ISO 23256:2023

Water quality — Detection of selected congeners of polychlorinated dibenzo-p-dioxins and polychlorinated biphenyls — Method using a flow immunosensor technique

This document specifies methods and principles for detection of selected congeners of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs) in water and wastewater using a flow immunosensor. The flow immunosensor utilizes antibodies specific to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and 3,3’,4,4’,5-pentachlorobiphenyl (3,3’,4,4’,5-PeCB), which have the highest toxic equivalent factor (TEF) value among the congeners of each of PCDDs and PCBs. The method is applicable to timely monitoring of selected congeners of 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB in water and wastewater to prioritize those for subsequent confirmatory determination. This document specifies practical methods and procedures for sampling, extraction, clean-up, measurement in a flow immunosensor, data processing and validation of measurement results. The combined use of automated instruments for extraction, clean-up, and flow immunosensing can reduce time-consumption and labour-intensity, while providing reproducible precise data. This method can provide the lower limit of quantification (LOQ) for 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB of 28 pg/l and 152 pg/l, respectively at 20 % or less of coefficient variation (CV) depending on sampling, extraction, clean-up and measurement conditions.

Qualité de l'eau — Détection d’une sélection de congénères de dibenzo-p-dioxines polychlorées et de biphényles polychlorés — Méthode utilisant la technique d'immunodétection en flux

General Information

• Status: Published
• Publication Date: 01-Jun-2023
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: ISO/TC 147/SC 2 - Physical, chemical and biochemical methods
• Drafting Committee: ISO/TC 147/SC 2/WG 79 - Flow immunosensor dioxins
• Current Stage: 6060 - International Standard published
• Start Date: 02-Jun-2023
• Due Date: 12-Jul-2022
• Completion Date: 02-Jun-2023

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 23256
First edition
2023-06
Water quality — Detection of selected
congeners of polychlorinated dibenzo-
p-dioxins and polychlorinated
biphenyls — Method using a flow
immunosensor technique
Qualité de l'eau — Détection d’une sélection de congénères de
dibenzo-p-dioxines polychlorées et de biphényles polychlorés —
Méthode utilisant la technique d'immunodétection en flux
Reference number
ISO 23256:2023(E)
© ISO 2023

---------------------- Page: 1 ----------------------
ISO 23256:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
  © ISO 2023 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 23256:2023(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms, definitions, and abbreviated terms . 1
3.1 Terms and definitions . 1
3.2 Abbreviated terms . 3
4 Principle . 4
4.1 Flow immunosensor . 4
4.2 Specific antibody . 5
5 Interferences . 5
6 Reagents . 5
7 Apparatus and materials .7
8 Sampling . 8
8.1 General . 8
8.1.1 General . 8
8.1.2 Bottles and material for sampling . 8
8.1.3 Bottles and material pre-cleaning . 8
8.1.4 Sampling . 8
8.2 Preparation of sample . 8
8.2.1 Extraction . 8
8.2.2 Clean-up. 9
8.2.3 Sample solutions . 9
9 Procedure .9
9.1 General . 9
9.2 Sample set up . 9
9.3 Measuring procedure . 10
10 Data processing .11
10.1 Measurement data . 11
10.2 Calibration curve . 11
10.3 Calculation of measured concentration .12
11 Validation . .13
11.1 General .13
11.2 Accuracy profile . 13
11.3 Range of quantitation . 14
11.4 Limit of detection . 15
11.5 E valuation of data .15
11.6 Quality control . 15
12 Test report .16
Annex A (informative) Specificity of mouse monoclonal antibodies used for the flow
immunosensor.17
Annex B (informative) Example of the automate sample clean-up and concentration device .18
Annex C (informative) Example of the flow immunosensor.20
Annex D (informative) Recoveries, calibration curves, ranges of quantitation of the
standard analytes 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB in drinking water .26
Annex E (informative) Measurement of 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB in river waters .30
iii
© ISO 2023 – All rights reserved

---------------------- Page: 3 ----------------------
ISO 23256:2023(E)
Annex F (informative) Interlaboratory trial of the measurement of 2,3,7,8-TCDD and
3,3’,4,4’,5-PeCB in water samples by the use of flow immunosensor method .33
Bibliography .36
iv
  © ISO 2023 – All rights reserved

---------------------- Page: 4 ----------------------
ISO 23256:2023(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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use
of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed
patent rights in respect thereof. As of the date of publication of this document, ISO had not received
notice of (a) patent(s) which may be required to implement this document. However, implementers are
cautioned that this may not represent the latest information, which may be obtained from the patent
database available at www.iso.org/patents. ISO shall not be held responsible for identifying any or all
such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2,
Physical, chemical and biochemical methods.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
© ISO 2023 – All rights reserved

---------------------- Page: 5 ----------------------
ISO 23256:2023(E)
Introduction
Persistent organic pollutants (POPs) including dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs)
and dioxin-like polychlorinated biphenyls (DL-PCBs) in water and wastewater are analysed by
instrumental methods such as gas chromatography/mass spectrometry (GC-MS) and high-resolution
gas chromatography/ high-resolution mass spectrometry (HRGC/HRMS). These methods are accurate
and precise, but labour-intensive, time-consuming, and costly. Alternatively, immunoassays are used
for monitoring of POPs. These methods have similar sensitivity and selectivity, but are more cost-
effective and able to manage large loads of sample well. The use of immunoassays is suitable for timely
detection of selected congeners of PCDDs and PCBs in water and wastewater in advance of subsequent
confirmatory methods.
Recently, automated immunoassay methods including a flow immunosensor have been developed
for detection of POPs in environmental samples. These methods reduced manual operations such as
pipetting, time-consumption and coefficient of variation. Therefore, practical use of these methods can
play an important role in timely, continuous cost-saving monitoring of water and wastewater in both
developed and developing countries. This method can be applicable to timely continuous monitoring of
selected congeners of dibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs) in water and
wastewater to prioritize those for subsequent confirmatory determination.
vi
  © ISO 2023 – All rights reserved

---------------------- Page: 6 ----------------------
INTERNATIONAL STANDARD ISO 23256:2023(E)
Water quality — Detection of selected congeners of
polychlorinated dibenzo-p-dioxins and polychlorinated
biphenyls — Method using a flow immunosensor technique
WARNING — The PCDDs, PCDFs and PCBs are among the most hazardous chemicals. Therefore, all
work with PCDDs, PCDFs and PCBs require the utmost care; the national safety measures which
correspond to those for hazardous substances shall be strictly adhered to. The responsibility of
the user is to establish appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably qualified staff.
1 Scope
This document specifies methods and principles for detection of selected congeners of polychlorinated
dibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs) in water and wastewater using a
flow immunosensor. The flow immunosensor utilizes antibodies specific to 2,3,7,8-tetrachlorodibenzo-
p-dioxin (2,3,7,8-TCDD) and 3,3’,4,4’,5-pentachlorobiphenyl (3,3’,4,4’,5-PeCB), which have the highest
toxic equivalent factor (TEF) value among the congeners of each of PCDDs and PCBs. The method is
applicable to timely monitoring of selected congeners of 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB in water and
wastewater to prioritize those for subsequent confirmatory determination.
This document specifies practical methods and procedures for sampling, extraction, clean-up,
measurement in a flow immunosensor, data processing and validation of measurement results. The
combined use of automated instruments for extraction, clean-up, and flow immunosensing can reduce
time-consumption and labour-intensity, while providing reproducible precise data. This method can
provide the lower limit of quantification (LOQ) for 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB of 28 pg/l and
152 pg/l, respectively at 20 % or less of coefficient variation (CV) depending on sampling, extraction,
clean-up and measurement conditions.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes and
sampling techniques
ISO 5667-3, Water quality — Sampling — Part 3: Preservation and handling of water samples
ISO 5667-6, Water quality — Sampling — Part 6: Guidance on sampling of rivers and streams
ISO 5667-10, Water quality — Sampling — Part 10: Guidance on sampling of waste water
3 Terms, definitions, and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
1
© ISO 2023 – All rights reserved

