Soil quality — Environmental availability of non-polar organic compounds — Determination of the potentially bioavailable fraction and the non-bioavailable fraction using a strong adsorbent or complexing agent

This document specifies an extraction method to determine the bioavailable (potential and environmental available) fraction and the non-bioavailable fraction of a contaminant in soil using a "receiver phase" for an organic contaminant with strong sorbing or complexing properties, for example, Tenax®[1] or cyclodextrin, respectively. NOTE 1 The bioavailable fraction is defined in ISO 17402 as environmental bioavailability. The method is applicable for non-polar organic contaminants with an aqueous solubility of NOTE 2 The method is theoretically applicable to non-polar organic contaminants with an aqueous solubility of 1 000 mg/l. The method has been often applied for compounds with a much lower solubility (Kow > 3) and less for compounds with a higher solubility. The applicability is therefore defined for compounds with an aqueous solubility of [1] Tenax® is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.

Qualité du sol — Disponibilité environnementale des composés organiques non polaires — Détermination de la fraction potentiellement biodisponible et de la fraction non biodisponible en utilisant un agent adsorbant fort ou un agent complexant

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Published
Publication Date
25-Jun-2020
Current Stage
6060 - International Standard published
Start Date
27-Apr-2020
Completion Date
26-Jun-2020
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INTERNATIONAL ISO
STANDARD 16751
First edition
2020-06
Soil quality — Environmental
availability of non-polar organic
compounds — Determination of the
potentially bioavailable fraction and
the non-bioavailable fraction using a
strong adsorbent or complexing agent
Qualité du sol — Disponibilité environnementale des composés
organiques non polaires — Détermination de la fraction
potentiellement biodisponible et de la fraction non biodisponible en
utilisant un agent adsorbant fort ou un agent complexant
Reference number
ISO 16751:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 16751:2020(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 16751:2020(E)
Contents Page

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

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

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

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

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

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

5 Reagents ........................................................................................................................................................................................................................ 3

6 Apparatus ..................................................................................................................................................................................................................... 3

7 Procedure..................................................................................................................................................................................................................... 4

7.1 Sample preparation ............................................................................................................................................................................ 4

7.2 Determining water content .......................................................................................................................................................... 5

7.3 Method A: Cyclodextrin ................................................................................................................................................................... 5

7.3.1 Extraction of the sample ........................................................................................................................................... 5

7.3.2 Phase separation .............................................................................................................................................................. 5

7.3.3 Extraction from receiver phase ........................................................................................................................... 5

7.4 Method B: Tenax® ............................................................................................................................................................................... 5

7.4.1 Extraction of the sample ........................................................................................................................................... 5

7.4.2 Phase separation .............................................................................................................................................................. 6

7.4.3 Extraction from receiver phase ........................................................................................................................... 6

7.5 Measurement ............................................................................................................................................................................................ 7

7.5.1 Potential bioavailable fraction ............................................................................................................................. 7

7.5.2 Non-bioavailable fraction ......................................................................................................................................... 7

7.6 Blank test ..................................................................................................................................................................................................... 7

8 Calculation .................................................................................................................................................................................................................. 7

8.1 Potential bioavailable fraction ................................................................................................................................................... 7

8.2 Non-bioavailable fraction .............................................................................................................................................................. 8

9 Expression of results ........................................................................................................................................................................................ 8

10 Validation ..................................................................................................................................................................................................................... 9

11 Test report ................................................................................................................................................................................................................... 9

Annex A (informative) Preparation and regeneration of Tenax .......................................................................................10

Annex B (informative) Illustrations....................................................................................................................................................................11

Annex C (informative) Validation study results .....................................................................................................................................14

Bibliography .............................................................................................................................................................................................................................25

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 16751:2020(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 on 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 the following

URL: www .iso .org/ iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 7, Soil

and site assessment.

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 16751:2020(E)
Introduction

The solubility of most non-polar organic contaminants is limited and they are sorbed to the soil

matrix. They may desorb and become available for organisms, which may result in an effect (toxicity,

degradation or bioaccumulation). Not all sorbed (adsorbed and absorbed) contaminants will desorb

and become available.

Extractions used in chemical analysis to measure the total concentration, release more contaminants

from the soil than are available. It is however also possible that contaminants are so strongly bounded

by the soil that they will not be released by chemical extraction. This strong sorption may also be

caused by incorporation of the contaminant (or a degradation or reaction product of the contaminant)

in the organic soil structure. The distribution of contaminants over sorption sites of varying sorption

strength is not constant in time and contaminants will shift, with increasing contact time, to the

stronger sorption sites.
Figure 1 shows schematically the differentiation between:

— extractable residues that are also bioavailable (i.e. the potentially bioavailable fraction);

— residues that are extractable by harsher extraction methods but are non-bioavailable;

— residues that are neither extractable nor bioavailable.

