Water quality — Determination of alkylmercury compounds in water — Method using gas chromatography-mass spectrometry (GC-MS) after phenylation and solvent extraction

This document specifies a method for the determination of alkylmercury compounds in filtered water samples by gas chromatography-mass spectrometry after phenylation and solvent extraction. This method is applicable to determination of individual methylmercury (MeHg) and ethylmercury (EtHg) compounds in surface water and waste water. The method can be applied to samples containing 0,2 μg/l to 10 μg/l of each compound as mercury mass. Depending on the matrix, the method may also be applicable to higher concentrations after suitable dilution of the sample or reduction in sample size.

Qualité de l'eau — Détermination des composés alkyl mercure dans l'eau — Méthode par chromatographie gazeuse et spectrométrie de masse (CG-SM) après phénylation et extraction par solvant

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INTERNATIONAL ISO
STANDARD 21863
First edition
2020-11
Water quality — Determination of
alkylmercury compounds in water —
Method using gas chromatography-
mass spectrometry (GC-MS) after
phenylation and solvent extraction
Qualité de l'eau — Détermination des composés alkyl mercure dans
l'eau — Méthode par chromatographie gazeuse et spectrométrie de
masse (CG-SM) après phénylation et extraction par solvant
Reference number
ISO 21863:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 21863:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 21863:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Interferences ............................................................................................................................................................................................................ 2

5.1 Interferences with sampling, sample storage and sample preparation ................................................ 2

5.2 Interferences with GC-MS .............................................................................................................................................................. 2

6 Reagents and standards ................................................................................................................................................................................ 2

7 Apparatus and materials.............................................................................................................................................................................. 4

8 Sample collection, preservation and storage.......................................................................................................................... 5

9 Procedure..................................................................................................................................................................................................................... 5

9.1 Sample preparation ............................................................................................................................................................................ 5

9.1.1 pH-adjustment of water sample ......................................................................................................................... 5

9.1.2 Phenylation and solvent extraction ................................................................................................................. 6

9.1.3 Dehydration of toluene extract ............................................................................................................................ 6

9.2 Preparation of samples for GC-MS ......................................................................................................................................... 6

9.3 Optimization of operating condition for GC-MS ......................................................................................................... 6

9.4 Identification of individual substances with GC-MS ............................................................................................... 6

9.5 Blank tests ................................................................................................................................................................................................... 7

10 Calibration .................................................................................................................................................................................................................. 7

10.1 General requirements ....................................................................................................................................................................... 7

10.2 Performance test of GC-MS ........................................................................................................................................................... 7

10.3 Calibration with internal standard ........................................................................................................................................ 8

10.3.1 General requirement .................................................................................................................................................... 8

10.3.2 Procedure of calibration ............................................................................................................................................ 8

10.4 Spike recovery test of target substances .......................................................................................................................... 9

11 Calculation .................................................................................................................................................................................................................. 9

11.1 Calculation of results after calibration with internal standards .................................................................. 9

11.2 Treatment of results lying outside the calibration range ................................................................................10

11.3 Quality checks for internal standardization ...............................................................................................................10

12 Expression of results .....................................................................................................................................................................................10

13 Test report ................................................................................................................................................................................................................11

Annex A (informative) Example of operating condition of GC-MS ......................................................................................12

Annex B (informative) Examples of mass chromatograms and mass spectra of phenylated

alkylmercury by GC-MS ...............................................................................................................................................................................13

Annex C (informative) The use of sodium tetrapropylborate as an alternative derivatizing

[4]

agent .........................................................................................................................................................................................................................16

Annex D (informative) The use of GC-AFS as an alternative detector ..............................................................................17

Annex E (informative) Performance data .....................................................................................................................................................20

Bibliography .............................................................................................................................................................................................................................21

© ISO 2020 – All rights reserved iii
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ISO 21863:2020(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.
iv © ISO 2020 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 21863:2020(E)
Introduction

This document specifies a method for the determination of alkylmercury compounds in water by gas

chromatography-mass spectrometry (GC-MS) after phenylation and solvent extraction.

Alkylmercury has high toxicity that causes Minamata disease in the heavy exposure as discovered

at Minamata City in Japan in 1956. Methylmercury in wastewater from an acetaldehyde acetic acid

manufacturing plant was identified as a causative substance. Subsequent investigation revealed that

ethylmercury poisoning has a similar toxic effect as methylmercury. Japanese government set an

effluent standard and an environment standard for alkylmercury.

Minamata Convention on Mercury was adopted by over 140 countries in 2013 for prevention of global

environmental pollution and health damage caused by mercury, and entered into force in 2017. The

convention states that each party shall identify the relevant point source categories and take measures

including the set of release limit values and the use of best available techniques and best environmental

practices. It should be noted that the released inorganic mercury is partially converted to alkylmercury

by biochemical processes of microorganism in water and sediment. Alkylmercury is concentrated in

biota through food chain, and consequently the risk to higher organism increases.