---------------------- Page: 7 ----------------------
ISO 23256:2023(E)
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1.1
analyte
selected congeners of polychlorinated dibenzo-p-dioxins and polychlorinated biphenyls which bind to
specific monoclonal antibodies (3.1.2)
Note 1 to entry: Annex A gives information of the specificity of mouse monoclonal antibodies used for the flow
immunosensor.
3.1.2
antibody
class of serum proteins that are induced by exposing to an immunogen and will bind specifically to
antigen/analyte (3.1.3)/(3.1.1) forming an antibody-antigen complex
Note 1 to entry: An antibody with a fluorescent label is used as a secondary antibody for binding to an antibody
specific to antigen/analyte.
3.1.3
antigen
molecule which selectively binds to an antibody (3.1.2)
Note 1 to entry: Antigens are analytes in the case of selected congeners of polychlorinated dibenzo-p-dioxins and
polychlorinated biphenyls.
3.1.4
conjugate
large molecule covalently coupled with a small molecule
Note 1 to entry: An antigen-mimic BSA conjugate is used for coating polymer beads.
3.1.5
cross-reactivity
ability of an antibody (3.1.2) to bind to certain molecules which structurally related to an antigen (3.1.3)
3.1.6
flocculation
physical process of contact and adhesions wherein the aggregates form larger-size clusters called flocs
being excluded from suspension for water treatment
3.1.7
flow immunosensor
immunosensor based on a kinetic exclusion assay (3.1.11) using a monoclonal antibody (3.1.2) in a
heterogeneous assay system for rapid detection of an analyte (3.1.1) in a number of samples
Note 1 to entry: The system of a flow immunosensor is operated automatically according to a program.
3.1.8
IgG
immunoglobulin-G
antibody molecule, consisting of two identical heavy(H)-chains and two identical light(L)-chains held
together with disulfide bonds
Note 1 to entry: Both H- and L-chains consist of the variable and constant regions. An antigen binding site is
presented in the variable region.
2
  © ISO 2023 – All rights reserved

---------------------- Page: 8 ----------------------
ISO 23256:2023(E)
3.1.9
immunoassay
immunochemical detection procedure based on specific antibody-antigen (3.1.2)-(3.1.3) binding theory
often using a tracer for the detection of a free or bound antibody
Note 1 to entry: In a heterogeneous immunoassay, one of immunoreagents is immobilized on a solid support and
requires a washing step to separate the bound and free immunoreagents.
3.1.10
inhibition concentration
IC
C
i50
analyte concentration which reduces the measuring signal of the zero standard (3.1.14), in the case of
50 % inhibition of the zero standard that expresses 50 values
3.1.11
kinetic exclusion assay
binding between antibody (3.1.2) and antigen (3.1.3) that reaches equilibrium in solution, and then
the three species such as the specific antibody, the antigen and the antibody-antigen complex that are
present
Note 1 to entry: A kinetic exclusion assay measures the concentration of the free antibody without perturbing
the equilibrium. The format of the assay is to measure a free specific antibody with a label.
3.1.12
monoclonal antibody
antibody (3.1.2) population, possessing identical selectivity and affinity produced by a single antibody–
producing cell line
Note 1 to entry: Monoclonal antibodies specific to selected congeners of polychlorinated dibenzo-p-dioxins and
polychlorinated biphenyls are prepared for measurement of these analytes.
3.1.13
procedure blank
solution prepared in the laboratory, using reagent water or other blank matrix
3.1.14
zero standard
analyte-free standard (blank) which is used for calibration
3.2 Abbreviated terms
BSA bovine serum albumin
CV coefficient of variation
DMSO dimethyl sulfoxide
GC-MS gas chromatography/mass spectrometry
HpCB heptachlorobiphenyl
HpCDD heptachlorodibenzo-p-dioxin
HpCDF heptachlorodibenzofuran
HRGC high-resolution gas chromatography
HRMS high-resolution mass spectrometry
3
© ISO 2023 – All rights reserved

---------------------- Page: 9 ----------------------
ISO 23256:2023(E)
HxCB hexachlorobiphenyl
HxCDD hexachlorodibenzo-p-dioxin
HxCDF hexachlorodibenzofuran
IgG immunoglobulin G
LED light emitting diode
LLE liquid-liquid extraction
LOD limit of detection
LOQ limit of quantification
OCDD octachlorodibenzo-p-dioxin
OCDF octachlorodibenzofuran
PBS phosphate-buffered saline
PCDD polychlorinated dibenzo-p-dioxin
PCDF polychlorinated dibenzofuran
PCB polychlorinated biphenyl
PeCB pentachlorobiphenyl
PeCDD pentachlorodibenzo-p-dioxin
PeCDF pentachlorodibenzofuran
POP persistent organic pollutant
PTFE polytetrafluoroethylene
SD standard deviation
SPE solid phase extraction
TCB tetrachlorobiphenyl
TCDD tetrachlorodibenzo-p-dioxin
TCDF tetrachlorodibenzofuran
TEF toxic equivalent factor
4 Principle
4.1 Flow immunosensor
A flow immunosensor based on kinetic exclusion assay principles consists of a flow cell containing
a capture reagent immobilized on a solid phase matrix such as azlactone, sepharose, polymethyl
methacrylate, polystyrene, or the like, along with an antibody specific for the analyte and a fluorescent
label used for detection. In many cases, the fluorescent label is attached to a second anti-species
antibody that recognizes and binds to the analyte specific antibody. In practice, the capture reagent
used on the solid phase is frequently either the analyte itself, an analogue of the analyte or a protein
4
  © ISO 2023 – All rights reserved