If a degradable substance enters a soil, part of it will degrade over time (curve a). The area between

curve a and c is extractable by exhaustive chemical procedures. For risk assessments, this part is

considered as the “total concentration” for which values are defined in many regulations. However,

only a part of this amount is bioavailable. The area between curves a and b is the bioavailable fraction

and the area between curves b and c is the non-bioavailable fraction. The method described in this

document enables the measurement of the potential bioavailable and the non-bioavailable fraction of a

contaminant in soil.
© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
ISO 16751:2020(E)
Key
X time
Y contaminant concentration
1 degradable
2 bioavailable
3 extractable, non-bioavailable
4 non-extractable: persistent residues
5 non-available fraction
NOTE For curves a, b and c see description in text above.

Figure 1 — Temporal changes in extractable/bioavailable fractions, extractable/non-

bioavailable fractions and non-extractable/non-bioavailable fractions of a non-polar organic

contaminant (modified from [1])

In the scientific research to bioavailability a large number of definitions and concepts are in use, which

reflect the discussion in the scientific world. However, a more clear and simple approach is necessary.

In regulation, organic contaminants are either bioavailable or non-bioavailable. To support decisions,

both should be measurable. Therefore, this document follows the approach of Ortega-Calvo et al. (2015)

[2]

as illustrated in Figure 2. In this approach all defined fractions are measurable as further explained

in Clause 4.
vi © ISO 2020 – All rights reserved
---------------------- Page: 6 ----------------------
ISO 16751:2020(E)

Figure 2 — Measurement of bioavailability of organic chemicals: a simplified scheme for use in

[9]
regulation

The colour boxes at the left of the biological membrane represent the distribution of pollutant molecules

among four classes (non-extractable, very slowly/slowly desorbing, rapidly desorbing and water-

dissolved) in soils and sediments. In the scheme in Figure 2, the bioavailable chemical is represented

by the rapidly desorbing and dissolved concentrations. The chemical methods able to measure the

pollutant present in each specific fraction are given in the grey boxes. The green box to the right of

the cell membrane represents the processes that occur within the organism exposed to the pollutant.

These biological processes can also serve as the basis for standard methods used for bioavailability

measurements.

As presented in Figure 2, the bioavailable fraction can be measured using the method described in this

document.
© ISO 2020 – All rights reserved vii
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INTERNATIONAL STANDARD ISO 16751:2020(E)
Soil quality — Environmental availability of non-polar
organic compounds — Determination of the potentially
bioavailable fraction and the non-bioavailable fraction
using a strong adsorbent or complexing agent
1 Scope

This document specifies an extraction method to determine the bioavailable (potential and

environmental available) fraction and the non-bioavailable fraction of a contaminant in soil using a

“receiver phase” for an organic contaminant with strong sorbing or complexing properties, for example,

®1)
Tenax or cyclodextrin, respectively.

NOTE 1 The bioavailable fraction is defined in ISO 17402 as environmental bioavailability.

The method is applicable for non-polar organic contaminants with an aqueous solubility of <100 mg/l.

The method is applicable for soil and soil-like material including (dredged) sediments.

NOTE 2 The method is theoretically applicable to non-polar organic contaminants with an aqueous solubility

of 1 000 mg/l. The method has been often applied for compounds with a much lower solubility (K > 3) and less

for compounds with a higher solubility. The applicability is therefore defined for compounds with an aqueous

solubility of <100 mg/l.
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 11074, Soil quality — Vocabulary

ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis —

Gravimetric method

ISO 14507, Soil quality — Pretreatment of samples for determination of organic contaminants

ISO 18512, Soil quality — Guidance on long and short term storage of soil samples

3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 11074, ISO 17402 and the

following apply.

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/

1) Tenax is an example of a suitable product available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO of this product.

© ISO 2020 – All rights reserved 1
---------------------- Page: 8 ----------------------
ISO 16751:2020(E)
3.1
potential bioavailable fraction

amount of contaminant present in the matrix that can be released from the solid phase to the aqueous

phase in a well-mixed water soil mixture and in presence of a receiving phase in a period of 20 h

Note 1 to entry: In ISO 17924 the term bioaccessibility is used, which is the fraction of a substance in soil or soil-

like material that is liberated in (human) gastrointestinal juices and thus available for absorption. This document

does not distinguish between bioavailability and bioaccessibility and uses the general term bioavailability. The

concept of bioavailability as followed in this document is described in the introduction of this document.

4 Principle

This method gives an estimation of the potential bioavailable and non-bioavailable fraction of organic

contaminants, i.e. the amount of the contaminant in the matrix that is potentially exchangeable with

the aqueous phase; specifically that, which is adsorbed/complexed by Tenax /cyclodextrin.

The extractable and non-bioavailable fraction of the contaminant left in the sample following the action

of Tenax /cyclodextrin can be subsequently measured with an exhaustive/harsh extraction technique

(designed to measure the total concentration) and in this way the non-bioavailable fraction of the

contaminant is assessed.

