BS EN ISO 12782-5:2012

BSI Standards Publication

Soil quality — Parameters

for geochemical modelling
of leaching and speciation
of constituents in soils and
materials
Part 5: Extraction of humic substances from
aqueous samples (ISO 12782-5:2012)

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raising standards worldwide™

BS EN ISO 12782-5:2012 BRITISH STANDARD

National foreword
This British Standard is the UK implementation of EN ISO
12782-5:2012.
The UK participation in its preparation was entrusted to Technical
Committee EH/4, Soil quality.
A list of organizations represented on this committee can be
obtained on request to its secretary.
This publication does not purport to include all the necessary
provisions of a contract. Users are responsible for its correct
application.
© The British Standards Institution 2012. Published by BSI Standards
Limited 2012
ISBN 978 0 580 66915 6
ICS 13.080.05
Compliance with a British Standard cannot confer immunity from
legal obligations.
This British Standard was published under the authority of the
Standards Policy and Strategy Committee on 30 June 2012.
Amendments issued since publication
Date Text affected
EUROPEAN STANDARD EN ISO 12782-5
NORME EUROPÉENNE
EUROPÄISCHE NORM June 2012

ICS 13.080.05

English Version

Soil quality - Parameters for geochemical modelling of leaching

and speciation of constituents in soils and materials - Part 5:
Extraction of humic substances from aqueous samples (ISO
12782-5:2012)

Qualité du sol - Paramètres pour la modélisation Bodenbeschaffenheit - Parameter zur geochemischen

géochimique de la lixiviation et de la spéciation des Modellierung der Elution und Speziation von Bestandteilen
constituants des sols et des matériaux - Partie 5: Extraction in Böden und Materialien - Teil 5: Extraktion von
des substances humiques des échantillons aqueux (ISO Huminstoffen aus wässrigen Proben (ISO 12782-5:2012)
12782-5:2012)

This European Standard was approved by CEN on 31 May 2012.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION

COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 12782-5:2012: E
worldwide for CEN national Members.
BS EN ISO 12782-5:2012
EN ISO 12782-5:2012 (E)

Foreword
This document (EN ISO 12782-5:2012) has been prepared by Technical Committee ISO/TC 190 "Soil quality"
in collaboration with Technical Committee CEN/TC 345 “Characterization of soils” the secretariat of which is
held by NEN.

This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by December 2012, and conflicting national standards shall be withdrawn
at the latest by December 2012.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.

According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland, Turkey and the United Kingdom.

Endorsement notice

The text of ISO 12782-5:2012 has been approved by CEN as a EN ISO 12782-5:2012 without any
modification.

3
 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Contents Page

Foreword............................................................................................................................................................................. iv
Introduction......................................................................................................................................................................... v
1 Scope....................................................................................................................................................................... 1
2 Normative references.......................................................................................................................................... 1
3 Terms and definitions.......................................................................................................................................... 1
4 Principle.................................................................................................................................................................. 3
5 Apparatus............................................................................................................................................................... 3
6 Reagents................................................................................................................................................................. 3
7 Sampling................................................................................................................................................................. 4
7.1 Laboratory sample............................................................................................................................................... 4
7.2 Test sample............................................................................................................................................................ 4
7.3 Test portion............................................................................................................................................................ 4
8 Procedure............................................................................................................................................................... 4
8.1 Preparation of DAX-8 resin................................................................................................................................ 4
8.2 Determination of total humic acid (HA), fulvic acid (FA) and hydrophilic organic carbon (Hy)
content in aqueous samples............................................................................................................................. 5
9 Eluate treatment and storage............................................................................................................................ 5
10 Analytical determination.................................................................................................................................... 5
11 Blank test................................................................................................................................................................ 5
12 Calculation............................................................................................................................................................. 6
12.1 General correction factors for the calculation of humic acid (HA), fulvic acid (FA), hydrophilic
organic carbon (Hy) and hydrophobic neutral organic carbon (HON) in aqueous samples........... 6
12.2 Concentration of total humic acid (HA), fulvic acid (FA), hydrophilic organic carbon (Hy) and
hydrophobic neutral organic carbon (HON) concentrations in aqueous samples............................ 7
13 Expression of results.......................................................................................................................................... 8
14 Test report.............................................................................................................................................................. 8
15 Performance characteristics............................................................................................................................. 8
Annex A (informative) Schematic representation of the fractionation procedure............................................. 9
Annex B (informative) Validation of procedure......................................................................................................... 11
Annex C (informative) Conditions regarding centrifugation.................................................................................. 16
Bibliography...................................................................................................................................................................... 19

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BS EN ISO 12782-5:2012
ISO 12782-5:2012(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.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.

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.

ISO 12782‑5 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 7, Soil and
site assessment.

ISO 12782 consists of the following parts, under the general title Soil quality — Parameters for geochemical
modelling of leaching and speciation of constituents in soils and materials:

— Part 1: Extraction of amorphous iron oxides and hydroxides with ascorbic acid

— Part 2: Extraction of crystalline iron oxides and hydroxides with dithionite

— Part 3: Extraction of aluminium oxides and hydroxides with ammonium oxalate/oxalic acid

— Part 4: Extraction of humic substances from solid samples

— Part 5: Extraction of humic substances from aqueous samples

iv  © ISO 2012 – All rights reserved

 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Introduction

In addition to leaching procedures for subsequent chemical and ecotoxicological testing of soil and other
materials including waste, predictive models are becoming indispensable tools in the environmental risk
assessment of these materials. Models are particularly required when the results of laboratory leaching tests
are to be translated to specific scenarios in the field, with regard to assessing the risks of both contaminant
migration and bioavailability.