---------------------- Page: 10 ----------------------
ISO 23256:2023(E)
conjugate of either the analyte or an analogue. To perform the assay, a suitable sample (which can be
extracted and/or concentrated from a large environmental sample) is mixed with the specific antibody
and the secondary antibody (C.2.6) with a fluorescent label, and then incubated for a period of time to
allow binding to occur. After incubation, the antibody mixture is flowed over the solid phase matrix and
free (not bound to analyte) antibody bound to the secondary antibody with a fluorescent label from the
solution binds to the solid phase capture reagent. A fluorescent signal is measured from the captured
antibody. The largest fluorescent signal occurs when there is zero analyte present and the presence of
[1][3]
analyte results in reduced signal levels .
4.2 Specific antibody
Specific antibodies (IgG) are selected for measurement of analytes in a flow immunosensor. Monoclonal
anti-2,3,7,8-TCDD antibody and anti-3,3’,4,4’,5-PeCB antibody show the highest affinity to 2,3,7,8-TCDD
and 3,3’,4,4’,5-PeCB, respectively as shown in Annex A. Each of these congeners is known to be present
in the environment and has the highest TEF value among the congeners of each of PCDDs and PCBs.
Thus, both antibodies are used as the representatives for detection of selected congeners of PCDDs
and PCBs in water and wastewater using a flow immunosensor. When antibodies specific for other
congeners or other related compounds (e.g. PCDFs) are available, the application of the immunosensor
described in 4.1 are readily expanded for detection of those related compounds or congeners.
5 Interferences
PCDDs, PCBs and other dioxin-like compounds are hydrophobic and largely present in water and
wastewater as adsorbed on solid particles. When these compounds are spiked into water samples
in containers, these compounds are quickly adsorbed on the surface of containers. The use of either
inappropriate sampling devices or a sampling flask, or both, can influence the test result because of
the possible adsorption of these compounds leading to false-negative results. On the other hand, these
compounds can be released into the sample from sampling flasks, especially when wares used are
contaminated with these compounds, and false-positive results can be generated. If filtered samples
are tested in order to remove the sample solid particles, the dioxins and dioxin-like compounds which
are adsorbed on particles cannot be detected.
Measurement conditions, for instance pH or sample components such as hormic acids, salinity and
solvents influencing the detection which so-called matrix effects can interfere with antibody binding.
The interference of matrix effects shall be assessed by spiking samples or reference samples with
known amounts of the analyte. In any case, extraction and clean-up are necessary to avoid interference.
In addition, air bubbles in measuring solutions and in a flow cell interfere not only antibody binding
but also fluorescence detection. Therefore, care shall be taken when preparing measuring solutions,
particularly by stirring gently at a room temperature.
Monoclonal antibodies specific to antigen/analyte are used for a flow immunosensor as shown in
Annex A. These antibodies cross-react against structurally related compounds and also bind to
non-specific compounds to show matrix effects. The extracted samples are each submitted to clean-
up process in an automate sample clean-up and concentration device as shown in Annex B, in which
a multiphase silica gel column works to eliminate polynuclear aromatic hydrocarbons and sulfur
compounds. In addition, the clean-up samples are also submitted to HRGC/HRMS analysis to confirm
the removal of matrices.
6 Reagents
Use reagents with a high purity for instance the grade “for residue analysis”. Information on type and
origin of antibodies as well as the cross-reactivity are stated in Annex A. Information on storage and
stability of the used reagents shall be requested according to the information of the supplier. If sensitivity
in a flow immunosensor is lower than expected, analyte-interfering substances such as matrices and
air bubbles shall be removed from samples and measuring solutions by suitable procedures.
5
© ISO 2023 – All rights reserved

---------------------- Page: 11 ----------------------
ISO 23256:2023(E)
6.1 Water, complying with grade 3 as defined in ISO 3696.
1)
For instance, a Milli-Q water is recommended for the preparation of a buffer and a regeneration
solution.
6.2 Sodium chloride, NaCl, >995 g/kg purity.
6.3 Potassium chloride, KCl, >990 g/kg purity.
6.4 Potassium dihydrogen phosphate, KH PO , >995 g/kg purity.
2 4
6.5 Sodium hydroxide, NaOH >970 g/kg purity.
6.6 Disodium hydrogen phosphate dodecahydrate, Na HPO ·12H O, >980 g/kg purity.
2 4 2
6.7 Organic solvents
Solvents used for spiking, extraction, clean-up and measurement in the following:
6.7.1 Propanone (acetone), >995 ml/l purity.
6.7.2 Toluene, >990 ml/l purity.
6.7.3 Hexane, >960 ml/l purity.
6.7.4 Dimethyl sulfoxide (DMSO), >995 ml/l purity.
6.7.5 Ethanol, >995 ml/l purity.
6.8 Standard solution.
The standard acetone solutions of 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB are each diluted with acetone for
spiking into a 3 l or more water sample and for confirmation of spiking concentration by HRGC/HRMS
analysis. For preparation of sample solutions, a part of each acetone solution of both standard chemicals
is diluted with DMSO.
6.9 Flocculant.
A flocculant consisting of activated charcoal powder, poly aluminium chloride, silica gel and sodium
carbonate is added into a water sample adjusted to pH 7 to pH 8 at a rate of 0,1 g/l. Then, flocs formed
are collected by filtration with a 0,45 µm of pore size of a glass fibre filter and then air-dried overnight
[4]
for extraction .
6.10 Phosphate-buffered saline.
Prepare 50 mmol/l of PBS at pH 7,4 by dissolving 2,9 g of Na HPO ·12H O, 0,2 g of KH PO , 8,0 g of NaCl
2 4 2 2 4
and 0,2 g of KCl in 1 000 ml of water (6.1).
Milli-Q is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does
1)
not constitute an endorsement by ISO of this product.

6
  © ISO 202
...

ISO/FDIS 23256:2023(E)
ISO/TC 147/SC
Date:
ISO/DIS 23256:2022(E)
TC /SC /2/WG 79
Secretariat: DIN
Water quality — Detection of selected congeners of polychlorinated dibenzo-p-
dioxins and polychlorinated biphenyls — Method using a flow immunosensor
technique
First edition
Date 2022-09-12: 2023-01-04

---------------------- Page: 1 ----------------------
ISO/FDIS 23256:2023(E)
© ISO 20222023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
Email: [e-mail]
Website: www.iso.org
Published in Switzerland.
© ISO 2023 – All rights reserved
ii

---------------------- Page: 2 ----------------------
ISO/FDIS 23256:2023(E)
Contents Page
Foreword . iv
Introduction . iv
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and abbreviated terms . 2
4 Principle . 5
5 Interferences . 5
6 Reagents . 6
7 Apparatus and materials . 9
8 Sampling . 9
9 Procedure . 11
10 Data processing . 13
11 Validation . 15
12 Test report . 17
Annex A (informative) Specificity of mouse monoclonal antibodies used for the flow
immunosensor . 18
Annex B (informative) Example of the automate sample clean-up and concentration device . 20
Annex C (informative) Example of the flow immunosensor . 22
Annex D (informative) Recoveries, calibration curves, ranges of quantitation of the standard
analytes 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB in a drinking water . 27
Annex E (informative) Measurement of 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB in river waters . 31
Annex F (informative) Interlaboratory trial of the measurement of 2,3,7,8-TCDD and
3,3’,4,4’,5-PeCB in water samples by the use of flow immunosensor method . 35
Bibliography . 38

© ISO 2023 – All rights reserved
iii

---------------------- Page: 3 ----------------------
ISO/FDIS 23256:2023(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 of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the World
Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2,
Physical, chemical and biochemical methods.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
© ISO 2023 – All rights reserved
iv

---------------------- Page: 4 ----------------------
ISO/FDIS 23256:2023(E)
Introduction
Persistent organic pollutants (POPs) including dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and
dioxin-like polychlorinated biphenyls (DL-PCBs) in water and wastewater are analysed by instrumental
methods such as GC-MS and HRGC/HRMS. These methods are accurate and precise, but labour-intensive,
time-consuming, and costly. Alternatively, immunoassays are used for monitoring of POPs. These
methods have similar sensitivity and selectivity, but are more cost-effective and able to manage large
loads of sample well. The use of immunoassays is suitable for timely detection of selected congeners of
PCDDs and PCBs in water and wastewater in advance of subsequent confirmatory methods.
Recently, automated immunoassay methods including a flow immunosensor have been developed for
detection of POPs in environmental samples. These methods reduced manual operations such as
pipetting, time-consumption and coefficient of variation. Therefore, practical use of these methods can
play an important role in timely, continuous cost-saving monitoring of water and wastewater in both
developed and developing countries. This method can be applicable to timely continuous monitoring of
selected congeners of PCDDs and PCBs in water and wastewater to prioritize those for subsequent
confirmatory determination.
© ISO 2023 – All rights reserved
v