Thus, in numerical terms, the total contaminant concentration in a sample is the sum of the bioavailable

concentration (established using a strong sorbent or complexing agent) and the non-bioavailable

concentration (established using a subsequent harsh extraction method performed on the residue that

is left after the matrix has been extracted using a strong sorbent or complexing agent):

cc=+ c (1)
tot,cont bionon-bio
where
c is the total contaminant concentration;
tot,cont
c is the bioavailable concentration;
bio
c is the non-bioavailable concentration.
non-bio

The soil, soil-like material or sediment sample with particle size <2 mm is extracted with water

containing a “receiver phase” for the organic contaminants. This phase is either a complexing agent

(cyclodextrin) or a strong adsorbent [Tenax ]. The solubility of non-polar compounds is limited and in

this method the receiver phase acts as an “infinite sink”. The measured amount, which is the amount

that desorbs from the soil or soil-like material during 20 h, reflects the fraction of contaminant that can

have effects on biotic systems and that can become mobile.

In the following step, the contaminants adsorbed are extracted from the receiver phase and determined

by appropriate analytical methods. The amount of contaminants left in the soil residue, the non-

bioavailable fraction, can be measured using a subsequent harsh/exhaustive extraction designed to

measure the total concentration. Formula (1) can then be used to determine the total contaminant

concentration in the sample (if desired).

NOTE 1 ISO 13859 and ISO 13876 are examples to measure the total concentration of respectively PAH and

PCB in soil and soil-like materials.

NOTE 2 Formula (1) shows the relationship between the “total contaminant concentration” the “bioavailable

concentration” and the “non-bioavailable concentration”. If two of the concentrations are known the third

concentration can be calculated through the use of Formula (1). For example, by measuring the “total

concentration” and the “non-bioavailable concentration”, the “bioavailable concentration” can be calculated.

This is allowed with homogeneous materials. If it is not known whether a material is homogeneous and the

bioavailable concentration is the concentration of interest, the bioavailable concentration needs to be measured.

2 © ISO 2020 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 16751:2020(E)
5 Reagents

Reagents used shall be of suitable grade – analytical grade or higher– if not specified otherwise. The

blank value of the reagents (including water) shall be negligible compared to the lowest concentration

of organic contaminants to be determined.

5.1 Demineralized water or deionized water or water of equivalent purity (5 < pH < 7,5) with a

conductivity of <0,5 mS/m in accordance with grade 3 specified in ISO 3696.

If biodegradation of the target compounds is to be expected, add sodium azide (5.2) to water to a final

concentration of 0,2 g/l. This will minimize the biological degradation of the target compounds. If

biodegradation is not to be expected, it is not necessary to add sodium azide. This is the case for some

persistent target compounds, e.g. PCB.

NOTE With some soil samples it can be difficult to obtain a proper separation between the soil, aqueous

phase and Tenax . The use of 0,001 mol/l or 0,01 mol/l CaCl (5.9) instead of water can improve this separation.

5.2 Sodium azide, [CAS No. 26628-22-8], NaN .

WARNING — Attention is drawn to the hazard deriving from the use of the sodium azide which

is acutely toxic.

5.3 Cyclodextrin, (hydroxypropyl-β-cyclodextrin) of >97 % purity of a Food Grade, Medicine Grade or

Pharmaceutical Grade.

NOTE Analytical Grade cyclodextrin is very expensive. For this method Food Grade has shown to be fit for

purpose.

5.4 Extraction solution of cyclodextrin, dissolve 100 mmol (=146 g) of cyclodextrin (5.3) per litre of

water (5.1).

NOTE If biodegradation of the target compounds is to be expected, add sodium azide (5.2) to this solution

to a final concentration of 0,2 g/l. This will minimize the biological degradation of the target compounds. If

biodegradation is not to be expected, it is not necessary to add sodium azide. This is the case for some persistent

target compounds, e.g. PCB

5.5 2,6-diphenyleneoxide polymer (Tenax® TA), 60 mesh to 80 mesh. See Annex A for the

preparation and regeneration of Tenax .
5.6 Petroleum ether, [CAS No. 8032-32-4], boiling range 40 °C to 60 °C.
5.7 Ethanol, [CAS No. 64-17-5], C H O.
2 6
5.8 Acetone, [CAS No. 67-64-1], C H O.
3 6
5.9 Calcium chloride, [CAS no.1035-04-8], CaCl
5.10 Sodium sulfate, [CAS No. 231-820-9], Na SO
2 4.
6 Apparatus

Use the following equipment. All materials that come into contact with the sample (or reagents) shall

not adsorb the contaminant of interest and shall not contaminate the sample. Glass and PTFE are

suitable materials for most contaminants.
6.1 Sieving equipment, with 2 mm nominal screen size.
© ISO 2020 – All rights reserved 3
---------------------- Page: 10 ----------------------
ISO 16751:2020(E)
6.2 Balance, accuracy 0,01 g.
6.3 Conical centrifuge tubes, with screwing caps.
6.4 Separation funnel, of suitable size.