In the past few years, geochemical models have been shown to be valuable tools to be combined with the
data obtained from characterization leaching standards, such as pH-dependence and percolation tests. These
models have the advantage of being based on fundamental thermodynamic parameters that have a general
validity. In order to enable extrapolation of laboratory leaching data to the mobility and/or bioavailability of
a constituent in a specific field scenario, these models require additional input parameters for specific soil
properties (see Figure 1).

Key
1 experiment
2 geochemical speciation modelling
3 available metal concentration
4 dissolved humic substances
5 reactive (solid) surfaces
6 database with stability constants
7 computer program
8 assumptions

Figure 1 — Relationships between experimental data, as obtained from laboratory

leaching/extraction tests, and geochemical modelling of the speciation of a heavy metal
in the environment (modified after M. Gfeller & R. Schulin, ETH, Zürich)

Characterization leaching standards provide information on the concentrations of the contaminant of interest as
a function of, in particular, pH and liquid/solid (L/S) ratio. In addition, a more complete analysis of the leachates
also provides information on the major ion composition and dissolved organic carbon (DOC), parameters that
are particularly important for the chemical speciation of constituents through processes such as precipitation,
complexation and competition for adsorption on reactive mineral and organic surfaces in the soil. As illustrated

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BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

in Figure  1, for the example of copper, geochemical modelling enables calculation of the metal distribution
among these different chemical species in the system of interest. This provides necessary information for risk-
assessment purposes, as these different chemical forms play distinct roles in the mobility and bioavailability
of the metal in the soil. In addition to information obtained from the leaching standards (in their current state of
development/definition), two additional types of information are required.

a) The “available” (sometimes also referred to as “active” or “exchangeable”) concentration of the constituent
in the solid phase, as opposed to the total concentration determined by acid destruction of the solid matrix.
This “available” concentration can be obtained by leaching at low pH, a condition that can be obtained by
extending the pH range in the pH-dependent leaching test (ISO/TS 21268‑4) down to pH ≈ 0,5 to pH ≈ 1.

b) The concentration of reactive organic and mineral surfaces in the soil, which constitute the major binding
(adsorption) sites for most constituents in the soil matrix.

The major reactive surfaces that control the binding of constituents by sorption processes to the soil matrix
are particulate organic matter and iron and aluminium (hydr)oxides. It is generally accepted that the reactivity
of these mineral and organic surfaces can strongly vary as a function of their specific surface area/crystallinity
[iron and aluminium (hydr)oxides] and composition (organic matter). When the results are intended to be used
for the above-described purposes of geochemical modelling in conjunction with leaching tests, it is important
that the methods be selective for reactive surfaces for which generic thermodynamic adsorption parameters
are also available for the most important major and trace elements.

These reactive surfaces have been identified in soils, as well as in a variety of other materials for which the
leaching of constituents is of relevance. It has been shown that the binding properties of these surfaces play
a generic role in the speciation and leaching of constituents among these different materials. As an example,
a similar geochemical modelling approach, using model input from the partial or complete ISO 12782 series,
has been successfully applied to different soils[4], amended soils[5][6], municipal incinerator bottom ash[7], steel
slag[8][9], bauxite residues[10], and recycled concrete aggregate[11]. Hence, the scope of the ISO 12782 series
extends from soils to materials including soil amendments and waste materials.

This part of ISO 12782 aims to determine important reactive organic surfaces in soil and materials, for which
generic thermodynamic adsorption parameters exist, i.e., humic and fulvic acids. The procedure is based on
Reference [12], while generic thermodynamic adsorption parameters for humic and fulvic acids are available
in References [13] and [14].

Thermodynamic parameters for adsorption models other than those used in References [13] and [14] are also
available in the literature and may also be used to model the binding of constituents to humic and fulvic acids.

The method[15] is based on a conventional isolation and purification method[12] that is also used by the
International Humic Substances Society (IHSS).

vi  © ISO 2012 – All rights reserved

 BS EN ISO 12782-5:2012
INTERNATIONAL STANDARD ISO 12782-5:2012(E)

Soil quality — Parameters for geochemical modelling of

leaching and speciation of constituents in soils and materials —

Part 5:
Extraction of humic substances from aqueous samples

1 Scope
This part of ISO 12782 specifies a procedure to determine the concentration of humic substances in aqueous
samples. These samples may be obtained as such or as eluates from leaching procedures applied to soil or
other materials. Other materials also include waste. The content of humic substances can be used as input in
geochemical models.

2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced document
(including any amendments) applies.

ISO 3696, Water for analytical laboratory use — Specification and test methods

ISO 5667-3, Water quality — Sampling — Part 3: Preservation and handling of water samples

ISO  8245, Water quality — Guidelines for the determination of total organic carbon (TOC) and dissolved
organic carbon (DOC)

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.

3.1
dissolved organic carbon
DOC
sum of organically bound carbon present in water originating from compounds (including cyanate and
thiocyanate) which will pass a membrane filter of pore size 0,45 µm

3.2
humic substance
HS
(partial) decomposition product from plant and animal tissue

NOTE 1 Humic substances form a series of relatively high-molecular-weight, brown-to-black-coloured substances

formed by secondary synthesis reactions.

NOTE 2 The term is used as a generic name to describe coloured material or its fractions (e.g. humic and fulvic acids)
obtained on the basis of solubility characteristics.

3.3
humic acid
HA
fraction of a humic substance that is not soluble in water under acidic conditions (pH <1 to 2) but is soluble at
higher pH values

NOTE Humic acids are dark brown to black in colour.