---------------------- Page: 5 ----------------------
FINAL DRAFT INTERNATIONAL STANDARDI ISO/FDIS 23256:2023(E)

Water quality — Detection of selected congeners of
polychlorinated dibenzo-p-dioxins and polychlorinated
biphenyls — Method using a flow immunosensor technique
WARNING — The PCDDs, PCDFs and PCBs are among the most hazardous chemicals. Therefore, all work
with PCDDs, PCDFs and PCBs require the utmost care; the national safety measures which correspond to
those for hazardous substances shall be strictly adhered to. The responsibility of the user is to establish
appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document be
carried out by suitably qualified staff.
1 Scope
This document specifies methods and principles for detection of selected congeners of polychlorinated
dibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs) in water and wastewater using a flow
immunosensor. The flow immunosensor utilizes antibodies specific to 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB,
which have the highest toxic equivalent factor (TEF) value among the congeners of each of PCDDs and
PCBs. The method is applicable to timely monitoring of selected congeners of 4,3,7,8--TCDD and
3,3’,4,4’,5--PeCB in water and wastewater to prioritize those for subsequent confirmatory determination.
This document specifies practical methods and procedures for sampling, extraction, clean--up,
measurement in a flow immunosensor, data processing and validation of measurement results. The
combined use of automated instruments for extraction, clean--up, and flow immunosensing can reduce
time-consumption and labour-intensity, while providing reproducible precise data. This method can
provide the lower limits of quantifications [LOQ] for 2, 3, 7, 8--TCDD and 3,3’,4,4’,5--PeCB of 28 pg/l and
152 pg/l, respectively at 20 % or less of coefficient variation (CV) depending on sampling, extraction,
clean-up, and measurement conditions.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 5667--1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes and
sampling techniques
ISO 5667--3, Water quality — Sampling — Part 3: Preservation and handling of water samples
ISO 5667--6, Water quality — Sampling — Part 6: Guidance on sampling of rivers and streams
ISO 5667--10, Water quality — Sampling — Part 10: Guidance on sampling of waste water
© ISO 2023 – All rights reserved
1

---------------------- Page: 6 ----------------------
ISO/FDIS 23256:2023(E)
3 Terms, definitions, and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1.1
analyte
selected congeners of PCDDspolychlorinated dibenzo-p-dioxins and PCBspolychlorinated biphenyls
which bind to specific monoclonal antibodies (3.1.2)
Note 1 to entry: Annex A gives information of the specific of mouse monoclonal antibodies used for the flow
immunosensor.
3.1.2
antibody
class of serum proteins that are induced by exposing to an immunogen and will bind specifically to
antigen/analyte (3.1.3/)/(3.1.1) forming an antibody-antigen complex
Note 1 to entry: An antibody with a fluorescent label is used as a secondary antibody for binding to an antibody
specific to antigen/analyte.
3.1.3
antigen
molecule which selectively binds to an antibody (3.1.2)
Note 1 to entry: Antigens are analytes in the case of selected congeners of PCDDs and PCBspolychlorinated dibenzo-
p-dioxins and polychlorinated biphenyls.
3.1.4
conjugate
large molecule covalently coupled with a small molecule
Note 1 to entry: An antigen-mimic BSA conjugate is used for coating polymer beads.
3.1.5
cross-reactivity
ability of an antibody (3.1.2) to bind to certain molecules which structurally related to an antigen (3.1.3)
[SOURCE: ISO 15089:2000, 3.9, Note 1]
3.1.6
flocculation
physical process of contact and adhesions wherein the aggregates form larger-size clusters called flocs
being excluded from suspension for water treatment
3.1.7
flow immunosensor
© ISO 2023 – All rights reserved
2

---------------------- Page: 7 ----------------------
ISO/FDIS 23256:2023(E)
immunosensor based on a kinetic exclusion assay (3.1.11) using a monoclonal antibody (3.1.2) in a
heterogeneous assay system for rapid detection of an analyte (3.1.1) in a number of samples
Note 1 to entry: The system of a flow immunosensor is operated automatically according to a program.
3.1.8
IgG
immunoglobulin-G
antibody molecule, consisting of two identical heavy(H)-chains and two identical light(L)-chains held
together with disulfide bonds
Note 1 to entry: Both H- and L-chains consist of the variable and constant regions. An antigen binding site is
presented in the variable region.
3.1.9
immunoassay
immunochemical detection procedure based on specific antibody--antigen (3.1.2-)-(3.1.3) binding theory
often using a tracer for the detection of a free or bound antibody (3.1.2)
Note 1 to entry: In a heterogeneous immunoassay, one of immunoreagents is immobilized on a solid support and
requires a washing step to separate the bound and free immunoreagents.
3.1.10
inhibition concentration
analyte concentration which reduces the measuring signal of the zero standard (analyte-free standard
(blank)),3.1.14), in the case of 50 % inhibition concentration 50 [IC 50], 50% of the zero standard that
expresses 50 values
[SOURCE: ISO 15089:2000, 3.18]
3.1.11
kinetic exclusion assay
binding between antibody (3.1.2) and antigen (3.1.3) reaches equilibrium in solution, and then three
species such as the specific antibody (3.1.2),, the antigen (3.1.3) and the antibody-antigen complex are
present
Note 1 to entry: A kinetic exclusion assay measures the concentration of the free antibody without perturbing the
equilibrium. The format of the assay is to measure a free specific antibody with a label.
3.1.12
monoclonal antibody
antibody (3.1.2) population, possessing identical selectivity and affinity produced by a single antibody–
producing cell line
Note 1 to entry: Monoclonal antibodies specific to selected congeners of PCDDs and PCBspolychlorinated dibenzo-
p-dioxins and polychlorinated biphenyls are prepared for measurement of these analytes.
3.1.13
procedure blank
solution prepared in the laboratory, using reagent water or other blank matrix
3.1.14
zero standard
analyte-free standard (blank) which is used for calibration
© ISO 2023 – All rights reserved
3

---------------------- Page: 8 ----------------------
ISO/FDIS 23256:2023(E)
3.2 Abbreviated terms
BSA bovine serum albumin
CV coefficient of variation
DMSO dimethyl sulfoxide
GC-MS gas chromatography/mass spectrometry
HRGC high-resolution gas chromatography
HRMS high-resolution mass spectrometry
IgG immunoglobulin G
LED light emitting diode
LLE liquid-liquid extraction
LOD limit of detection
LOQ limit of quantification
PBS phosphate-buffered saline
PCDD polychlorinated dibenzo-p-dioxin
PCDF polychlorinated dibenzofuran
PCB polychlorinated biphenyl
POP persistent organic pollutant
PTFE polytetrafluoroethylene
SD standard deviation
SPE solid phase extraction
TEF toxic equivalent factor
4 Principle
4.1 Flow immunosensor
A flow immunosensor based on kinetic exclusion assay principles (3.1.11) consists of a flow cell
containing a capture reagent immobilized on a solid phase matrix such as azlactone, sepharose,
polymethyl methacrylate, polystyrene, or the like, along with an antibody specific for the analyte and a
fluorescent label used for detection. In many cases, the fluorescent label is attached to a second-anti-
species antibody that recognizes and binds to the analyte specific antibody. In practice, the capture
reagent used on the solid phase is frequently either the analyte itself, an analogue of the analyte, or a
protein conjugate of either the analyte or an analogue. To perform the assay a suitable sample (which can
be extracted and/or concentrated from a large environmental sample) is mixed with the specific antibody
and the secondary antibody (C.2.6) with a fluorescent label, and then incubated for a period of time to
allow binding to occur. After incubation, the antibody mixture is flowed over the solid phase matrix and
free (not bound to analyte) antibody bound to the secondary antibody with a fluorescent label from the
solution binds to the solid phase capture reagent. A fluorescent signal is measured from the captured
antibody. The largest fluorescent signal will occur when there is zero analyte present and the presence
[1][3]
of analyte results in reduced signal levels .
© ISO 2023 – All rights reserved
4