NOTE The hole in the tap of the separation funnel used for the Tenax extraction needs to be large enough

for the soil particles to leave the funnel. Otherwise no separation between Tenax and the sample is possible.

6.5 Shaking machine, which limits breakdown of sample particles, e.g. an end-over-end shaker,

capable of (20 ± 2) r/min or other mild agitation method or a horizontal movement shaker, capable to

have 150 r/min to 180 r/min.
6.6 Centrifuge, capable to centrifuge the centrifuge tubes (6.3).
6.7 Crushing equipment, jaw crusher or cutting device.
6.8 Appropriate glassware and plastic ware.
6.9 Kuderna Danish sample concentrator.
6.10 Collection vessel.
6.11 Folded filter.
7 Procedure
7.1 Sample preparation

The sample shall be pretreated in the laboratory in accordance with ISO 14507, but with the following

restrictions:

— Intensive pretreatment like grinding may have an effect on the environmental availability of

contaminants, therefore grinding is not allowed.

— In general, the test portion to be prepared shall have a grain size less than or equal to 2 mm, but on

no account the material shall be ground to reach this grain size.
— Remove stones, shells and any material not representative for the sample.

— Sieve the sample using a sieve (6.1). If necessary, press the material by hand through the sieve.

Instead of using ISO 14507, also freeze-drying according ISO 16720 may be used to pretreat the sample.

Some soils (e.g. peat and some sediments) are difficult to sieve. In these cases, remove stones, shells and

material not representative for the sample (e.g. plant material) by hand and process the sample without

sieving.

If the sample cannot be sieved at all because of its water content, reduce the water content until the

laboratory sample can be sieved. In the case of drying, the drying temperature shall not exceed 30 °C.

If a rapidly degradable fraction is to be expected, reduction of water content by air drying is not allowed.

In that case the original collected sample shall be forced by hand through a 2 mm sieve or the sample

can be freeze-dried.
4 © ISO 2020 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 16751:2020(E)

If samples shall be stored, store them in accordance with ISO 18512 and in such a way that processes

that have an effect on the bioavailability (biodegradation, change in organic matter composition) are

prevented.
7.2 Determining water content

Determine the water content of the test portion obtained after sample preparation (7.1) as specified in

ISO 11465.
7.3 Method A: Cyclodextrin
7.3.1 Extraction of the sample

Using a balance (6.2) weigh a test portion equivalent to (4 ± 1) g of dry material and place this in an

appropriate centrifuge tube (e.g. 50 ml) (6.3). Add 40 ml of the cyclodextrin extraction solution (5.4).

Place the tubes on the end-over-end shaker or the horizontal shaker (6.5) in the dark for 20 h at a

temperature of (20 ± 2) °C. Use appropriate glassware and plastic ware (6.8) in order to make phase

separation possible.

Use a mild agitation at a frequency that ensures that the ingredients are well mixed, and the breakdown

of soil particles is limited (6.5).
7.3.2 Phase separation

Centrifuge the tubes during 15 min at least at 2 000 g to obtain phase separation (clear supernatant).

Remove a portion of the supernatant, by pipetting (e.g. 10 ml) for further analysis.

The residual extraction pellet contains the non-bioavailable fraction of the contaminant. This pellet can

be used for further analysis to determine the non-bioavailable fraction of the contaminant (see 7.5.2).

NOTE Depending on the sample it can be necessary to use a centrifuge with a higher g-value and longer

centrifuge time. It is possible to get the same separation efficiency at other centrifugation conditions (shorter

centrifugation time at higher centrifugation speed or extended centrifugation time at lower centrifugation

speed). ISO 12782-1:2012, Annex A, provides a number of principles that need to be considered in order to ensure

reproducibility of the centrifugation when deviating from the recommended centrifugation procedure.

7.3.3 Extraction from receiver phase

Extract the 10 ml aliquot of the complexing aqueous cyclodextrin phase (see 7.3.2) with 5 ml petroleum

ether (5.6). Shake during at least 1 min. Repeat twice collecting all three aliquots of petroleum ether

into the same flask.
7.4 Method B: Tenax®
7.4.1 Extraction of the sample

Using a balance (6.2) weigh a test portion equivalent to (4 ± 1) g of dry material and place it in the

separation funnel (6.4) or a centrifuge tube (6.3), both of ~100 ml. Add 70 ml water (5.1). Add (1,5 ± 0,1) g

Tenax (5.5). Place the separation funnel on a horizontal shaking machine (6.5) in the dark for 20 h at a

temperature of (20 ± 2) °C.

1,5 g of Tenax is not sufficient to measure the availability of mineral oil. If it is necessary to measure

®[10]
the availability of mineral oil, use 4 g instead of 1,5 g of Tenax .

NOTE Some soils can obstruct the separation funnel (6.4). In such cases it is preferable to use a centrifuge

tube (6.3).