This document will be beneficial to evaluate the risk of alkylmercury from water and to control the

anthropogenic releases of alkylmercury from the relevant point sources.
© ISO 2020 – All rights reserved v
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INTERNATIONAL STANDARD ISO 21863:2020(E)
Water quality — Determination of alkylmercury
compounds in water — Method using gas chromatography-
mass spectrometry (GC-MS) after phenylation and solvent
extraction

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document 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.

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 a method for the determination of alkylmercury compounds in filtered water

samples by gas chromatography-mass spectrometry after phenylation and solvent extraction.

This method is applicable to determination of individual methylmercury (MeHg) and

ethylmercury (EtHg) compounds in surface water and waste water.

The method can be applied to samples containing 0,2 μg/l to 10 μg/l of each compound as mercury

mass. Depending on the matrix, the method may also be applicable to higher concentrations after

suitable dilution of the sample or reduction in sample size.
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 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 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
© ISO 2020 – All rights reserved 1
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ISO 21863:2020(E)
4 Principle

Alkylmercury compounds dissolved in a water sample are phenylated with sodium tetraphenylborate

after adjusting the sample solution at pH 5,0 with acetate buffer. The phenylated mercury compounds

are extracted from the water samples into toluene by liquid-liquid extraction. An internal standard is

added to the sample solution prior to pH-adjustment of sample preparation. The phenylated mercury

compounds in the toluene extracts are determined by gas chromatography-mass spectrometry (GC-MS).

5 Interferences
5.1 Interferences with sampling, sample storage and sample preparation

Potential sources of mercury or alkylmercury contamination during sampling, sample storage and

sample preparation include: labware, containers, sampling equipment, reagents, reagent water,

atmospheric dirt and dust, and human contact.

Only fluoropolymer or borosilicate glass containers shall be used for sample storage because mercury

vapor can diffuse in or out of sample solution, if other materials are used.

Apparatus or parts which may come into contact with either water sample or the extract shall be

non-metallic and free from target substances to be determined and interfering substances. All

apparatus and labware shall be cleaned using the cleaning procedure in this method.

Phenylation and solvent extraction are prone to be interfered with large amounts of coexisting

substances. For the majority of natural water samples, this type of interference should not be significant,

but severe interference may be found in some waste water samples which contain high concentration

of coexisting substances. Some organosulfur compounds (e.g. L-cysteine) binding alkylmercury also

interfere with the phenylation. The user should know the level of coexisting substances on typical water

samples using appropriate methods and if severe interferences are indicated, the level of interferences

should be assessed by recovery tests with spiking a standard solution of alkylmercury into water

samples.

If the coexisting substances in a water sample are precipitated with tetraphenylborate during

phenylation step, the precipitates may interfere with the solvent extraction procedure causing

problems in phase separation. For example, potassium of high concentration (> several hundred mg/l)

in water precipitates tetraphenylborate, that leads to interfere with the phase separation. In these

cases, filter the water sample through a glass fibre filter (7.2.2), and then add a new solution of sodium

tetraphenylborate and toluene.
5.2 Interferences with GC-MS

Substances that co-elute with the target alkylmercury or the internal standard may interfere with the

determination. These interferences may lead to incompletely resolved signals and may, depending on

their magnitude, affect accuracy and precision of the analytical results. Non-symmetrical peaks and

peaks broader than the corresponding peaks of the reference substance suggest interferences.

Interferences may also be caused by carry-over contamination mainly from the injection system of

the GC, especially when analysing a sample with low concentration after a sample with much higher

concentration of alkylmercury. A memory test, by injecting toluene, is useful to check the extent of

carry-over.
6 Reagents and standards

Unless otherwise indicated, reagents of purity grade "for analysis" or "for residue analysis" are used as

reagents.
6.1 Water, grade 1, as specified in ISO 3696.
2 © ISO 2020 – All rights reserved
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ISO 21863:2020(E)
6.2 Hydrochloric acid, c(HCl) = 10 mol/l, ultra-pure grade.
6.3 Acetic acid, c(CH COOH) = 17 mol/l, ultra-pure grade.
6.4 Sodium hydroxide solution, c(NaOH) = 3 mol/l, ultra-pure grade.
6.5 Acetate buffer solution

An aliquot of 11,5 ml of acetic acid (6.3) and 42,5 ml of sodium hydroxide solution (6.4) is dissolved in

water (6.1) to give a final volume of 1,0 l. The final concentration of the acetate is 0,2 mol/l.

6.6 Sodium tetraphenylborate solution, ρ = 20 g/l.

Sodium tetraphenylborate reagent is of purity grade for gas chromatographic analysis, or equivalent.