---------------------- Page: 9 ----------------------
ISO/FDIS 23256:2023(E)
4.2 Specific antibody
Specific antibodies (IgGsIgG) are selected for measurement of analytes in a flow immunosensor.
Monoclonal anti-2,3,7,8--TCDD antibody and anti-3,3’,4,4’,5--PeCB antibody show the highest affinity to
2,3,7,8--TCDD and 3,3’,4,4’,5--PeCB, respectively as shown in Annex A. Each of these congeners is known
to be present in the environment and has the highest TEF value among the congeners of each of PCDDs
and PCBs. Thus, both antibodies are used as the representatives for detection of selected congeners of
PCDDs and PCBs in water and wastewater using a flow immunosensor. When antibodies specific for other
congeners or other related compounds (e.g. PCDFs) are available, the application of the immunosensor
described in 4.1 are readily expanded for detection of those related compounds or congeners.
5 Interferences
PCDDs, PCBs and other dioxin--like compounds are hydrophobic and largely present in water and
wastewater as adsorbed on solid particles. When these compounds are spiked into water samples in
containers, these compounds are quickly adsorbed on the surface of containers. The use of inappropriate
sampling devices and/or sampling flask can influence the test result because of the possible adsorption
of these compounds leading to false-negative results. On the other hand, these compounds can be
released into the sample from sampling flasks, especially when wares used are contaminated with these
compounds, and false-positive results can be generated. If filtered samples are tested in order to remove
the sample solid particles, the dioxins and dioxin-like compounds which are adsorbed on particles can
notcannot be detected.
Measurement conditions, for instance pH or sample components such as hormic acids, salinity and
solvents influencing the detection which so-called matrix effects can interfere with antibody binding. The
interference of matrix effects shall be assessed by spiking samples or reference samples with known
amounts of the analyte. In any case, extraction and clean-up are necessary to avoid interference. In
addition, air bubbles in measuring solutions and in a flow cell interfere not only antibody binding but also
fluorescence detection. Therefore, it shall be taken care to prepare measuring solutions particularly by
stirring gently at a room temperature.
Monoclonal antibodies specific to antigen/analyte are used for a flow immunosensor as shown in
Annex A. These antibodies cross--react against structurally related compounds and also bind to non--
specific compounds to show matrix effects. The extracted samples are each submitted to clean--up
process in an automate sample clean-up and concentration device as shown in Annex B, in which a
multiphase silica gel column works to eliminate polynuclear aromatic hydrocarbons and sulfur
compounds. In addition, the clean-up samples are also submitted to HRGC/HRMS analysis to confirm
removal of matrices
6 Reagents
Use reagents with a high purity for instance the grade “for residue analysis”. Information on type and
origin of antibodies as well as the cross-reactivity are stated in Annex A. Information on storage and
stability of the used reagents shall be requested according to the information of the supplier. If sensitivity
in a flow immunosensor is lower than expected, analyte--interfering substances such as matrices and air
bubbles shall be removed from samples and measuring solutions by suitable procedures.
6.1 Water, complying with grade 3 as defined in ISO 3696.
For instance, a Milli-Q water is recommended for preparation of buffer and regeneration solution.
6.2 Sodium chloride, NaCl, > 995 g/kg purity.
6.3 Potassium chloride, KCl, > 990 g/kg purity.
© ISO 2023 – All rights reserved
5

---------------------- Page: 10 ----------------------
ISO/FDIS 23256:2023(E)
6.4 Sodium hydroxide, NaOH, > 970 g/kg purity.
6.5 Monosodium phosphate, NaH PO , > 980 g/kg purity.
2 4
6.6 Sodium dihydrogen phosphate dodecahydrate, Na HPO ·12H O, >980 g/kg purity.
2 4 2
6.7 Organic solvents
Solvents used for spiking, extraction, clean--up, and measurement in the following:
6.87.1 Propanone (acetone), >995 ml/l purity.
6.97.2 Toluene, >990 ml/l purity.
6.107.3 Hexane, >960 ml/l purity.
6.117.4 Dimethyl sulfoxide, DMSO, >995 ml/l purity.
6.127.5 Ethanol, >995 ml/l purity.
6.138 Standard solution.
The standard acetone solutions of 2,3,7,8--TCDD and 3,3’,4,4’,5--PeCB are each diluted with acetone for
spiking into a 3 l or more water sample and for confirmation of spiking concentration by HRGC/HRMS
analysis. For preparation of sample solutions, a part of each acetone solution of both standard chemicals
is diluted with DMSO.
6.149 Flocculant.
A flocculant consisting of activated charcoal powder, poly aluminium chloride, silica gel and sodium
carbonate is added into a water sample adjusted to pH 7 to pH 8 at a rate of 0,1 g/l. Then, flocs formed
are collected by filtration with a 0,45 µm of pore size of a glass fibre filter and then air-dried overnight
[4]
for extraction .
6.1510 Phosphate-buffered saline.
Prepare 50 mmol/l of PBS at pH 7,4 by dissolving 2,9 g of Na HPO ·12H O, 0,2 g of KH PO , 8,0 g of NaCl
2 4 2 2 4
and 0,2 g of KCl in 1 000 ml of water (6.1).
6.1611 Sample buffer.
Used for sample preparation, with PBS (6.15) containing 1 g/l of BSA and 0,2 g/l of NaN , passed through
3
a 0,45 μm of pore size of a polyvinylidene difluoride filter. The buffer prepared in a glass bottle is kept in
a refrigerator.
6.1712 Measuring buffer.
PBS containing 1 g/l of BSA, 0,2 g/l of NaN and 55 g/l of DMSO, passed through a 0,45 μm polyvinylidene
3
difluoride filter. The buffer in a glass bottle is kept in a refrigerator.
6.1813 Correcting solution.
Prepare 0,6 ng/ml of 2,3,7,8--TCDD in DMSO and 1,6 ng/ml of 3,3’,4,4’,5--PeCB in DMSO by the dilution
of each of the standard chemicals in DMSO. The solution prepared in a glass bottle is kept in a refrigerator.
An antigen-mimic, 3-[-[6-(-(2,4,5--trichlorophenoxy) hexanoylaminol] propionic acid, is also used as a
reference at a concentration of 9,5 ng/l in DMSO.
© ISO 2023 – All rights reserved
6

---------------------- Page: 11 ----------------------
ISO/FDIS 23256:2023(E)
6.1914 Regeneration solution.
Prepare 1 g/l of NaOH (6.4) and 55 g/l of DMSO in water passed through a 0,45 μm polyvinylidene
difluoride filter. The solution prepared in a glass bottle is kept in refrigerator.
6.2015 Cleaning solution, Prepare 20 ml/l ethanol (volume fraction) in a glass bottle.
© ISO 2023 – All rights reserved
7