Use a mild agitation at a frequency that ensures that the ingredients are well mixed, and the breakdown

of soil particles is limited, e.g. 150 r/min to180 r/min.
© ISO 2020 – All rights reserved 5
---------------------- Page: 12 ----------------------
ISO 16751:2020(E)
7.4.2 Phase separation
7.4.2.1 Separation funnel

If a separation funnel is used in 7.4.1, then drain the aqueous phase including soil or soil-like material

carefully from the separation funnel (6.4). Tenax -polymers (beads) are hydrophobic and will float on

water and Tenax will be attached to the wall during phase separation. Remove all visible soil or soil-

like material by rinsing the Tenax with water (5.1) and drain the water (see Annex B for an example).

Collect the aqueous phase including soil or soil-like material in a suitable centrifuge tube. Centrifuge

the tubes during 15 min at least at 2 000 g to obtain the pellet with res
...

INTERNATIONAL ISO
STANDARD 16751
First edition
Soil quality — Environmental
availability of non-polar organic
compounds — Determination of the
potentially bioavailable fraction and
the non-bioavailable fraction using a
strong adsorbent or complexing agent
Qualité du sol — Disponibilité environnementale des composés
organiques non polaires — Détermination de la fraction
potentiellement biodisponible et de la fraction non biodisponible en
utilisant un agent adsorbant fort ou un agent complexant
PROOF/ÉPREUVE
Reference number
ISO 16751:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 16751:2020(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 PROOF/ÉPREUVE © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 16751:2020(E)
Contents Page

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

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

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

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

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

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

5 Reagents ........................................................................................................................................................................................................................ 3

6 Apparatus ..................................................................................................................................................................................................................... 3

7 Procedure..................................................................................................................................................................................................................... 4

7.1 Sample preparation ............................................................................................................................................................................ 4

7.2 Determining water content .......................................................................................................................................................... 5

7.3 Method A: Cyclodextrin ................................................................................................................................................................... 5

7.3.1 Extraction of the sample ........................................................................................................................................... 5

7.3.2 Phase separation .............................................................................................................................................................. 5

7.3.3 Extraction from receiver phase ........................................................................................................................... 5

7.4 Method B: Tenax® ............................................................................................................................................................................... 5

7.4.1 Extraction of the sample ........................................................................................................................................... 5

7.4.2 Phase separation .............................................................................................................................................................. 6

7.4.3 Extraction from receiver phase ........................................................................................................................... 6

7.5 Measurement ............................................................................................................................................................................................ 7

7.5.1 Potential bioavailable fraction ............................................................................................................................. 7

7.5.2 Non-bioavailable fraction ......................................................................................................................................... 7

7.6 Blank test ..................................................................................................................................................................................................... 7

8 Calculation .................................................................................................................................................................................................................. 7

8.1 Potential bioavailable fraction ................................................................................................................................................... 7

8.2 Non-bioavailable fraction .............................................................................................................................................................. 8

9 Expression of results ........................................................................................................................................................................................ 8

10 Validation ..................................................................................................................................................................................................................... 9

11 Test report ................................................................................................................................................................................................................... 9

Annex A (informative) Preparation and regeneration of Tenax .......................................................................................10

Annex B (informative) Illustrations....................................................................................................................................................................11

Annex C (informative) Validation study results .....................................................................................................................................14

Bibliography .............................................................................................................................................................................................................................25

© ISO 2020 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO 16751:2020(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 on 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 the following

URL: www .iso .org/ iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 7, Soil

and site assessment.

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.
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ISO 16751:2020(E)
Introduction

The solubility of most non-polar organic contaminants is limited and they are sorbed to the soil

matrix. They may desorb and become available for organisms, which may result in an effect (toxicity,

degradation or bioaccumulation). Not all sorbed (adsorbed and absorbed) contaminants will desorb

and become available.

Extractions used in chemical analysis to measure the total concentration, release more contaminants

from the soil than are available. It is however also possible that contaminants are so strongly bounded

by the soil that they will not be released by chemical extraction. This strong sorption may also be

caused by incorporation of the contaminant (or a degradation or reaction product of the contaminant)

in the organic soil structure. The distribution of contaminants over sorption sites of varying sorption

strength is not constant in time and contaminants will shift, with increasing contact time, to the

stronger sorption sites.
Figure 1 shows schematically the differentiation between:

— extractable residues that are also bioavailable (i.e. the potentially bioavailable fraction);

— residues that are extractable by harsher extraction methods but are non-bioavailable;

— residues that are neither extractable nor bioavailable.

If a degradable substance enters a soil, part of it will degrade over time (curve a). The area between

curve a and c is extractable by exhaustive chemical procedures. For risk assessments, this part is

considered as the “total concentration” for which values are defined in many regulations. However,

only a part of this amount is bioavailable. The area between curves a and b is the bioavailable fraction

and the area between curves b and c is the non-bioavailable fraction. The method described in this

document enables the measurement of the potential bioavailable and the non-bioavailable fraction of a

contaminant in soil.
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ISO 16751:2020(E)
Key
X time
Y contaminant concentration
1 degradable
2 bioavailable
3 extractable, non-bioavailable
4 non-extractable: persistent residues
5 non-available fraction
NOTE For curves a, b and c see description in text above.