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BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

3.4
fulvic acid
FA
fraction of a humic substance that is soluble in water under all pH conditions

NOTE 1 Fulvic acids remain in solution after removal of humic acid by acidification.

NOTE 2 Fulvic acids are light yellow to yellow-brown in colour.

3.5
hydrophilic organic carbon
Hy
organic carbon compound consisting of non-humic and humic-like substances

NOTE In this part of ISO  12782, Hy is essentially regarded as the extractable organic carbon fraction that is not
identified as humic acid, fulvic acid or hydrophobic neutral organic carbon in accordance with the procedure specified in
Clause 8. Hydrophilic organic carbon generally consists of molecules with a lower molecular weight and higher COOH/C
ratios than humic acids and fulvic acids. Examples of compounds are: oxidized carbohydrates with carboxylic acid groups,
low-molecular-weight carboxylic acids, and sugar phosphates.

3.6
hydrophobic neutral organic carbon
HON
difference between the amount of adsorbed fulvic acid and hydrophilic organic carbon and the amount of
desorbed fulvic acid

NOTE Hydrophobic neutral organic carbon can include non-humic and humic-like compounds.

3.7
laboratory sample
sample intended for laboratory inspection or testing

[ISO 11074:2005]

3.8
test sample
sample, prepared from the laboratory sample, from which the test portions are removed for testing or for
analysis; this portion of material, resulting from the laboratory sample by means of an appropriate method of
sample pretreatment, and having the size (volume/mass) necessary for the desired testing or analysis

NOTE Adapted from ISO 11074:2005.

3.9
test portion
analytical portion
quantity of material, of proper size, for measurement of the concentration or other property of interest, removed
from the test sample

NOTE 1 The test portion may be taken from the primary sample or from the laboratory sample directly if no preparation
of sample is required (e.g. with liquids), but usually it is taken from the prepared test sample.

NOTE 2 A unit or increment of proper homogeneity, size, and fineness, needing no further preparation, may be a test portion.

[ISO 11074:2005]

3.10
soil material
excavated soil, dredged material, manufactured soil, treated soil and fill material and other relevant materials,
including soil amendments and waste materials

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 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

4 Principle
Specific dissolved organic carbon species are isolated based on defined operational conditions. Humic acids
are precipitated at pH 1 and fulvic acids (and the hydrophobic organic neutral fraction) are adsorbed onto DAX-
8 resin. The organics remaining in solution after resin addition are classified as hydrophilic organic substances.
The DOC concentrations are measured after every step, from which the individual concentrations of humic and
fulvic acids, hydrophobic organic neutrals, and hydrophilic organic substances, are calculated.

5 Apparatus
The following apparatus shall be used. All materials that come in contact with the sample (material or reagents)
should not contaminate the compounds to be determined or adsorb the compounds of interest.

5.1 Balance, with an accuracy of 0,1 g.

5.2 Usual laboratory glass or plastic ware, rinsed in accordance with ISO 5667‑3.

5.3 pH-meter, with a measurement accuracy of at least ±0,05 pH units.

5.4 End-over-end shaking machine (5 min-1 to 10 min-1).

NOTE Other shaking methods can be used, provided that they can be shown to provide equivalent results.

5.5 Filtration apparatus, either a vacuum filtration device (between 2,5 kPa and 4,0 kPa) or a high-pressure
filtration apparatus (<0,5 MPa). Cleaning is compulsory.

5.6 Filters, pore size 20 µm, for use in the Büchner-funnel filtration device (5.7).

5.7 Büchner-funnel filtration device.

5.8 Membrane filters, for the filtration device, fabricated from inert material with a pore size of 0,45  µm.
Filters shall be pre-washed with demineralized water in order to remove DOC.

5.9 Soxhlet extraction device.

5.10 Soxhlet extraction thimbles, glass-fibre extraction thimbles for Soxhlet extraction device (5.9).

5.11 Centrifuge, preferably at 3 000g. For other appropriate conditions, see Annex C.

5.12 Centrifuge bottles, e.g. polycarbonate, of capacity 250  ml, cleaned with distilled and demineralized
water and diluted acid (HNO3) before use.

5.13 Crushing equipment: jaw crusher or cutting device.

5.14 Sieving equipment, with a nominal screen size of 2 mm or 4 mm.

6 Reagents
The reagents used shall be of analytical grade and the water used shall comply with grade 3 in accordance
with ISO 3696.

6.1 Demineralized water, deionized water or water of equivalent purity (5 < pH < 7,5) with a conductivity
< 0,5 mS/m according to grade 3 specified in ISO 3696.

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BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

6.2 Potassium hydroxide, c(KOH) = 0,1 mol/l and 1 mol/l.

6.3 Hydrochloric acid, c(HCl) = 0,1 mol/l to 6 mol/l.

6.4 Sodium hydroxide, c(NaOH) = 0,1 mol/l to 5 mol/l.

6.5 Acetonitrile, (CH3CN), suitable for liquid chromatography.

6.6 Methanol, (CH3OH), suitable for liquid chromatography.

6.7 DAX-8 resin, e.g. Sigma-Aldrich1).

NOTE Various documented methods for HS isolation and purification make use of XAD-8 resin to adsorb HA and/or
FA. This resin is no longer commercially available; therefore, the comparability of the substitute resin DAX-8 was tested.
See Annex B for information.

6.8 Nitric acid, c(HNO3) = 0,1 mol/l.

7 Sampling

7.1 Laboratory sample

The laboratory sample shall consist of a volume to at least 50 ml.