---------------------- Page: 12 ----------------------
ISO/FDIS 23256:2023(E)
6.2116 Antibody solution.
Antibody solution is prepared to mix with each antibody which is bind specifically to 2,3,7,8--TCDDs or
3,3’,4,4’,5--PeCB and the second with a fluorescent label as a secondary antibody (C.2.6) in sample
buffer (6.16). A fluorescent label should have a high intensity of emission and absorption at certain
wavelength which can be detected by a photodetector of an immunosensor.
1
Anti-2,3,7,8--TCDD antibody solution (D48 kit ) contains 0,12 µg/ml IgG of anti--2,3,7,8--TCDD antibody
and 0,06 µg/ml of secondary antibody in sample buffer (6.16). Anti--3,3’,4,4’,5--PeCB antibody solution
2
[P126 kit ] contains 0,10 µg IgG/ml of anti-3,3',4,4',5--PeCB antibody and 0,05 µg/ml of secondary
antibody (C.2.6.)) in sample buffer. D48 kit and P126 kit are each used for detection of 2,3,7,8--TCDD and
3,3’4,4’,5--PeCB, respectively. Each of both solutions in a glass bottle is covered with an aluminium foil
for protection against light and kept in a refrigerator.
7 Apparatus and materials
All water sample containers are rinsed with acetone, toluene, and hexane in turn, and drydried prior to
[5]
use. Apparatus usingused for extraction, clean up and concentration are referred to in ISO 18073 and
[6]
ISO 17858 .
7.1 Flow immunosensor (4.1)).
7.2 Vial, add a containing mixed antibody solution (6.2116) and either samples or correcting
solution (6.1813) and then mix gently.
7.3 Flow cell.
Stuffing with polymer beads coated with the antigen-mimic BSA conjugate (C.2.7) in a column. There are
two kinds of flow cell, a disassembly type and assembly types. A disassembly type flow cell is supplied
pre-filled with polymer beads coated with the antigen-mimic BSA conjugate (C.2.7) and the entire flow
cell is removed and replaced after several measurements. For an assembly types, the flow cell remains in
service much longer and the beads are automatically filled for each measurement.
8 Sampling
8.1 General
8.1.1 Introduction
((Remark SC 2 CM: After having accepted the changes in the track change version, all subclause
numbers level 3 appear not bold in Clause 8; no idea why not))
8.1.1 General
This document describes specific requirements for the sampling and samples with respect to the
determination of dioxins or dioxin-like compounds in water samples. These compounds are hydrophobic
and are present in water and wastewater as adsorbed on solid particles as well as the surface of wares

1
D48 kit is the trade name a product supplied by Seeds Tec Co., Ltd. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products
may be used if they can be shown to lead to the same results.
2
P126 kit is the trade name of a product supplied by Seeds Tec Co., Ltd. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
© ISO 2023 – All rights reserved
8

---------------------- Page: 13 ----------------------
ISO/FDIS 23256:2023(E)
used. Thus, collection, extraction, and clean-up procedures suitable for these compounds are requested.
For general information about sampling and samples, ISO 5667--1, ISO 5667--3, ISO 5667--6 and
ISO 5667--10 shall be considered.
8.1.2 Bottles and material for sampling
Use clean glass bottles preferably with polytetrafluoroethylene (PTFE) -lined caps. To avoid
photodegradation of compounds of interest, preferably use amber glass bottles. If transparent glass
bottles are used, wrap the bottles in aluminium foil or store them in a dark container.
8.1.3 Bottles and material pre-cleaning
After the routine cleaning procedure, additionally clean bottles and bottles and caps as follows: rinse the
clean bottles and the caps three times with a minimum amount of acetone. Let the residual acetone
evaporate (e.g. drying oven). Close the bottles immediately after drying. Rinse all glassware, spatulas etc.
getting in contact with the sample three times with a minimum amount of acetone. Let the residual
acetone evaporate.
8.1.4 Sampling
Use a 10 l bucket made from stainless steel for collection of water samples including drinking waters and
river waters. A 3 l or more water sample is added into a 3,8 l of amber bottle and adjusted to pH 7 to pH 8
with Na CO . For river waters, surface water is collected at a central area of a stream by the use of a 10 l
2 3
of stainless bucket. A 3 l or more river water is added into a 3,8 l amber bottle, adjusted to pH 7 to pH 8
with Na CO , and stored in a cold and dark room at 4 °C to 25 °C.
2 3
8.2 Preparation of sample
8.2.1 Extraction
For measurement of a pg/l LOQ level of dioxins and dioxin-like compounds in water samples, a 3 l or
more water sample is submitted to extraction with suitable solvents such as dichloromethane and
[4]
toluene, liquid-liquid extraction (LLE), solid phase extraction (SPE)LLE, SPE or flocculation and
3
®
Soxhlet extraction.
For the purpose of efficient collection of extremely low concentrations of dioxins and dioxin--like
compounds in a large volume of water samples, a flocculant (6.14) should be used. A 300 mg of flocculant
in 3 ml of a hexane-rinse
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 23256
ISO/TC 147/SC 2
Water quality — Detection of selected
Secretariat: DIN
congeners of polychlorinated dibenzo-
Voting begins on:
2023-01-19 p-dioxins and polychlorinated
biphenyls — Method using a flow
Voting terminates on:
2023-03-16
immunosensor technique
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 SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 23256:2023(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. © ISO 2023

---------------------- Page: 1 ----------------------
ISO/FDIS 23256:2023(E)
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 23256
ISO/TC 147/SC 2
Water quality — Detection of selected
Secretariat: DIN
congeners of polychlorinated dibenzo-
Voting begins on:
p-dioxins and polychlorinated
biphenyls — Method using a flow
Voting terminates on:
immunosensor technique
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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.
RECIPIENTS OF THIS DRAFT ARE INVITED TO
ISO copyright office
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
CP 401 • Ch. de Blandonnet 8
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
CH-1214 Vernier, Geneva
DOCUMENTATION.
Phone: [phone]
IN ADDITION TO THEIR EVALUATION AS
Reference number
Email: [e-mail]
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 23256:2023(E)
Website: www.iso.org
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
Published in Switzerland
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
DARDS TO WHICH REFERENCE MAY BE MADE IN
ii
  © ISO 2023 – All rights reserved
NATIONAL REGULATIONS. © ISO 2023

---------------------- Page: 2 ----------------------
ISO/FDIS 23256:2023(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms, definitions, and abbreviated terms . 1
3.1 Terms and definitions . 1
3.2 Abbreviated terms . 3
4 Principle . 4
4.1 Flow immunosensor . 4
4.2 Specific antibody . 4
5 Interferences . 4
6 Reagents . 5
7 Apparatus and materials .7
8 Sampling . 7
8.1 General . 7
8.1.1 General . 7
8.1.2 Bottles and material for sampling . 7
8.1.3 Bottles and material pre­cleaning . 7
8.1.4 Sampling . 8
8.2 Preparation of sample . 8
8.2.1 Extraction . 8
8.2.2 Clean­up. 8
8.2.3 Sample solutions . 8
9 Procedure .9
9.1 General . 9
9.2 Sample set up . 9
9.3 Measuring procedure . 9
10 Data processing .10
10.1 Measurement data . 10
10.2 Calibration curve . 11
10.3 Calculation of measured concentration .12
11 Validation . .12
11.1 General .12
11.2 Accuracy profile . 12
11.3 Range of quantitation .13
11.4 Limit of detection . 14
11.5 E valuation of data . 14
11.6 Quality control . 14
12 Test report .15
Annex A (informative) Specificity of mouse monoclonal antibodies used for the flow
immunosensor.16
Annex B (informative) Example of the automate sample clean-up and concentration device .17
Annex C (informative) Example of the flow immunosensor .19
Annex D (informative) Recoveries, calibration curves, ranges of quantitation of the
standard analytes 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB in drinking water .25
Annex E (informative) Measurement of 2,3,7,8-TCDD and 3,3’,4,4’,5-PeCB in river waters .29
iii
© ISO 2023 – All rights reserved