Figure 1 — Temporal changes in extractable/bioavailable fractions, extractable/non-

bioavailable fractions and non-extractable/non-bioavailable fractions of a non-polar organic

contaminant (modified from [1])

In the scientific research to bioavailability a large number of definitions and concepts are in use, which

reflect the discussion in the scientific world. However, a more clear and simple approach is necessary.

In regulation, organic contaminants are either bioavailable or non-bioavailable. To support decisions,

both should be measurable. Therefore, this document follows the approach of Ortega-Calvo et al. (2015)

[2]

as illustrated in Figure 2. In this approach all defined fractions are measurable as further explained

in Clause 4.
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ISO 16751:2020(E)

Figure 2 — Measurement of bioavailability of organic chemicals: a simplified scheme for use in

[9]
regulation

The colour boxes at the left of the biological membrane represent the distribution of pollutant molecules

among four classes (non-extractable, very slowly/slowly desorbing, rapidly desorbing and water-

dissolved) in soils and sediments. In the scheme in Figure 2, the bioavailable chemical is represented

by the rapidly desorbing and dissolved concentrations. The chemical methods able to measure the

pollutant present in each specific fraction are given in the grey boxes. The green box to the right of

the cell membrane represents the processes that occur within the organism exposed to the pollutant.

These biological processes can also serve as the basis for standard methods used for bioavailability

measurements.

As presented in Figure 2, the bioavailable fraction can be measured using the method described in this

document.
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INTERNATIONAL STANDARD ISO 16751:2020(E)
Soil quality — Environmental availability of non-polar
organic compounds — Determination of the potentially
bioavailable fraction and the non-bioavailable fraction
using a strong adsorbent or complexing agent
1 Scope

This document specifies an extraction method to determine the bioavailable (potential and

environmental available) fraction and the non-bioavailable fraction of a contaminant in soil using a

“receiver phase” for an organic contaminant with strong sorbing or complexing properties, for example,

®1)
Tenax or cyclodextrin, respectively.

NOTE 1 The bioavailable fraction is defined in ISO 17402 as environmental bioavailability.

The method is applicable for non-polar organic contaminants with an aqueous solubility of <100 mg/l.

The method is applicable for soil and soil-like material including (dredged) sediments.

NOTE 2 The method is theoretically applicable to non-polar organic contaminants with an aqueous solubility

of 1 000 mg/l. The method has been often applied for compounds with a much lower solubility (K > 3) and less

for compounds with a higher solubility. The applicability is therefore defined for compounds with an aqueous

solubility of <100 mg/l.
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 11074, Soil quality — Vocabulary

ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis —

Gravimetric method

ISO 14507, Soil quality — Pretreatment of samples for determination of organic contaminants

ISO 18512, Soil quality — Guidance on long and short term storage of soil samples

3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 11074, ISO 17402 and the

following apply.

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/

1) Tenax is an example of a suitable product available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO of this product.

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ISO 16751:2020(E)
3.1
potential bioavailable fraction

amount of contaminant present in the matrix that can be released from the solid phase to the aqueous

phase in a well-mixed water soil mixture and in presence of a receiving phase in a period of 20 h

Note 1 to entry: In ISO 17924 the term bioaccessibility is used, which is the fraction of a substance in soil or soil-

like material that is liberated in (human) gastrointestinal juices and thus available for absorption. This document

does not distinguish between bioavailability and bioaccessibility and uses the general term bioavailability. The

concept of bioavailability as followed in this document is described in the introduction of this document.

4 Principle

This method gives an estimation of the potential bioavailable and non-bioavailable fraction of organic

contaminants, i.e. the amount of the contaminant in the matrix that is potentially exchangeable with

the aqueous phase; specifically that, which is adsorbed/complexed by Tenax /cyclodextrin.

The extractable and non-bioavailable fraction of the contaminant left in the sample following the action

of Tenax /cyclodextrin can be subsequently measured with an exhaustive/harsh extraction technique

(designed to measure the total concentration) and in this way the non-bioavailable fraction of the

contaminant is assessed.

Thus, in numerical terms, the total contaminant concentration in a sample is the sum of the bioavailable

concentration (established using a strong sorbent or complexing agent) and the non-bioavailable

concentration (established using a subsequent harsh extraction method performed on the residue that

is left after the matrix has been extracted using a strong sorbent or complexing agent):

cc=+ c (1)
tot,cont bionon-bio
where
c is the total contaminant concentration;
tot,cont
c is the bioavailable concentration;
bio
c is the non-bioavailable concentration.
non-bio

The soil, soil-like material or sediment sample with particle size <2 mm is extracted with water

containing a “receiver phase” for the organic contaminants. This phase is either a complexing agent

(cyclodextrin) or a strong adsorbent [Tenax ]. The solubility of non-polar compounds is limited and in

this method the receiver phase acts as an “infinite sink”. The measured amount, which is the amount

that desorbs from the soil or soil-like material during 20 h, reflects the fraction of contaminant that can

have effects on biotic systems and that can become mobile.