7.2 Test sample

The samples shall be tested on filtered (0,45 µm) liquids. The test samples can be of diverse origins and should be
stored in a refrigerator. To avoid changes in the samples during storage, NaN3 (0,1 % in solution) can be added.

Samples that have been spiked with NaN3 should be processed in a fume hood. NaN3 decomposes under
acidic conditions, releasing toxic gasses.

7.3 Test portion

Based on eluate volume requirements for analysis, the test portion size shall be 50 ml (with a tolerance of ±10 %).

8 Procedure

8.1 Preparation of DAX-8 resin

Clean every new batch of DAX-8 resin (6.7) to remove organic impurities with five 0,1  mol/l hydrochloric
acid (6.3) extractions (for 24  h). Renew the solution after each extraction. Repeat this cycle with 0,1  mol/l
sodium hydroxide (6.4). Then, clean the resin thoroughly by Soxhlet extractions (5.9) with acetonitrile (6.5) and
methanol (6.6), each for 24 h. The cleaned resin is stored in methanol (6.6) until use.

Prior to use, remove the methanol by placing the DAX-8 resin (6.7) in a Büchner funnel (5.7) with a filter (5.6)
and wash the resin under vacuum with water (6.1) that has a volume 20 times that of the resin. Subsequently,
rinse the resin similarly with 0,1 mol/l hydrochloric acid (6.3) having 10 times the resin volume.

NOTE It has been demonstrated[15] that 250 g of DAX-8 resin (6.7) can be cleaned sufficiently by rinsing with 2 l of
water (6.1) and 1 l of 0,1 mol/l hydrochloric acid (6.3). This cleaning sequence can be used to obtain a DOC-free (DOC
generally < 2 mg C/l) and acidic (pH 1) resin.

1) DAX-8 resin from Sigma‑Aldrich 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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 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

8.2 Determination of total humic acid (HA), fulvic acid (FA) and hydrophilic organic carbon
(Hy) content in aqueous samples
Weigh the test portion (7.3) in a centrifuge bottle (5.12). Acidify the test portion (Mw) with 1 mol/l hydrochloric
acid (6.3) to pH 1 to 2. Adjust the solution volume to 200 ml (L/S = 10) with 0,1 mol/l hydrochloric acid (6.3)
and record the total volume of added hydrochloric acid (1 mol/l and 0,1 mol/l) (V6). Close the centrifuge bottle,
equilibrate the suspension by continuous shaking (5.4) for 1  h and centrifuge for 30  min at 3  000g or at
appropriate centrifugation conditions as given in Annex  C. Remove the supernatant (FAHyHON1) from the
residue by decantation into a 250  ml bottle (5.2) and record the water volume (V7). Store the sample in a
refrigerator until DAX-8 treatment (see below).

Neutralize the test portion that remains in the centrifuge bottle with 1 mol/l sodium hydroxide (6.4) to pH = 7,0.
Add 0,1 mol/l sodium hydroxide under a N2 atmosphere to a final volume of 200 ml (L/S = 10). Check that the final
pH is ≥ 12 to ensure high HA solubility. If necessary, add 1 mol/l sodium hydroxide. Close the centrifuge bottle,
equilibrate the suspension overnight by continuous shaking (5.4) and centrifuge the suspension for 30 min at
3 000g or at appropriate centrifugation conditions as given in Annex C. Remove the supernatant from the residue
by decanting into a clean 250 ml centrifuge bottle (5.12) and record the volume of the decanted eluate (V1).

Acidify the supernatant to precipitate HA by adding 6 mol/l hydrochloric acid (6.3) (V2) while continuously stirring
until a pH of 1,0 is reached. Allow the suspension to stand overnight and centrifuge for 30 min at 3 000g or under
appropriate centrifugation conditions as given in Annex C. Remove the supernatant by decantation (V8) into a
250 ml bottle (5.2). Store the solution (FAHyHON2) in a refrigerator for DAX-8 treatment. Re‑dissolve the HA
that remains in the centrifuge bottle in 0,1 mol/l potassium hydroxide (6.2) (V5) and analyse for DOC (DOCHA).

Filter the stored solutions (FAHyHON1,2) over a membrane filter (5.5, 5.8) and analyse DOC (Clause 10)
(DOCFAHyHON1,2). Transfer 50 ml (V4,i) of the filtered solutions to (separate) 100 ml bottles (5.2) and add 10 g of
moist DAX-8 (8.1) (mDAX,i) to both samples. Equilibrate for 1 h by continuous shaking (5.4) and filter (5.6, 5.7)
the suspensions. Analyse DOC (Clause 10) in both solutions (DOCHy1,2). In order to desorb FA, transfer the
filtered resins to separate 50 ml bottles (5.2), add 20 ml of 0,1 mol/l potassium hydroxide (6.2) and equilibrate
by continuous shaking for 1 h (5.4). Filter (5.6, 5.7) the suspensions and collect the filtrates in 100 ml bottles.
Transfer the filtered resins to 50 ml bottles (5.2), add 20 ml of 1,0 mol/l potassium hydroxide (6.2) and repeat the
desorption of FA in three additional steps (1 h each). The pH should be > 11 and can be adjusted with 1 mol/l
potassium hydroxide (6.2) if necessary. Collect the four fractions of filtered potassium hydroxide in the same
100 ml bottle, record the total volume (V9,10,i) and analyse DOC (Clause 10) (DOCFA,i).

A schematic overview of the procedure is given in Annex A.

9 Eluate treatment and storage

Preserve the eluate sub-samples depending on the elements to be analysed and store them in accordance
with the requirements in ISO 5667-3.