Two grams of the reagent is dissolved in water (6.1) to give a final volume of 100 ml. The solution

should be immediately used within 1 d after the preparation because the phenylation activity reduces

significantly after this time.
6.7 Toluene, C H CH .
6 5 3
6.8 Methanol, CH OH.
6.9 Sodium sulfate, Na SO , anhydrous, powdered.
2 4
6.10 Methylmercury chloride, CH HgCl.
6.11 Ethylmercury chloride, C H HgCl.
2 5
6.12 2,4,6-trichloroanisole-d3, C H D Cl O.
7 2 3 3
6.13 Poly(ethylene glycol) 300 solution, PEG300, ρ = 100 g/l.

Dissolve 1 g of poly(ethylene glycol) 300 of reagent grade in 10 ml of toluene (6.7) in a stoppered test

tube (7.5).

6.14 Stock solutions of methylmercury chloride and ethylmercury chloride, ρ = 1 000 mg/l.

Dissolve approximately 0,125 g, accurately weighed, of methylmercury chloride or 0,132 g, accurately

weighed, of ethylmercury chloride in 100 ml of methanol (6.8) or reagent water (6.1) containing 5 ml/l

acetic acid (6.3) and 2 ml/l HCl (6.2) in a fluoropolymer bottle. Each solution should contain approx.

1 000 mg/l CH Hg or C H Hg as Hg. It is also recommended to use commercially available certified

3 2 5
standard solutions of methylmercury chloride or ethylmercury chloride.
6.15 Standard solutions of alkylmercury, ρ = 10 mg/l or 1 mg/l.

Dilute 100 μl or 10 μl of the stock solution (6.14) to 10 ml of methanol (6.8) or reagent water (6.1)

containing 5 ml/l acetic acid (6.3) and 2 ml/l HCl (6.2). Each solution contains 10 mg/l or 1 mg/l

CH Hg or C H Hg as Hg. It was reported that the amount of the alkylmercury in the standard solution

3 2 5

using acetic acid and HCl has been maintained over a year when stored in a fluoropolymer bottle in a

refrigerator (see Reference [2]).
6.16 Reference solutions of alkylmercury for calibration

Prepare a minimum of five reference solutions with different concentrations for calibration. Pour 100 ml

of reagent water (6.1) in a narrow-neck flat-bottomed flask (7.3) and add stepwise from 10 μl to 100 μl

© ISO 2020 – All rights reserved 3
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ISO 21863:2020(E)

of the alkylmercury standard solution (6.15) directly into the water using a microliter syringe (7.7)

without contacting it on the wall of the flask. Put the stopper on the flask and stir the solution gently.

The concentrations of alkylmercury are adjusted for the calibration from 0,2 μg/l to 10 μg/l as Hg.

6.17 Check solutions of phenylated alkylmercury for performance of GC-MS

Treat the reference solution of alkylmercury at 10 μg/l (6.16) using the procedure as outlined in 9.1. The

final concentration in the solvent extract should be 200 μg/l of phenylated alkylmercury species due

to the preconcentration factor of the liquid-liquid extraction employed. Dilute the final extracts with

toluene (6.7) at an appropriate ratio to give the concentrations of phenylated alkylmercury ranging

between 4 μg/l and 200 μg/l as Hg. Add 2 μl of PEG300 solution (6.13) in the proportion of 1 ml of

toluene. The addition of PEG300 is required to ensure good chromatographic behaviour of phenylated

alkylmercury. These solutions are used for checking the performance of the GC-MS, such as sensitivity,

linearity of calibration, and resolution of target peaks before conducting an analysis of water samples.

6.18 Internal standard solution, ρ = 40 mg/l.

Weigh 10 mg of 2,4,6-trichloroanisole-d3 (6.12) in a 10 ml volumetric flask and make up to the mark

with methanol. The standard solutions that are commercially available can also be used. Dilute this

solution in the ratio of 1:25 with methanol.
6.19 Operating gases for GC-MS, helium, purity ≥999,99 mmol/mol.
7 Apparatus and materials

7.1 Sample collection bottles, fluoropolymer or borosilicate glass, of capacity 125 ml to 1 000 ml,

with such materials or fluoropolymer-lined cap.

New bottles should be cleaned by heating from 65 °C to 75 °C in 4 mol/l HCl for at least 48 h. After

cooling, they are rinsed three or more times with reagent water, filled with reagent water containing

4 ml/l HCl (6.2) and capped, and placed in a plastic box until use. Rinse the bottles with reagent

water just prior to use for sampling. Bottle blanks should be analysed as described in 9.5 to verify the

effectiveness of the cleaning procedures.
7.2 Filter

7.2.1 Filter for sampling, cellulose acetate or cellulose nitrate, of pore size 0,45 μm.

7.2.2 Filter for solvent extraction, borosilicate glass fibre, diameter of fibres 0,75 μm to 1,5 μm.

7.3 Narrow-neck flat-bottomed flasks, borosilicate glass, of capacity 150 ml to 200 ml, with glass

stoppers, used for phenylation and solvent extraction.

