the first two peaks in
nonylphenols are baseline separated when measured at ion 207, see Table 2.
7 Sample storage and sample pretreatment
7.1 Sample storage
Store the samples in a dark place in a freezer or in a refrigerator. The samples can be stored up to three
weeks in a freezer at a temperature of (–18 ± 3) °C or up to seven days in the refrigerator at (4 ± 3) °C.
Determine the content of dry matter in the sample according to EN 15934.
NOTE Sludge samples with unusually high amounts of nonylphenol polyethoxylates (NPPEO) relative to the analytes
can only be stored for seven days in the freezer at (–18 ± 3) °C.
7.2 Sample pretreatment
Pretreat the samples according to EN 16179 if not otherwise specified.
Different pretreatment procedures are used for the different matrices. This is presented in Table 1.
Table 1 — Pretreatment methods used prior to nonylphenol analysis
Matrix Freeze drying No drying
(EN ISO 16720)
a
Sludge dry matter > 2 % x x
b
Sludge dry matter < 2 % x
c
Soil x x
Treated biowaste x x
a
Sludge samples with more than 2 % dry matter can be analysed as wet samples, or they can be analysed after freeze-drying.
b
Sludge samples with less than 2 % dry matter can only be analysed after freeze-drying.
c
Soil and treated bio-waste samples can be analysed as wet samples (field-moist samples), or they can be analysed after freeze-
drying.

8 Procedure
8.1 Extraction
8.1.1 General
The wash of organic phase (extraction solvent) with water may be carried out directly in the extraction flask
with the sample present.
Other extraction techniques than described in this Technical Specification, like ultrasonic extraction,
microwave or pressurised liquid extraction may be suitable. If other extraction techniques are applied, the
comparability to the method described in this Technical Specification shall be proven.
9

---------------------- Page: 11 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
8.1.2 Extraction of wet sludge samples
Wet sludge samples are extracted as follows:
 Weigh between 10 g and 50 g of the test sample (depending on dry matter content) and place it in a
100 ml screw cap flask (6.2.1). The sample should preferably contain between 2 g and 3 g dry matter.
 Add 100 µl of internal standard solution (5.12) equal to 2 µg of 4-n-NP and 10 µg of 4-n-NP2EO.
 Add 10 ml of acetone (5.2), close the screw cap and shake thoroughly by hand.
 Add 10 ml of hexane-like solvent (5.3), close the screw cap again and place the flask on the shaking
device (6.3). The flask shall be placed in horizontal position with the movement along the flask.
 Shake for at least 2 h with (250 ± 20) strokes per minute.
 Transfer the organic phase to another 100 ml flask. If an emulsion is present, this shall be included.
 Add water and shake to wash the extract. Use 5 ml of water per ml hexane-like solvent (5.3).
 Transfer the extract (enough for the subsequent analysis) to a test tube (6.2.3) and dry the extract by
adding anhydrous sodium sulfate (5.4).
The extract is now ready for further treatment described in 8.2 to 8.4.
8.1.3 Extraction of freeze-dried sludge samples
Freeze-dried sludge samples are extracted as follows:
 Weigh 2 g to 3 g of the test sample and place it in a 100 ml screw cap flask (6.2.1).
 Add 100 µl of internal standard solution (5.12) equal to 2 µg of 4-n-NP and 10 µg of 4-n-NP2EO.
 Add 5 ml of water (approximately 2 ml per g of dry sample) and shake the sample by hand.
 Add 10 ml of acetone (5.2), close the screw cap and shake thoroughly by hand.
 Add 10 ml of hexane-like solvent (5.3), close the screw cap again and place the flask on a reciprocating
shaker (6.3). The flask shall be placed in horizontal position with the movement along the flask.
 Shake for at least 2 h with (250 ± 20) strokes per minute.
 Transfer the organic phase to another flask. If an emulsion is present, this shall be included.
 Add water and shake to wash the extract. Use 5 ml of water per ml hexane-like solvent (5.3).
 Transfer the extract (enough for the subsequent analysis) to a test tube (6.2.3) and dry the extract by
adding anhydrous sodium sulfate (5.4).
The extract is now ready for further treatment described in 8.2 to 8.4.
8.1.4 Extraction of soil and treated biowaste samples
Soil and treated biowaste samples are usually extracted wet without drying the sample before extraction.
These samples are extracted as follows:
10

---------------------- Page: 12 ----------------------

SIST-TS CEN/TS 16182:2012
CEN/TS 16182:2012 (E)
 Weigh between 20 g and 40 g of the test sample (depending on dry matter content) and place it in a
100 ml screw cap flask (6.2.1). The sample shall contain between 10 g and 20 g dry matter.
 Add 100 µl of internal standard solution (5.12) equal to 2 µg of 4-n-NP and 10 µg of 4-n-NP2EO.
 Add 10 ml of water and shake the sample by hand.
 Add 30 ml of acetone (5.2) to the test sample, close the screw cap and shake thoroughly by hand.
 Add 30 ml of hexane-like solvent (5.3), close the screw cap again and place the flask on a reciprocating
shaker (6.3). The flask shall be placed in horizontal position with the movement along the flask.
 Shake for at least 2 h with (250 ± 20) strokes per minute.
 Transfer the organic phase to a 250 ml flask. If an emulsion is present, this shall be included.
 Add water and shake to wash the extract. Use 5 ml of water per millilitre of hexane-like solvent (5.3).
 Transfer the extract (enough for the subsequent analysis) to a test tube (6.2.3) and dry the extract by
adding anhydrous sodium sulfate (5.4).
The extract is now ready for further treatment described in 8.2 to 8.4.
8.1.5 Extraction of freeze-dried soil and treated biowaste samples
Freeze-dried soil and treated biowaste samples are extracted as follows:
 Weigh 10 g to 20 g of the test sample and place it in a 100 ml screw cap flask (6.2.1).
 Add 100 µl of internal standard solution (5.12) equal to 2 µg of 4-n-NP and 10 µg of 4-n-NP2EO.
 Add 10 ml to 20 ml of water (approximately 1 ml per g of dry sample) and shake the sample by hand.
 Add 20 ml of acetone (5.2), close the screw cap and shake thoroughly by hand.
 Add 20 ml of hexane-like solvent (5.3), close the screw cap again and place the flask on a reciprocating
shaker (6.3). The flask shall be placed in horizontal position with the movement along the flask.
 Shake for at least 2 h with (250 ± 20) strokes per minute.
 Transfer the organic phase to a 100 ml to 250 ml flask. If an emulsion is present, this shall be included.
 Add water and shake to wash the extract. Use 5 ml of water per ml hexane-like solvent (5.3).
 Transfer the extract (enough for the subsequent analy
...