10 Analytical determination
Analyse the samples in accordance with ISO 8245.

11 Blank test
Perform a blank test to determine the DOC contribution from each batch of cleaned DAX-8 resin (8.1). Add 10 g
of moist DAX-8 resin (mDAX,BL) to 50 ml of 0,1 mol/l hydrochloric acid (6.3) (V4,BL) after previous DOC analysis
(Clause 10) (DOCBL1). Allow the resin to settle for 5 min after 1 h of equilibration by continuous shaking (5.4),
and measure DOC (Clause 10) (DOCBL2).

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BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

12 Calculation

12.1 General correction factors for the calculation of humic acid (HA), fulvic acid (FA), hydro-
philic organic carbon (Hy) and hydrophobic neutral organic carbon (HON) in aqueous samples
Correction factor for acid addition used in HA precipitation:

(V1 + V2 )
f1 = (1)
V1

Correction factors for moisture content from DAX-8, f 2,i can be used for aqueous samples (i = L) and the blank
experiments (i = BL):

mDAX,i × wm,DAX × 0, 01
f 2,i = + 1 (2)
V 4,i

DOC contribution of DAX-8 in blank (BL) experiment, in milligrams of carbon per litre (mg C/l):

(
BlDAX = DOCBL2 ⋅ f 2,BL − DOCBL1 (3) )
where

V1 is the sample volume used for the determination of HA, FA, Hy and HON in aqueous samples,
in millilitres (ml);

V2 is the added volume of hydrochloric acid for precipitation of HA, in millilitres (ml);

mDAX,i is the wet mass of DAX-8 applied for the adsorption of FA or used in the blank experiment, in
grams (g) (see the last paragraph of this subclause);

wm,DAX is the moisture content of the cleaned DAX-8, as a percentage (%);

V4,i is the sample volume taken into account for the DAX-8 adsorption experiment, in millilitres (ml),
after removal of HA, or the amount of 0,1 mol/l hydrochloric acid used in the blank experiment
(see the last paragraph of this subclause). i =  L for aqueous samples and i =  BL in the blank
experiments;

DOCBL1 is the DOC concentration in the 0,1  mol/l hydrochloric acid solution used in the blank
experiment (mg C/l); when DOCBL1 < DTL, DOCBL1 = 0;

DOCBL2 is the DOC concentration in the 0,1 mol/l hydrochloric acid in the blank experiment after 1 h of
equilibration with DAX-8 (mg C/l).

For simplicity, it is recommended that both mDAX,i and V4,i be kept constant (10 g and 50 ml, respectively) in
both the samples (i =  L) and the blank experiments (i =  BL). In this case, the factor f 2,i will be constant in all
calculations.

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 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

12.2 Concentration of total humic acid (HA), fulvic acid (FA), hydrophilic organic carbon (Hy)
and hydrophobic neutral organic carbon (HON) concentrations in aqueous samples
Concentration of HA in solution, in milligrams of carbon per litre (mg C/l), corrected for DOC concentration of
remaining water in HA pellet after centrifugation (containing small concentration of FA + Hy). Only apply the
correction if V1 – V3 > 0.

DOCHA × V5 (V1 − V3 ) × DOCFAHyHON × f 1

HA = − (4)
V1 1 000

Concentration of Hy in solution, in milligrams of carbon per litre (mg C/l):

Hy = DOCHy × f 2,L − BlDAX (5)

Concentration of FA + HON in solution, in milligrams of carbon per litre (mg C/l):

( )
 FA + HON  = DOCFAHyHON × f 1 − Hy (6)

Concentration of DOCFA after correction of blank DAX-8 contribution, in milligrams of carbon per litre (mg C/l).

Only apply if DOCFA,measured,i – DOCBL,DAX > 0, otherwise 0 is used.

DOCFA,i = DOCFA,measured,i − DOCBL,DAX (7)

Concentration of FA in solution, in milligrams of carbon per litre (mg C/l):

4
∑ DOCFA,i × V11,i
FA = i=1 (8)
V 4,L

Concentration of HON in solution, in milligrams of carbon per litre (mg C/l):

HON =  FA + HON  − FA (9)

where

DOCHA is the measured DOC concentration of HA, in milligrams of carbon per litre (mg C/l);

V5 is the added volume of potassium hydroxide to dissolve the HA fraction, in millilitres

(ml);

V3 is the volume of supernatant after acid precipitation and centrifugation, in millilitres

(ml);

V11,i is the added volume(s) of potassium hydroxide to dissolve FA from DAX-8 in

aqueous samples; volumes are registered separately (i = 1 to 4);

DOCFAHyHON is the measured DOC concentration of the sample after removal of HA, in milligrams
of carbon per litre (mg C/l);

DOCHy is the measured DOC concentration of the sample after equilibration with DAX-8, in
milligrams of carbon per litre (mg C/l);

DOCFA,measured,i is the DOC concentration(s) in 0,1 mol/l potassium hydroxide after dissolution of FA

from DAX-8 in aqueous samples, in milligrams of carbon per litre (mg C/l). DOC
concentrations are registered separately (i = 1 to 4).

© ISO 2012 – All rights reserved  7

BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

13 Expression of results
Report the concentration of HA, FA, Hy and HON in the sample, in milligrams of carbon per litre (mg C/l).

14 Test report
The test report shall include at least the following details:

a) a reference to this part of ISO 12782;

b) any information necessary for the complete identification of the sample;

c) a reference to the method used for the analytical determination, i.e. ISO 8245;

d) the result of the determination;

e) any details that are optional or deviations from the specifications of this part of ISO 12782, and any effects
which may have affected the results.