7.4 Volumetric pipettes, capacity 100 ml, used for measuring the volume of water sample

with ±0,08 ml tolerance.

7.5 Stoppered test tubes, capacity 10 ml, used for pre-examination to adjust the pH of the buffered

solutions (9.1.1) or for dehydration of the extracts (9.1.3).

7.6 Vials for GC-MS, made of amber glass, capacity 2 ml, with fluoropolymer-lined screw-cap.

7.7 Microlitre syringe, made of borosilicate glass.
7.8 Disposable Pasteur pipettes, made of borosilicate glass.
4 © ISO 2020 – All rights reserved
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ISO 21863:2020(E)
7.9 Balances, analytical type capable of weighing 0,1 mg.
7.10 pH meter, with combination glass electrode.

7.11 Magnetic stirrer, with PTFE-coated (PTFE = polytetrafluoroethylene) stirring bar of suitable size.

7.12 Gas chromatograph mass spectrometer (GC-MS), with electron impact ionization.

An example of operating condition of GC-MS is given in Annex A.
7.13 GC column

The column shall be capable of resolving the phenylated alkylmercury and inorganic mercury(II)

compounds (phenylmethylmercury, phenylethylmercury and diphenylmercury) listed in Table 1.

Examples of the columns are shown in Annex A.

Table 1 — Phenylated alkylmercury and inorganic mercury(II) compounds and internal

standard and selected diagnostic ions for identification and quantification in mass

spectrometric detection
Name of mercury Name of mercury Molecular Selected ions for Selected ions for
species in water compounds after formula after identification quantification
samples phenylation phenylation m/z m/z
Methylmercury Phenylated CH HgC H 200, 202, 217, 279, 292, 294 292, 294
3 6 5
methylmercury
Ethylmercury Phenylated C H HgC H 200, 202, 231, 279, 306, 308 306, 308
2 5 6 5
ethylmercury
Inorganic mercury(II) Diphenylmercury C H HgC H 200, 202, 279, 354, 356 354, 356
6 5 6 5
2,4,6-trichloroanisole- C H D Cl O 213, 215 213, 215
7 2 3 3
d3 (internal standard)
8 Sample collection, preservation and storage

Sample bottles should be stored in clean polyethylene bags until analysis. Samples are collected into

rigorously cleaned fluoropolymer or borosilicate glass bottles as specified in ISO 5667-1 and ISO 5667-3

and References [2] and [3]. Collected samples are filtered through a 0,45 μm filter (7.2.1) and preserved

at about pH 1,4 by adding an appropriate amount of HCl (6.2), which can be changed depending on

the acidity of the sample, e.g. 4 ml of HCl per litre of neutral sample. Samples may be shipped to the

laboratory unpreserved if they are kept dark and maintained at 0 °C to 4 °C from the time of collection

until preservation. The samples shall be acid-preserved within 48 h of sampling. Methylmercury in

acid-preserved samples is stable for at least six months, if kept dark and cool (Reference [2]).

9 Procedure
9.1 Sample preparation
9.1.1 pH-adjustment of water sample

Measure 100 ml of the acid-preserved water sample with a volumetric pipette (7.4) and transfer into

a narrow-neck flat-bottomed flask (7.3). The exact volume of the water sample can also be calculated

from its weight and density.

Following sample transfer, neutralize the acid-preserved water sample by adding an appropriate

amount of NaOH solution (6.4), add 5 ml of acetate buffer solution (6.5) and 5 µl of an internal standard

solution (6.18) using a microliter syringe (7.7) without contacting it on the wall of the flask and

© ISO 2020 – All rights reserved 5
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ISO 21863:2020(E)

adjust the pH of the solutions with diluted HCl or NaOH solution at pH 5,0 ± 0,1. To minimize mercury

contamination, do not dip a pH glass electrode into the sample; take a small aliquot of the water sample

with a clean pipet to a test tube (7.5) and examine the pH for estimating required amounts of acid or

alkali for pH adjustment. In the separate pH test, record the volume used for pH test to calculate exact

sample volume for phenylation. It is also recommended to use a pH meter which can measure a sample

with a small volume (e.g. 0,1 ml) by placing the solution onto the flat sensor in a measuring scoop.

9.1.2 Phenylation and solvent extraction

Add 1 ml of sodium tetraphenylborate solution (6.6). Cover the flask with stopper and swirl gently to mix.

Add 5 ml of toluene and place a magnetic stirring bar in the flask and stir the water sample vigorously

using a magnetic stirrer (7.11) for about 60 min at room temperature, and then allow to stand for

about 10 min.