In the following step, the contaminants adsorbed are extracted from the receiver phase and determined

by appropriate analytical methods. The amount of contaminants left in the soil residue, the non-

bioavailable fraction, can be measured using a subsequent harsh/exhaustive extraction designed to

measure the total concentration. Formula (1) can then be used to determine the total contaminant

concentration in the sample (if desired).

NOTE 1 ISO 13859 and ISO 13876 are examples to measure the total concentration of respectively PAH and

PCB in soil and soil-like materials.

NOTE 2 Formula (1) shows the relationship between the “total contaminant concentration” the “bioavailable

concentration” and the “non-bioavailable concentration”. If two of the concentrations are known the third

concentration can be calculated through the use of Formula (1). For example, by measuring the “total

concentration” and the “non-bioavailable concentration”, the “bioavailable concentration” can be calculated.

This is allowed with homogeneous materials. If it is not known whether a material is homogeneous and the

bioavailable concentration is the concentration of interest, the bioavailable concentration needs to be measured.

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ISO 16751:2020(E)
5 Reagents

Reagents used shall be of suitable grade – analytical grade or higher– if not specified otherwise. The

blank value of the reagents (including water) shall be negligible compared to the lowest concentration

of organic contaminants to be determined.

5.1 Demineralized water or deionized water or water of equivalent purity (5 < pH < 7,5) with a

conductivity of <0,5 mS/m in accordance with grade 3 specified in ISO 3696.

If biodegradation of the target compounds is to be expected, add sodium azide (5.2) to water to a final

concentration of 0,2 g/l. This will minimize the biological degradation of the target compounds. If

biodegradation is not to be expected, it is not necessary to add sodium azide. This is the case for some

persistent target compounds, e.g. PCB.

NOTE With some soil samples it can be difficult to obtain a proper separation between the soil, aqueous

phase and Tenax . The use of 0,001 mol/l or 0,01 mol/l CaCl (5.9) instead of water can improve this separation.

5.2 Sodium azide, [CAS No. 26628-22-8], NaN .

WARNING — Attention is drawn to the hazard deriving from the use of the sodium azide which

is acutely toxic.

5.3 Cyclodextrin, (hydroxypropyl-β-cyclodextrin) of >97 % purity of a Food Grade, Medicine Grade or

Pharmaceutical Grade.

NOTE Analytical Grade cyclodextrin is very expensive. For this method Food Grade has shown to be fit for

purpose.

5.4 Extraction solution of cyclodextrin, dissolve 100 mmol (=146 g) of cyclodextrin (5.3) per litre of

water (5.1).

NOTE If biodegradation of the target compounds is to be expected, add sodium azide (5.2) to this solution

to a final concentration of 0,2 g/l. This will minimize the biological degradation of the target compounds. If

biodegradation is not to be expected, it is not necessary to add sodium azide. This is the case for some persistent

target compounds, e.g. PCB

5.5 2,6-diphenyleneoxide polymer (Tenax® TA), 60 mesh to 80 mesh. See Annex A for the

preparation and regeneration of Tenax .
5.6 Petroleum ether, [CAS No. 8032-32-4], boiling range 40 °C to 60 °C.
5.7 Ethanol, [CAS No. 64-17-5], C H O.
2 6
5.8 Acetone, [CAS No. 67-64-1], C H O.
3 6
5.9 Calcium chloride, [CAS no.1035-04-8], CaCl
5.10 Sodium sulfate, [CAS No. 231-820-9], Na SO
2 4.
6 Apparatus

Use the following equipment. All materials that come into contact with the sample (or reagents) shall

not adsorb the contaminant of interest and shall not contaminate the sample. Glass and PTFE are

suitable materials for most contaminants.
6.1 Sieving equipment, with 2 mm nominal screen size.
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ISO 16751:2020(E)
6.2 Balance, accuracy 0,01 g.
6.3 Conical centrifuge tubes, with screwing caps.
6.4 Separation funnel, of suitable size.

NOTE The hole in the tap of the separation funnel used for the Tenax extraction needs to be large enough

for the soil particles to leave the funnel. Otherwise no separation between Tenax and the sample is possible.