15 Performance characteristics
The performance characteristics of the method are described in Reference [15].

8  © ISO 2012 – All rights reserved

 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Annex A
(informative)

Schematic representation of the fractionation procedure

© ISO 2012 – All rights reserved  9

BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Key
1 centrifugation
2 filtration
3 starting point for solid samples (see ISO 12782-4)
4 starting point for liquid samples
5 HA dissolution 0,1 M KOH
6 TOC analysis
7 DAX-8 addition, 1 h equilibration
8 FA dissolution 0,1 M KOH, 1 h
9 5 min settling

Figure A.1 — Schematic representation of the fractionation procedure

10  © ISO 2012 – All rights reserved

 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Annex B
(informative)

Validation of procedure

B.1 General
The validation of the procedure specified in this part of ISO 12782 is also described in Reference [15]. Several
choices made in the development of the procedure are described in this annex.

B.2 Notes on the use of cleaned DAX-8

The average moisture content (wm,DAX) of the cleaned moist resin was (60,3 ± 3,4) % (n = 5). Small changes
in moisture content have a limited effect on the final results, so this average value was used to correct the
measured FA and Hy concentrations for the resin’s water content.

After severe cleaning of DAX-8, a blank DOC concentration of about 2 mg C/l was found in the procedure.
The molecular size of the residual DOC in cleaned DAX-8 was < 100 u (atomic mass units), based on high-
performance size-exclusion chromatography. Therefore, it is assumed that the residual DOC originates from
resin bleeding rather than from residual FA.

B.3 Preliminary investigations of DAX-8 and XAD-8 performance

The adsorption of FA on XAD-8 and DAX-8 was studied at different resin additions and the adsorption time
was measured. Moreover, the reversibility of the adsorption process was measured because FA are generally
identified on the basis of their adsorption and subsequent desorption from XAD-8[12][16][17][18]. The results
of these preliminary investigations are given in B.2 and this subclause. It should be noted that the DAX-8
resin tends to adsorb slightly greater amounts (up to 5 %) of FA as compared to XAD-8. These results are
consistent with Reference [19]. These studies concluded that the XAD-8 and DAX-8 resins isolate mixtures
of components with generally similar structural compositions, although the content of aliphatics within the
extracted HS is slightly greater for DAX-8. It should be noted that the results in Reference [19] were based on
mixtures of HA and FA, whereas this annex only focuses on the equivalence for FA sorption and desorption. It
is concluded that XAD-8 and DAX-8 are equivalent with regard to the estimation of FA concentrations and that
the use of this procedure with DAX-8 is compatible with the standard procedures recommended by the IHSS.

See Figures B.1 and B.2.

© ISO 2012 – All rights reserved  11

BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Key
X amount of moist XAD-8/DAX-8, in grams (g)
Y FA adsorbed, as a percentage (%)

NOTE The circles represent experiments with XAD-8 resin whereas the squares show the results with DAX-8 resin. The
vertical line indicates the amount of resin selected for the standard procedure.

Figure B.1 — Adsorption of Elliot soil FA (28 mg C/l) as a function

of the amount of XAD-8 or DAX-8 resin (moist)

a) b)

Key Key
X adsorption time, in hours (h) X desorption step
Y FA adsorbed, as a percentage (%) Y FA adsorbed, as a percentage (%)

NOTE The experiments were performed with 10 g of moist resin for 50 ml of sample solution. The vertical line indicates
the selected adsorption time for the standard procedure.

Figure B.2 — Adsorption of FA as a function of time [a)] and desorption

characteristics of XAD-8 and DAX-8 [b)] for Elliot soil FA (28 mg C/l)

B.4 Concentration-dependent precipitation behaviour of HA

Figure B.3 reflects the HA recovery from precipitation as a function of the measured HA concentrations. The
fitted relationships for solid and aquatic HA can be used to approximately account for the precipitation efficiency.
Please note that these general relationships might not be adequate enough in specific samples.

12  © ISO 2012 – All rights reserved

 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

a) b) c)

Key
X HA measured (mg C/l)
Y1 recovery, as a percentage (%) of HA
Y2 recovery, as a percentage (%) of FA
Y3 recovery, as a percentage (%) of Hy

NOTE The open symbols represent isolated aquatic HA: Suwannee river (circle), Landfill leachate (square) and
Zwanenwater (triangle). The solid line is the fitted curve [y  =  10,46 ln(x) +  32,93, r 2 = 0,86, n  =  3; r 2: coefficient of
determination] based on data from peat, soil, compost and the HA derived from the landfill waste mixture; the dashed line
is the fitted HA concentration dependency [y = 8,28 ln(x) + 12,88, r 2 = 0,52, n = 27] based on the data from three aquatic
HA samples. Graphs b) and c) show the percentage of DOC (originating from HA) measured as FA and Hy, respectively,
as a function of the measured HA.

Figure B.3 — Recovery of purified HA as a function of the measured HA concentration for the peat
(black square), Elliot soil (black triangle), Elliot soil after high-speed centrifugation (black diamond),
compost (black circle) and the landfill waste mixture (+) solid-source materials (Graph a)
(HON was not detected in these samples)

B.5 Chemical characterization of HA precipitates obtained at different concentrations

Additional experiments were performed with HA isolated from compost, to chemically characterize the fractions
obtained at different concentrations in the batch method. HA fractions precipitated at five different initial
concentrations (10 mg to 100 mg C/l) were again purified by dialysis, freeze-dried and chemically characterized
by high-precision size-exclusion chromatography (HPSEC), ultraviolet/visible (UV/VIS) absorbance (254 nm
to 665 nm) and elemental analysis (C, H, N, O). The results are shown in Tables B.1 (where ε is the molar
extinction coefficient) and B.2. The HPSEC chromatograms are shown in Figure  B.4. The E465/E665 ratio
seems to be higher in the original sample; this property is not reflected in the other isolates. It is unclear why
the absorbance of the original sample at 665 nm is relatively low in comparison with all other results, although it
should be noted that very little absorption is measured at this wavelength. The other chemical properties seem
to be comparable with each other, irrespective of the sample. The HPSEC chromatograms (Figure B.4) show
(other than the peak height due to different concentrations) no significant differences in the apparent molecular
size distribution. It is therefore concluded that these techniques reveal no significant differences between the
HA fractions that were precipitated from low to high concentrations (0,5 mg to 100 mg C/l).