---------------------- Page: 3 ----------------------
ISO/FDIS 23256:2023(E)
Annex F (informative) Interlaboratory trial of the measurement of 2,3,7,8-TCDD and
3,3’,4,4’,5-PeCB in water samples by the use of flow immunosensor method .32
Bibliography .35
iv
  © ISO 2023 – All rights reserved

---------------------- Page: 4 ----------------------
ISO/FDIS 23256:2023(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 non­governmental, 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 of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2,
Physical, chemical and biochemical methods.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
© ISO 2023 – All rights reserved

---------------------- Page: 5 ----------------------
ISO/FDIS 23256:2023(E)
Introduction
Persistent organic pollutants (POPs) including dibenzo­p-dioxins (PCDDs), dibenzofurans (PCDFs) and
dioxin-like polychlorinated biphenyls (DL-PCBs) in water and wastewater are analysed by instrumental
methods such as GC­MS and HRGC/HRMS. These methods are accurate and precise, but labour­intensive,
time-consuming, and costly. Alternatively, immunoassays are used for monitoring of POPs. These
methods have similar sensitivity and selectivity, but are more cost-effective and able to manage large
loads of sample well. The use of immunoassays is suitable for timely detection of selected congeners of
PCDDs and PCBs in water and wastewater in advance of subsequent confirmatory methods.
Recently, automated immunoassay methods including a flow immunosensor have been developed
for detection of POPs in environmental samples. These methods reduced manual operations such as
pipetting, time-consumption and coefficient of variation. Therefore, practical use of these methods can
play an important role in timely, continuous cost-saving monitoring of water and wastewater in both
developed and developing countries. This method can be applicable to timely continuous monitoring
of selected congeners of PCDDs and PCBs in water and wastewater to prioritize those for subsequent
confirmatory determination.
vi
  © ISO 2023 – All rights reserved

---------------------- Page: 6 ----------------------
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 23256:2023(E)
Water quality — Detection of selected congeners of
polychlorinated dibenzo-p-dioxins and polychlorinated
biphenyls — Method using a flow immunosensor technique
WARNING — The PCDDs, PCDFs and PCBs are among the most hazardous chemicals. Therefore, all
work with PCDDs, PCDFs and PCBs require the utmost care; the national safety measures which
correspond to those for hazardous substances shall be strictly adhered to. The responsibility of
the user is to establish appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably qualified staff.
1 Scope
This document specifies methods and principles for detection of selected congeners of polychlorinated
dibenzo­p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs) in water and wastewater using a
flow immunosensor. The flow immunosensor utilizes antibodies specific to 2,3,7,8-TCDD and 3,3’,4,4’,5-
PeCB, which have the highest toxic equivalent factor (TEF) value among the congeners of each of PCDDs
and PCBs. The method is applicable to timely monitoring of selected congeners of 4,3,7,8-TCDD and
3,3’,4,4’,5-PeCB in water and wastewater to prioritize those for subsequent confirmatory determination.
This document specifies practical methods and procedures for sampling, extraction, clean-up,
measurement in a flow immunosensor, data processing and validation of measurement results. The
combined use of automated instruments for extraction, clean-up, and flow immunosensing can reduce
time-consumption and labour-intensity, while providing reproducible precise data. This method can
provide the lower LOQ for 2, 3, 7, 8-TCDD and 3,3’,4,4’,5-PeCB of 28 pg/l and 152 pg/l, respectively at
20 % or less of coefficient variation (CV) depending on sampling, extraction, clean-up and measurement
conditions.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 5667­1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes and
sampling techniques
ISO 5667­3, Water quality — Sampling — Part 3: Preservation and handling of water samples
ISO 5667­6, Water quality — Sampling — Part 6: Guidance on sampling of rivers and streams
ISO 5667­10, Water quality — Sampling — Part 10: Guidance on sampling of waste water
3 Terms, definitions, and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
1
© ISO 2023 – All rights reserved

---------------------- Page: 7 ----------------------
ISO/FDIS 23256:2023(E)
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1.1
analyte
selected congeners of polychlorinated dibenzo-p-dioxins and polychlorinated biphenyls which bind to
specific monoclonal antibodies (3.1.2)
Note 1 to entry: Annex A gives information of the specific of mouse monoclonal antibodies used for the flow
immunosensor.
3.1.2
antibody
class of serum proteins that are induced by exposing to an immunogen and will bind specifically to
antigen/analyte (3.1.3)/(3.1.1) forming an antibody-antigen complex
Note 1 to entry: An antibody with a fluorescent label is used as a secondary antibody for binding to an antibody
specific to antigen/analyte.
3.1.3
antigen
molecule which selectively binds to an antibody (3.1.2)
Note 1 to entry: Antigens are analytes in the case of selected congeners of polychlorinated dibenzo-p-dioxins and
polychlorinated biphenyls.
3.1.4
conjugate
large molecule covalently coupled with a small molecule
Note 1 to entry: An antigen-mimic BSA conjugate is used for coating polymer beads.
3.1.5
cross-reactivity
ability of an antibody (3.1.2) to bind to certain molecules which structurally related to an antigen (3.1.3)
3.1.6
flocculation
physical process of contact and adhesions wherein the aggregates form larger-size clusters called flocs
being excluded from suspension for water treatment
3.1.7
flow immunosensor
immunosensor based on a kinetic exclusion assay (3.1.11) using a monoclonal antibody (3.1.2) in a
heterogeneous assay system for rapid detection of an analyte (3.1.1) in a number of samples
Note 1 to entry: The system of a flow immunosensor is operated automatically according to a program.
3.1.8
IgG
immunoglobulin-G
antibody molecule, consisting of two identical heavy(H)-chains and two identical light(L)-chains held
together with disulfide bonds
Note 1 to entry: Both H- and L-chains consist of the variable and constant regions. An antigen binding site is
presented in the variable region.
2
  © ISO 2023 – All rights reserved

---------------------- Page: 8 ----------------------
ISO/FDIS 23256:2023(E)
3.1.9
immunoassay
immunochemical detection procedure based on specific antibody-antigen (3.1.2)­(3.1.3) binding theory
often using a tracer for the detection of a free or bound antibody
Note 1 to entry: In a heterogeneous immunoassay, one of immunoreagents is immobilized on a solid support and
requires a washing step to separate the bound and free immunoreagents.
3.1.10
inhibition concentration
analyte concentration which reduces the measuring signal of the zero standard (3.1.14), in the case of
50 % inhibition of the zero standard that expresses 50 values
3.1.11
kinetic exclusion assay
binding between antibody (3.1.2) and antigen (3.1.3) reaches equilibrium in solution, and then three
species such as the specific antibody, the antigen and the antibody-antigen complex are present
Note 1 to entry: A kinetic exclusion assay measures the concentration of the free antibody without perturbing
the equilibrium. The format of the assay is to measure a free specific antibody with a label.
3.1.12
monoclonal antibody
antibody (3.1.2) population, possessing identical selectivity and affinity produced by a single antibody–
producing cell line
Note 1 to entry: Monoclonal antibodies specific to selected congeners of polychlorinated dibenzo-p-dioxins and
polychlorinated biphenyls are prepared for measurement of these analytes.
3.1.13
procedure blank
solution prepared in the laboratory, using reagent water or other blank matrix
3.1.14
zero standard
analyte-free standard (blank) which is used for calibration
3.2 Abbreviated terms
BSA bovine serum albumin
CV coefficient of variation
DMSO dimethyl sulfoxide
GC­MS gas chromatography/mass spectrometry
HRGC high-resolution gas chromatography
HRMS high-resolution mass spectrometry
IgG immunoglobulin G
LED light emitting diode
LLE liquid-liquid extraction
LOD limit of detection
LOQ limit of quantification
3
© ISO 2023 – All rights reserved