Pour reagent water (6.1) gently along the inside wall of the flask until the toluene layer rises to narrow-

neck part, and transfer the toluene layer using a disposable Pasteur pipette (7.8) to a stoppered test

tube (7.5).
9.1.3 Dehydration of toluene extract

To remove water from the extract, add 2 g of sodium sulfate (6.9) to the toluene extract in the stoppered

test tube (7.5).
9.2 Preparation of samples for GC-MS

Transfer a supernatant aliquot (approx. 1 ml) of the dehydrated toluene to a vial for GC-MS (7.6) and

add 2 μl of PEG300 solution (6.13). The addition of PEG300 is required to deactivate the active site

in the injection port and the column and to ensure good chromatographic behaviour of phenylated

alkylmercury especially at low concentrations.
9.3 Optimization of operating condition for GC-MS

Optimize the operating conditions of the GC-MS system in electron impact ionization mode in

accordance with the manufacturer’s i
...

DRAFT INTERNATIONAL STANDARD
ISO/DIS 21863
ISO/TC 147/SC 2 Secretariat: DIN
Voting begins on: Voting terminates on:
2020-02-05 2020-04-29
Water quality — Determination of alkylmercury
compounds in water — Method using gas chromatography-
mass spectrometry (GC-MS) after phenylation and solvent
extraction

Qualité de l'eau — Détermination des composés alkyl mercure dans l'eau — Méthode par chromatographie

gazeuse et spectrométrie de masse (CG-SM) après phénylation et extraction par solvant

ICS: 13.060.50
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
This document is circulated as received from the committee secretariat.
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 21863:2020(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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ISO/DIS 21863:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/DIS 21863:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Interferences ............................................................................................................................................................................................................ 2

5.1 Interferences with sampling, sample storage and sample preparation ................................................ 2

5.2 Interferences with GC-MS .............................................................................................................................................................. 2

6 Reagents and standards ................................................................................................................................................................................ 2

7 Apparatus and materials.............................................................................................................................................................................. 4

8 Sample collection, preservation and storage.......................................................................................................................... 5

9 Procedure..................................................................................................................................................................................................................... 5

9.1 Sample preparation ............................................................................................................................................................................ 5

9.1.1 pH-adjustment of water sample ......................................................................................................................... 5

9.1.2 Phenylation and solvent extraction ................................................................................................................. 6

9.1.3 Dehydration of toluene extract ............................................................................................................................ 6

9.2 Preparation of samples for GC-MS ......................................................................................................................................... 6

9.3 Optimization of operating condition for GC-MS ......................................................................................................... 6

9.4 Identification of individual substances with GC-MS ............................................................................................... 6

9.5 Blank tests ................................................................................................................................................................................................... 7

10 Calibration .................................................................................................................................................................................................................. 7

10.1 General requirements ....................................................................................................................................................................... 7

10.2 Performance test of GC-MS ........................................................................................................................................................... 7

10.3 Calibration with internal standard ........................................................................................................................................ 8

10.3.1 General requirement .................................................................................................................................................... 8

10.3.2 Procedure of calibration ............................................................................................................................................ 8

10.4 Spike recovery test of target substances .......................................................................................................................... 9

11 Calculation .................................................................................................................................................................................................................. 9

11.1 Calculation of results after calibration with internal standards .................................................................. 9

11.2 Treatment of results lying outside the calibration range ................................................................................10

11.3 Quality checks for internal standardization ...............................................................................................................10

12 Expression of results .....................................................................................................................................................................................10

13 Test report ................................................................................................................................................................................................................11

Annex A (informative) Example of operating condition of GC-MS ......................................................................................12

Annex B (informative) Examples of mass chromatograms and mass spectra of phenylated

alkylmercury by GC-MS ...............................................................................................................................................................................13

Annex C (informative) The use of sodium tetrapropylborate as an alternative derivatizing

[4]

agent .........................................................................................................................................................................................................................16

Annex D (informative) The use of GC-AFS as an alternative detector ..............................................................................17

Annex E (informative) Performance data .....................................................................................................................................................20

Bibliography .............................................................................................................................................................................................................................22

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ISO/DIS 21863:2020(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).

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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.
iv © ISO 2020 – All rights reserved
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ISO/DIS 21863:2020(E)
Introduction

This document specifies a method for the determination of alkylmercury compounds in water by gas

chromatography-mass spectrometry (GC-MS) after phenylation and solvent extraction.

Alkylmercury has high toxicity that causes Minamata disease in the heavy exposure as discovered

at Minamata city in Japan in 1956. Methylmercury in wastewater from an acetaldehyde acetic acid

manufacturing plant was identified as a causative substance. Subsequent investigation revealed that

ethylmercury poisoning has a similar toxic effect as methylmercury. Japanese government set an

effluent standard and an environment standard for alkylmercury.