© ISO 2012 – All rights reserved  13

BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Table B.1 — UV/VIS characteristics of previously purified compost HA (original) and reprecipitated

compost HA samples that were subsequently dissolved at different concentrations
(Aromaticity was calculated from the absorbance at 254 nm according to Reference [20].)

E465/
E300/
Precipitation DOC 254 280 300 400 465 665 E665 ε (254) Aromaticity
E400
ratio
(mg nm nm nm nm nm nm
Concentration (- ) ( -) mol/l·cm %
C/l) (abs) (abs) (abs) (abs) (abs) (abs)
Original 15,1 0,767 0,668 0,577 0,233 0,124 0,018 2,5 6,9 611 33,3
10 mg C/l 13,5 0,731 0,626 0,541 0,235 0,142 0,058 2,3 2,5 556 34,5
20 mg C/l 15,2 0,769 0,677 0,585 0,261 0,154 0,058 2,2 2,7 534 33,4
30 mg C/l 13,6 0,743 0,645 0,553 0,25 0,145 0,054 2,2 2,7 568 35,1
50 mg C/l 14,4 0,767 0,675 0,584 0,25 0,145 0,055 2,3 2,6 561 34,8
100 mg C/l 14,7 0,803 0,703 0,608 0,255 0,15 0,058 2,4 2,6 572 35,4

Table B.2 — Elemental analysis of previously purified compost HA (original) and reprecipitated

compost HA samples that were subsequently dissolved at different concentrations. Elemental
composition was determined on a dry-matter basis
(The original material was isolated and purified with the conventional isolation procedure.)

Precipitation C H N O Sum CHNO O/C ratio

concentration % % % % % (-)
Original 55,78 4,82 7,77 27,71 96,1 0,61
10 mg C/l 53,31 4,46 6,84 29,17 93,8 0,53
20 mg C/l 54,52 4,83 7,4 29,04 95,8 0,57
30 mg C/l 53,35 4,74 7,26 20,55 85,9 0,56
50 mg C/l 53,86 4,77 7,19 27,1 92,9 0,55
100 mg C/l 54,27 4,79 7,3 27,12 93,5 0,61

14  © ISO 2012 – All rights reserved

 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Key
X time, in min
Y absorption, in mAU (milli- absorbance unit)

Figure B.4 — HPSEC chromatograms of previously purified compost HA (original) and reprecipitated

compost HA samples that were subsequently dissolved at different concentrations

© ISO 2012 – All rights reserved  15

BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Annex C
(informative)

Conditions regarding centrifugation

C.1 General
According to this part of ISO 12782, the first step of the solid/liquid separation shall be done by centrifugation.
It is recommended centrifuging at 3 000g for 30 min.

However, 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). In order to ensure reproducibility of the centrifugation, the following principles shall be considered
when deviating from the recommended centrifugation procedure.

Generally the relative centrifugal force (Fc,r, in g) depends on the rotor speed, n (revolutions per minute, min−1)
and the rotor radius, r (in cm), and is calculated using Equation (C.1):

2
Fc,r = 0, 000 01118 × ( n ) × r (C.1)

Drawing on this, each rotor has a specific k-factor that describes its pelleting efficiency. The lower the k‑factor,
the more efficient the pelleting will be.

This factor can be used to determine the time required for reproducible centrifugation at different rotor speeds.
The k-factor can be calculated using Equation (C.2):

2, 53 × ( ln rmax − ln rmin )
k= × 1011 (C.2)
2
(n)
where

k is the rotor-specific factor;

rmax is the maximum radius from the axis, in centimetres (cm);

rmin is the minimum radius from the axis, in centimetres (cm);

n is the rotor speed, in revolutions per minute (min -1).

To calculate the time needed at different rotor speeds to get the same centrifugation result, the k-factors for
both rotor speeds shall be calculated. Using Equation (C.3), the times can be determined:

ka
ta = tb ⋅ (C.3)
kb

where

ka is the rotor-specific factor at rotor speed a;

k b is the rotor-specific factor at rotor speed b;

t a is the centrifugation time for rotor speed a, needed to achieve the same separation efficiency as for
rotor speed b in the time tb, in minutes (min);

tb is the centrifugation time for rotor speed b, in minutes (min).

16  © ISO 2012 – All rights reserved

 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

C.2 Example for calculation

Rotor dimension: rmax = 9 cm, rmin = 3 cm, r  = 6 cm

n needed for Fc,r = 3 000g [see Equation (C.1)]:

3 000
n= = 6 694 min −1
0, 000 011 18 × 6

Desired n: 3 000 min -1

Question: 6 694 min -1 for 30 min correspond to 3 000 min -1 for x min.