6.5 Shaking machine, which limits breakdown of sample particles, e.g. an end-over-end shaker,

capable of (20 ± 2) r/min or other mild agitation method or a horizontal movement shaker, capable to

have 150 r/min to 180 r/min.
6.6 Centrifuge, capable to centrifuge the centrifuge tubes (6.3).
6.7 Crushing equipment, jaw crusher or cutting device.
6.8 Appropriate glassware and plastic ware.
6.9 Kuderna Danish sample concentrator.
6.10 Collection vessel.
6.11 Folded filter.
7 Procedure
7.1 Sample preparation

The sample shall be pretreated in the laboratory in accordance with ISO 14507, but with the following

restrictions:

— Intensive pretreatment like grinding may have an effect on the environmental availability of

contaminants, therefore grinding is not allowed.

— In general, the test portion to be prepared shall have a grain size less than or equal to 2 mm, but on

no account the material shall be ground to reach this grain size.
— Remove stones, shells and any material not representative for the sample.

— Sieve the sample using a sieve (6.1). If necessary, press the material by hand through the sieve.

Instead of using ISO 14507, also freeze-drying according ISO 16720 may be used to pretreat the sample.

Some soils (e.g. peat and some sediments) are difficult to sieve. In these cases, remove stones, shells and

material not representative for the sample (e.g. plant material) by hand and process the sample without

sieving.

If the sample cannot be sieved at all because of its water content, reduce the water content until the

laboratory sample can be sieved. In the case of drying, the drying temperature shall not exceed 30 °C.

If a rapidly degradable fraction is to be expected, reduction of water content by air drying is not allowed.

In that case the original collected sample shall be forced by hand through a 2 mm sieve or the sample

can be freeze-dried.
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ISO 16751:2020(E)

If samples shall be stored, store them in accordance with ISO 18512 and in such a way that processes

that have an effect on the bioavailability (biodegradation, change in organic matter composition) are

prevented.
7.2 Determining water content

Determine the water content of the test portion obtained after sample preparation (7.1) as specified in

ISO 11465.
7.3 Method A: Cyclodextrin
7.3.1 Extraction of the sample

Using a balance (6.2) weigh a test portion equivalent to (4 ± 1) g of dry material and place this in an

appropriate centrifuge tube (e.g. 50 ml) (6.3). Add 40 ml of the cyclodextrin extraction solution (5.4).

Place the tubes on the end-over-end shaker or the horizontal shaker (6.5) in the dark for 20 h at a

temperature of (20 ± 2) °C. Use appropriate glassware and plastic ware (6.8) in order to make phase

separation possible.

Use a mild agitation at a frequency that ensures that the ingredients are well mixed, and the breakdown

of soil particles is limited (6.5).
7.3.2 Phase separation

Centrifuge the tubes during 15 min at least at 2 000 g to obtain phase separation (clear supernatant).

Remove a portion of the supernatant, by pipetting (e.g. 10 ml) for further analysis.

The residual extraction pellet contains the non-bioavailable fraction of the contaminant. This pellet can

be used for further analysis to determine the non-bioavailable fraction of the contaminant (see 7.5.2).

NOTE Depending on the sample it can be necessary to use a centrifuge with a higher g-value and longer

centrifuge time. It is possible to get the same separation efficiency at other centrifugation conditions (shorter

centrifugation time at higher centrifugation speed or extended centrifugation time at lower centrifugation

speed). ISO 12782-1:2012, Annex A, provides a number of principles that need to be considered in order to ensure

reproducibility of the centrifugation when deviating from the recommended centrifugation procedure.

7.3.3 Extraction from receiver phase

Extract the 10 ml aliquot of the complexing aqueous cyclodextrin phase (see 7.3.2) with 5 ml petroleum

ether (5.6). Shake during at least 1 min. Repeat twice collecting all three aliquots of petroleum ether

into the same flask.
7.4 Method B: Tenax®
7.4.1 Extraction of the sample

Using a balance (6.2) weigh a test portion equivalent to (4 ± 1) g of dry material and place it in the

separation funnel (6.4) or a centrifuge tube (6.3), both of ~100 ml. Add 70 ml water (5.1). Add (1,5 ± 0,1) g

Tenax (5.5). Place the separation funnel on a horizontal shaking machine (6.5) in the dark for 20 h at a

temperature of (20 ± 2) °C.

1,5 g of Tenax is not sufficient to measure the availability of mineral oil. If it is necessary to measure

®[10]
the availability of mineral oil, use 4 g instead of 1,5 g of Tenax .

NOTE Some soils can obstruct the separation funnel (6.4). In such cases it is preferable to use a centrifuge

tube (6.3).

Use a mild agitation at a frequency that ensures that the ingredients are well mixed, and the breakdown

of soil particles is limited, e.g. 150 r/min to180 r/min.
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ISO 16751:2020(E)
7.4.2 Phase separation
7.4.2.1 Separation funnel

If a separation funnel is used in 7.4.1, then drain the aqueous phase including soil or soil-like material

carefully from the separation funnel (6.4). Tenax -polymers (beads) are hydrophobic and will float on

water and Tenax will be attached to the wall during phase separation. Remove all visible soil or soil-

like material by rinsing the Tenax with water (5.1) and drain the water (see Annex B for an example).

Collect th
...

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