Minamata Convention on Mercury was adopted by over 140 countries in 2013 for prevention of global

environmental pollution and health damage caused by mercury, and entered into force in 2017. The

convention states that each party shall identify the relevant point source categories and take measures

including the set of release limit values and the use of best available techniques and best environmental

practices. It should be noted that the released inorganic mercury is partially converted to alkylmercury

by biochemical processes of microorganism in water and sediment. Alkylmercury is concentrated in

biota through food chain, and consequently the risk to higher organism increases.

This document will be beneficial to evaluate the risk of alkylmercury from water and to control the

anthropogenic releases of alkylmercury from the relevant point sources.
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DRAFT INTERNATIONAL STANDARD ISO/DIS 21863:2020(E)
Water quality — Determination of alkylmercury
compounds in water — Method using gas chromatography-
mass spectrometry (GC-MS) after phenylation and solvent
extraction

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document 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.

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 a method for the determination of alkylmercury compounds in filtered water

samples by gas chromatography-mass spectrometry after phenylation and solvent extraction.

This method is applicable to determination of individual methylmercury (MeHg) and

ethylmercury (EtHg) compounds in surface water and waste water.

The method can be applied to samples containing 0,2 μg/l to 10 μg/l of each compound as mercury

mass. Depending on the matrix, the method may also be applicable to higher concentrations after

suitable dilution of the sample or reduction in sample size.
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 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 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at http:// www .iso .org ./ obp
— IEC Electropedia: available at http:// www .electropedia .org/
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ISO/DIS 21863:2020(E)
4 Principle

Alkylmercury compounds dissolved in a water sample are phenylated with sodium tetraphenylborate

after adjusting the sample solution at pH 5,0 with acetate buffer. The phenylated mercury compounds

are extracted from the water samples into toluene by liquid-liquid extraction. An internal standard is

added to the sample solution prior to pH-adjustment of sample preparation. The phenylated mercury

compounds in the toluene extracts are determined by gas chromatography-mass spectrometry (GC-MS).

5 Interferences
5.1 Interferences with sampling, sample storage and sample preparation

Potential sources of mercury or alkylmercury contamination during sampling, sample storage and

sample preparation include: labware, containers, sampling equipment, reagents, reagent water,

atmospheric dirt and dust, and human contact.

Only fluoropolymer or borosilicate glass containers shall be used for sample storage because mercury

vapor can diffuse in or out of sample solution, if other materials are used.

Apparatus or parts which may come into contact with water sample or the extract shall be non-metallic

and free from target substances to be determined and interfering substances. All apparatus and

labware shall be cleaned using the cleaning procedure in this method.

Phenylation and solvent extraction are prone to be interfered with large amounts of coexisting

substances. For the majority of natural water samples, this type of interference should not be significant,

but severe interference may be found in some waste water samples which contain high concentration

of coexisting substances. Some organosulfur compounds (e.g. L-cysteine) binding alkylmercury also

interfere with the phenylation. The user should know the level of coexisting substances on typical water

samples using appropriate methods and if severe interferences are indicated, the level of interferences

should be assessed by recovery tests with spiking a standard solution of alkylmercury into water

samples.

If the coexisting substances in a water sample are precipitated with tetraphenylborate during

phenylation step, the precipitates may interfere with the solvent extraction procedure causing

problems in phase separation. For example, potassium of high concentration (> several hundred mg/l)

in water precipitates tetraphenylborate, that leads to interfere with the phase separation. In these

cases, filter the water sample through a glass fibre filter (7.2.2), and then add a new solution of sodium

tetraphenylborate and toluene.
5.2 Interferences with GC-MS

Substances that co-elute with the target alkylmercury or the internal standard may interfere with the

determination. These interferences may lead to incompletely resolved signals and may, depending on

their magnitude, affect accuracy and precision of the analytical results. Non-symmetrical peaks and

peaks broader than the corresponding peaks of the reference substance suggest interferences.

Interferences may also be caused by carry-over contamination mainly from the injection system

of GC, especially when analysing a sample with low concentration after a sample with much higher

concentration of alkylmercury. A memory test, by injecting toluene, is useful to check the extent of

carry-over.
6 Reagents and standards

Unless otherwise indicated, reagents of purity grade "for analysis" or "for residue analysis" are used as

reagents.
6.1 Water, grade 1, as specified in ISO 3696.
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ISO/DIS 21863:2020(E)
6.2 Hydrochloric acid, c(HCl) = 10 mol/l, ultra-pure grade.
6.3 Acetic acid, c(CH COOH) = 17 mol/l, ultra-pure grade.
6.4 Sodium hydroxide solution, c(NaOH) = 3 mol/l, ultra-pure grade.
6.5 Acetate buffer solution

An aliquot of 11,5 ml of acetic acid (6.3) and 42,5 ml of sodium hydroxide solution (6.4) is dissolved in

water (6.1) to give a final volume of 1,0 l. The final concentration of the acetate is 0,2 mol/l.