Calculation of k-factors [see Equation (C.2)]:

a) 6 694 min -1:

2, 53 × ( ln 9 − ln 3 )
k 6 694 = × 1011 = 6 203
2
6 694

a) 3 000 min-1:

2, 53 × ( ln 9 − ln 3 )
k 3 000 = × 1011 = 30 883
2
3 000

Time needed at 3 000 min -1 [see Equation (C.3)]:

30 883
t 3 000 = 30 × = 149, 4 min
6 203

Answer: at 3 000 min -1 the centrifugation time shall be 149 min.

Figure  C.1 shows a plot of the centrifugation time, t, versus the rotation speed, n, for the rotor dimension
described above (based on 3 000g for 30 min).

© ISO 2012 – All rights reserved  17

BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Key
X centrifugation time, in min
Y rotor speed, in r/min

Figure C.1 — Plot of the centrifugation time, t, versus the rotation speed, n,

for the rotor dimension described above (based on 3 000g for 30 min)

18  © ISO 2012 – All rights reserved

 BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

Bibliography

[1] ISO 11074:2005, Soil quality — Vocabulary

[2] ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis —
Gravimetric method

[3] ISO/TS 21268-4, Soil quality — Leaching procedures for subsequent chemical and ecotoxicological
testing of soil and soil materials — Part 4: Influence of pH on leaching with initial acid/base addition

[4] Dijkstra, J.J.; Meeussen, J.C.L.; Comans, R.N.J. Evaluation of a generic multi-surface sorption model

for inorganic soil contaminants. Environ. Sci. Technol. 2009, 43, pp. 6196-6201

[5] Khai, N.M.; Oborn, I.; Hillier, S.; Gustafsson, J.P. Modeling of metal binding in tropical Fluvisols and
Acrisols treated with biosolids and wastewater. Chemosphere, 2008, 70, pp. 1338-1346

[6] Carter,  C.M.; Van  der Sloot,  H.A.; Cooling,  D. pH dependent extraction of soils and soil
amendments to understand the factors controlling element mobility - New approach to assess soil and
soil amendments. Eur. J. Soil Sci. 2009, 60, pp. 622-637

[7] Dijkstra, J.J.; Van Zomeren, A.; Meeussen, J.C.L.; Comans, R.N.J. Effect of Accelerated Aging of

MSWI Bottom Ash on the Leaching Mechanisms of Copper and Molybdenum. Environ. Sci. Technol.
2006, 40, pp. 4481-4487

[8] Apul,  D.S.; Gardner,  K.H.; Eighmy,  T.T.; Fallman,  A.; Comans,  R.N.J. Simultaneous application
dissolution/precipitation and surface complexation/surface precipitation modeling to contaminant
leaching. Environ. Sci. Technol. 2005, 39, pp. 5736-5741

[9] Huijgen, W.J.J.; Comans, R.N.J. Carbonation of steel slag for CO2 sequestration: leaching of products
and reaction mechanism. Environ. Sci. Technol. 2006, 40, pp. 2790-2796

[10] Carter, C.M.; Van der Sloot, H.A.; Cooling, D.; Van Zomeren, A.; Matheson, T. Characterization of

untreated and neutralized bauxite residue for improved waste management. Environ. Eng. Sci. 2008,
25, pp. 475-489

[11] Engelsen, C.J.; Van der Sloot, H.A.; Wibetoe, G.; Petkovic, G.; Stoltenberg -Hansson, E.; Lund, W.

Release of major elements from recycled concrete aggregates and geochemical modelling. Cement
and concrete research. 2009, 39, pp. 446-459

[12] Thurman,  E.M., Malcolm,  R.L. Preparative isolation of aquatic humic substances, Environ. Sci.
Technol., 1981, 15, pp. 463-466

[13] Milne, C.J., Kinniburgh, D.G., Tipping, E. Generic NICA-Donnan model parameters for proton binding
by humic substances, Environ. Sci. Technol., 2001, 35, pp. 2049-2059

[14] Milne,  C.J., Kinniburgh,  D.G., van Riemsdijk,  W.H., Tipping,  E. Generic NICA-Donnan model
parameters for metal-ion binding by humic substances, Environ. Sci. Technol., 2003, 37, pp. 958-971

[15] van Zomeren, A., Comans, R.N.J. Measurement of humic and fulvic acid concentrations and dissolution
properties by a rapid batch procedure, Environ. Sci. Technol., 2007, 41, pp. 6755-6761

[16] Swift, R.S. Organic matter characterization. In Methods of soil analysis. Part 3. Chemical methods;
Sparks, D.L., Ed.; Soil Science Society of America: Madison, WI, 1996; pp. 1011-1069

[17] Hayes, M.H.B.; Swift, R.S. Genesis, isolation, composition and structures of soil humic substances.
In Soil colloids and their associations in aggregates; de Boodt, M. F., Ed.; Plenum Press: New York,
1990; pp. 245-305

[18] Aiken,  G.R., Thurman,  E.M., Malcolm,  R.L. Comparison of XAD macroporous resins for the
concentration of fulvic acid from aqueous solution, Anal. Chem., 1979, 51, pp. 1799-1803

© ISO 2012 – All rights reserved  19

BS EN ISO 12782-5:2012
ISO 12782-5:2012(E)

[19] Peuravuori, J., Lehtonen, T., Pihlaja, K. Sorption of aquatic humic matter by DAX-8 and XAD-8 resins
- Comparative study using pyrolysis gas chromatography 5, Anal. Chim. Acta, 2002, 471, pp. 219-226

[20] Chin, Y.P., Aiken, G.R., O’Loughlin, E. Molecular weight, polydispersity, and spectroscopic properties
of aquatic humic substances, Environ. Sci. Technol., 1994, 28, pp. 1853-1858

[21] Gfeller, M., Schulin, R. ETH Zürich, http://www.randomdots.net

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