---------------------- Page: 9 ----------------------
ISO/FDIS 23256:2023(E)
PBS phosphate­buffered saline
PCDD polychlorinated dibenzo-p-dioxin
PCDF polychlorinated dibenzofuran
PCB polychlorinated biphenyl
POP persistent organic pollutant
PTFE polytetrafluoroethylene
SD standard deviation
SPE solid phase extraction
TEF toxic equivalent factor
4 Principle
4.1 Flow immunosensor
A flow immunosensor based on kinetic exclusion assay principles consists of a flow cell containing
a capture reagent immobilized on a solid phase matrix such as azlactone, sepharose, polymethyl
methacrylate, polystyrene, or the like, along with an antibody specific for the analyte and a fluorescent
label used for detection. In many cases, the fluorescent label is attached to a second-anti-species
antibody that recognizes and binds to the analyte specific antibody. In practice, the capture reagent
used on the solid phase is frequently either the analyte itself, an analogue of the analyte or a protein
conjugate of either the analyte or an analogue. To perform the assay a suitable sample (which can be
extracted and/or concentrated from a large environmental sample) is mixed with the specific antibody
and the secondary antibody (C.2.6) with a fluorescent label, and then incubated for a period of time to
allow binding to occur. After incubation, the antibody mixture is flowed over the solid phase matrix and
free (not bound to analyte) antibody bound to the secondary antibody with a fluorescent label from the
solution binds to the solid phase capture reagent. A fluorescent signal is measured from the captured
antibody. The largest fluorescent signal occur when there is zero analyte present and the presence of
[1][3]
analyte results in reduced signal levels .
4.2 Specific antibody
Specific antibodies (IgG) are selected for measurement of analytes in a flow immunosensor. Monoclonal
anti-2,3,7,8-TCDD antibody and anti-3,3’,4,4’,5-PeCB antibody show the highest affinity to 2,3,7,8-TCDD
and 3,3’,4,4’,5-PeCB, respectively as shown in Annex A. Each of these congeners is known to be present
in the environment and has the highest TEF value among the congeners of each of PCDDs and PCBs.
Thus, both antibodies are used as the representatives for detection of selected congeners of PCDDs
and PCBs in water and wastewater using a flow immunosensor. When antibodies specific for other
congeners or other related compounds (e.g. PCDFs) are available, the application of the immunosensor
described in 4.1 are readily expanded for detection of those related compounds or congeners.
5 Interferences
PCDDs, PCBs and other dioxin-like compounds are hydrophobic and largely present in water and
wastewater as adsorbed on solid particles. When these compounds are spiked into water samples in
containers, these compounds are quickly adsorbed on the surface of containers. The use of inappropriate
sampling devices and/or sampling flask can influence the test result because of the possible adsorption
of these compounds leading to false­negative results. On the other hand, these compounds can be
released into the sample from sampling flasks, especially when wares used are contaminated with
these compounds, and false-positive results can be generated. If filtered samples are tested in order
4
  © ISO 2023 – All rights reserved

---------------------- Page: 10 ----------------------
ISO/FDIS 23256:2023(E)
to remove the sample solid particles, the dioxins and dioxin-like compounds which are adsorbed on
particles cannot be detected.
Measurement conditions, for instance pH or sample components such as hormic acids, salinity and
solvents influencing the detection which so-called matrix effects can interfere with antibody binding.
The interference of matrix effects shall be assessed by spiking samples or reference samples with
known amounts of the analyte. In any case, extraction and clean-up are necessary to avoid interference.
In addition, air bubbles in measuring solutions and in a flow cell interfere not only antibody binding
but also fluorescence detection. Therefore, it shall be taken care to prepare measuring solutions
particularly by stirring gently at a room temperature.
Monoclonal antibodies specific to antigen/analyte are used for a flow immunosensor as shown in
Annex A. These antibodies cross-react against structurally related compounds and also bind to
non-specific compounds to show matrix effects. The extracted samples are each submitted to clean-
up process in an automate sample clean­up and concentration device as shown in Annex B, in which
a multiphase silica gel column works to eliminate polynuclear aromatic hydrocarbons and sulfur
compounds. In addition, the clean-up samples are also submitted to HRGC/HRMS analysis to confirm
removal of matrices
6 Reagents
Use reagents with a high purity for instance the grade “for residue analysis”. Information on type and
origin of antibodies as well as the cross-reactivity are stated in Annex A. Information on storage and
stability of the used reagents shall be requested according to the information of the supplier. If sensitivity
in a flow immunosensor is lower than expected, analyte-interfering substances such as matrices and
air bubbles shall be removed from samples and measuring solutions by suitable procedures.
6.1 Water, complying with grade 3 as defined in ISO 3696.
For instance, a Milli­Q water is recommended for preparation of buffer and regeneration solution.
6.2 Sodium chloride, NaCl, > 995 g/kg purity.
6.3 Potassium chloride, KCl, > 990 g/kg purity.
6.4 Sodium hydroxide, NaOH, > 970 g/kg purity.
6.5 Monosodium phosphate, NaH PO , > 980 g/kg purity.
2 4
6.6 Sodium dihydrogen phosphate dodecahydrate, Na HPO ·12H O, >980 g/kg purity.
2 4 2
6.7 Organic solvents
Solvents used for spiking, extraction, clean-up, and measurement in the following:
6.7.1 Propanone (acetone), >995 ml/l purity.
6.7.2 Toluene, >990 ml/l purity.
6.7.3 Hexane, >960 ml/l purity.
6.7.4 Dimethyl sulfoxide, DMSO, >995 ml/l purity.
6.7.5 Ethanol, >995 ml/l purity.
5
© ISO 2023 – All rights reserved

---------------------- Page: 11 ----------------------
ISO/FDIS 23256:2023(E)
6.8 Standard solution.
The standard acetone solutions of 2,3,7,8­TCDD and 3,3’,4,4’,5­PeCB are each diluted with acetone for
spiking into a 3 l or more water sample and for confirmation of spiking concentration by HRGC/HRMS
analysis. For preparation of sample solutions, a part of each acetone solution of both standard chemicals
is diluted with DMSO.
6.9 Flocculant.
A flocculant consisting of activated charcoal powder, poly aluminium chloride, silica gel and sodium
carbonate is added into a water sample adjusted to pH 7 to pH 8 at a rate of 0,1 g/l. Then, flocs formed
are collected by filtration with a 0,45 µm of pore size of a glass fibre filter and then air-dried overnight
[4]
for extraction .
6.10 Phosphate-buffered saline.
Prepare 50 mmol/l of PBS at pH 7,4 by dissolving 2,9 g of Na HPO ·12H O, 0,2 g o
...