6.6 Sodium tetraphenylborate solution, ρ = 20 g/l.

Sodium tetraphenylborate reagent is of purity grade for gas chromatographic analysis, or equivalent.

Two grams of the reagent is dissolved in water (6.1) to give a final volume of 100 ml. The solution should

be immediately used within 1 d after the preparation because the phenylation activity is not kept long.

6.7 Toluene, C H CH .
6 5 3
6.8 Methanol, CH OH.
6.9 Sodium sulfate, Na SO , anhydrous, powdered.
2 4
6.10 Methylmercury chloride, CH HgCl.
6.11 Ethylmercury chloride, C H HgCl.
2 5
6.12 2,4,6-trichloroanisole-d3, C H D Cl O.
7 2 3 3

6.13 Stock solutions of methylmercury chloride and ethylmercury chloride, ρ = 1 000 mg/l.

Dissolve approximately 0,125 g, accurately weight, of methylmercury chloride or 0,132 g, accurately

weight, of ethylmercury chloride in 100 ml of methanol (6.8) or reagent water (6.1) containing 0,5 %

volume fraction acetic acid (6.3) and 0,2 % volume fraction HCl (6.2) in a fluoropolymer bottle. Each

solution should contain approx. 1 000 mg/l CH Hg or C H Hg as Hg. It is also recommended to use

3 2 5

commercially available certified standard solutions of methylmercury chloride or ethylmercury

chloride.
6.14 Standard solutions of alkylmercury, ρ = 10 mg/l or 1 mg/l.

Dilute 100 μl or 10 μl of the stock solution (6.13) with 10 ml of methanol (6.8) or reagent water (6.1)

containing 0,5 % volume fraction acetic acid (6.3) and 0,2 % volume fraction HCl (6.2). Each solution

contains 10 mg/l or 1 mg/l CH Hg or C H Hg as Hg. It was reported that the amount of the alkylmercury

3 2 5

in the standard solution using acetic acid and HCl has been maintained over a year when stored in a

fluoropolymer bottle in a refrigerator (Reference [2]).
6.15 Reference solutions of alkylmercury for calibration

Pour 100 ml of reagent water (6.1) in a narrow-neck flat-bottomed flask (7.3) and add stepwise from

10 μl to 100 μl of the alkylmercury standard solution (6.14) directly into the water using a microliter

syringe (7.7) without contacting it on the wall of the flask. Put the stopper on the flask and stir the

solution gently. The concentrations of alkylmercury are adjusted for the calibration from 0,2 μg/l to

10 μg/l as Hg.
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ISO/DIS 21863:2020(E)
6.16 Check solutions of phenylated alkylmercury for performance of GC-MS

Treat the reference solution of alkylmercury at 10 μg/l (6.15) using the procedure as outlined in 9.1. The

final concentration in the solvent extract should be 200 μg/l of phenylated alkylmercury species due

to the preconcentration factor of the liquid-liquid extraction employed. Dilute the final extracts with

toluene (6.7) at an appropriate ratio to give the concentrations of phenylated alkylmercury ranging

between 4 μg/l and 200 μg/l as Hg. Add 2 μl of PEG300 solution (6.18) in the proportion of 1 ml of

toluene. The addition of PEG300 is required to ensure good chromatographic behaviour of phenylated

alkylmercury. These solutions are used for checking the performance of GC-MS, such as sensitivity,

linearity of calibration, and resolution of target peaks before conducting an analysis of water samples.

6.17 Internal standard solution, ρ = 40 mg/l.

Weight 10 mg of 2,4,6-trichloroanisole-d3 (6.12) in a 10 ml volumetric flask and make up to the mark

with methanol. The standard solutions that are commercially available can also be used. Dilute this

solution in the ratio of 1:25 with methanol.
6.18 Poly(ethylene glycol) 300 solution, PEG300, ρ = 100 g/l.

Dissolve 1 g of poly(ethylene glycol) 300 of reagent grade in 10 ml of toluene (6.7) in a stoppered test

tube (7.5).
6.19 Operating gases for GC-MS, helium, purity ≥ 99,999 %.
7 Apparatus and materials

7.1 Sample collection bottles, fluoropolymer or borosilicate glass, of capacity 125 ml to 1 000 ml,

with such materials or fluoropolymer-lined cap.

New bottles should be cleaned by heating from 65 °C to 75 °C in 4 mol/l HCl for at least 48 h. After

cooling, they are rinsed three or more times with reagent water, filled with reagent water containing

0,4 % volume fraction HCl (6.2) and capped, and placed in a plastic box until use. Rinse the bottles with

reagent water just prior to use for sampling. Bottle blanks should be analysed as described in 9.5 to

verify the effectiveness of the cleaning procedures.
7.2 Filter

7.2.1 Filter for sampling, cellulose acetate or cellulose nitrate, of pore size 0,45 μm.

7.2.2 Filter for solvent extraction, borosilicate glass fibre, diameter of fibres 0,75 μm to 1,5 μm.

7.3 Narrow-neck flat-bottomed flasks, borosilicate glass, of capacity 150 ml to 200 ml, with glass

stoppers, used for phenylation and solvent extraction.

7.4 Graduated cylinders, capacity 100 ml, used for measuring the volume of water sample with ± 1 %

accuracy.

7.5 Stoppered test tubes, capacity 10 ml, used for pre-examination to adjust the pH of the buffered

solutions (9.1.1) or for dehydration of the extracts (9.1.3).

7.6 Vials for GC-MS, made of amber glass, capacity 2 ml, with fluoropolymer-lined screw-cap.

7.7 Microlitre syringe, made of borosilicate glass.
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ISO/DIS 21863:2020(E)
7.8 Disposable Pasteur pipettes, made of borosilicate glass.
7.9 Balances, analytical type capable of weighing 0,1 mg.
7.10 pH meter, with combination glass electrode.

7.11 Magnetic stirrer, with PTFE-coated (PTFE = polytetrafluoroethylene) stirring bar of suitable size.

7.12 Gas chromatograph mass spectrometer (GC-MS), with electron impact ionization.

An example of operating condition of GC-MS is given in Annex A.
7.13 GC column

The column shall be capable of resolving the phenylated alkylmercury and inorganic mercury(II)

compounds (phenylmethylmercury, phenylethylmercury and diphenylmercury) listed in Table 1.

Examples of the columns are shown in Annex A.

Table 1 — Phenylated alkylmercury and inorganic mercury(II) compounds and internal

standard and selected diagnostic ions for identification and quantification in mass

spectrometric detection

Name of mercury Name of mercury Molecular Selected ions for identifi- Selected ions for

species in water compounds after formula after cation quantification
samples phenylation phenylation m/z m/z
Methylmercury Phenylated meth- CH HgC H 200, 202, 217, 279, 292, 294 292, 294
3 6 5
ylmercury
Ethylmercury Phenylated ethyl- C H HgC H 200, 202, 231, 279, 306, 308 306, 308
2 5 6 5
mercury
Inorganic mercu- Diphenylmercury C H HgC H 200, 202, 279, 354, 356 354, 356
6 5 6 5
ry(II)
2,4,6-trichloroan- C H D Cl O 213, 215 213, 215
7 2 3 3
isole-d3 (internal
standard)
8 Sample collection, preservation and storage

Samples are collected into rigorously cleaned fluoropolymer or borosilicate glass bottles as specified

in the documents listed in Clause 2 and References [2] and [3]. Collected samples are filtered through

a 0,45 μm filter (7.2.1) and preserved at about pH 1,4 by adding an appropriate amount of HCl (6.2),

which can be changed depending on the acidity of the sample, e.g. 4 ml of HCl per liter of neutral sample.

Samples may be shipped to laboratory unpreserved if they are kept dark and maintained at 0 °C to

4 °C from the time of collection until preservation. The samples shall be acid-preserved within 48 h of

sampling. Sample bottles should be stored in clean polyethylene bags until analysis. Methylmercury in

acid-preserved samples is stable for at least six months, if kept dark and cool (Reference [2]).

9 Procedure
9.1 Sample preparation
9.1.1 pH-adjustment of water sample

Measure 100 ml of the acid-preserved water sample with a graduated cylinder (7.4) and transfer into

a narrow-neck flat-bottomed flask (7.3). The exact volume of the water sample can also be calculated

from its weight and density.
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ISO/DIS 21863:2020(E)

Following sample transfer, neutralize the acid-preserved water sample by adding an appropriate

amount of NaOH solution (6.4), add 5 ml of acetate buffer solution (6.5) and 5 µl of an internal standard

solution (6.17) using a microliter syringe (7.7) without contacting it on the wall of the flask and

adjust the pH of the solutions with diluted HCl or NaOH solution at pH 5,0 ± 0,1. To minimize mercury

contamination, do not dip a pH glass electrode into the sample; take a small aliquot of the water sample

with a clean pipet to a test tube (7.5) and examine the pH for estimating required amounts of acid or

alkali for pH adjustment. In the separate pH test, record the volume used for pH test to calculate exact

sample volume for phenylation. It is also recommended to use a pH meter which can measure a sample

with a small volume (e.g. 0,1 ml) by placing the solution onto the flat sensor in a measuring scoop.

9.1.2 Phenylation and solvent extraction

Add 1 ml of sodium tetraphenylborate solution (6.6). Cover the flask with stopper and swirl gently to mix.

Add 5 ml of toluene and place a magnetic stirring bar in the flask and stir the water sample vigorously

using a magnetic stirrer (7.11) for about 60 min at room temperature, and then allow to stand for

about 10 min.

Pour reagent water (6.1) gently along the inside wall of the flask until the toluene layer rises to narrow-

neck
...

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