EN ISO 5815-1:2019

SLOVENSKI STANDARD
01-november-2019
Nadomešča:
SIST EN 1899-1:2000
SIST EN 1899-2:2000
Kakovost vode - Določevanje biokemijske potrebe po kisiku po n dneh (BPKn) - 1.
del: Metoda razredčevanja in cepljenja z dodatkom aliltiosečnine (ISO 5815-1:2019)
Water quality - Determination of biochemical oxygen demand after n days (BODn) - Part
1: Dilution and seeding method with allylthiourea addition (ISO 5815-1:2019)
Wasserbeschaffenheit - Bestimmung des biochemischen Sauerstoffbedarfs nach n
Tagen (BSBn) - Teil 1: Verdünnungs- und Impfverfahren mit Zugabe von Allylthioharnstoff
(ISO 5815-1:2019)
Qualité de l'eau - Détermination de la demande biochimique en oxygène après n jours
(DBOn) - Partie 1: Méthode par dilution et ensemencement avec apport d'allylthiourée
(ISO 5815-1:2019)
Ta slovenski standard je istoveten z: EN ISO 5815-1:2019
ICS:
13.060.50 Preiskava vode na kemične Examination of water for
snovi chemical substances
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 5815-1
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2019
EUROPÄISCHE NORM
ICS 13.060.50 Supersedes EN 1899-2:1998
English Version
Water quality - Determination of biochemical oxygen
demand after n days (BODn) - Part 1: Dilution and seeding
method with allylthiourea addition (ISO 5815-1:2019)
Qualité de l'eau - Détermination de la demande Wasserbeschaffenheit - Bestimmung des
biochimique en oxygène après n jours (DBOn) - Partie biochemischen Sauerstoffbedarfs nach n Tagen (BSBn)
1: Méthode par dilution et ensemencement avec apport - Teil 1: Verdünnungs- und Impfverfahren mit Zugabe
d'allylthiourée (ISO 5815-1:2019) von Allylthioharnstoff (ISO 5815-1:2019)
This European Standard was approved by CEN on 26 July 2019.

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, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 5815-1:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 5815-1:2019) has been prepared by Technical Committee ISO/TC 147 "Water
quality" in collaboration with Technical Committee CEN/TC 230 “Water analysis” the secretariat of
which is held by DIN.
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 March 2020, and conflicting national standards shall
be withdrawn at the latest by March 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 1899-2:1998.
According to the CEN-CENELEC Internal Regulations, the national standards organizations 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, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 5815-1:2019 has been approved by CEN as EN ISO 5815-1:2019 without any
modification.
INTERNATIONAL ISO
STANDARD 5815-1
Second edition
2019-07
Water quality — Determination of
biochemical oxygen demand after n
days (BOD ) —
n
Part 1:
Dilution and seeding method with
allylthiourea addition
Qualité de l'eau — Détermination de la demande biochimique en
oxygène après n jours (DBO ) —
n
Partie 1: Méthode par dilution et ensemencement avec apport
d'allylthiourée
Reference number
ISO 5815-1:2019(E)
©
ISO 2019
ISO 5815-1:2019(E)
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
ii © ISO 2019 – All rights reserved

ISO 5815-1:2019(E)
Contents Page
Foreword .iv
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Reagents . 3
6 Apparatus . 6
7 Sampling and preservation . 6
8 Interferences . 7
8.1 General . 7
8.2 Presence of free and/or combined chlorine . 7
8.3 Presence of algae . 7
8.4 Presence of peroxides and peroxide compounds. 8
9 Procedure. 8
9.1 General . 8
9.2 Pretreatment . 8
9.2.1 Neutralization of the sample . 8
9.2.2 Homogenization . 9
9.3 Preparation of test solutions . 9
9.4 Calculation of dilutions . 9
9.4.1 Empirical determination of the dilutions . 9
9.4.2 Determination of dilutions via the factors R of the TOC, the permanganate
index or the COD .10
9.4.3 Calculation of dilution stages via the COD .11
9.5 Blank value determination .11
9.6 Determination of dissolved oxygen .11
9.6.1 Measurement of dissolved oxygen using iodometric method (in
accordance with ISO 5813) .11
9.6.2 Measurement of dissolved oxygen using probes (in accordance with
ISO 5814 or ISO 17289) .12
9.7 Control analysis .12
10 Calculation and indication of the results .13
10.1 Examination of test solutions for valid oxygen consumption during test .13
10.2 Calculation of biochemical oxygen demand after n days (BOD ) .13
n
10.3 Validity criteria .14
11 Test report .14
Annex A (normative) Influence of incubation periods and temperatures .15
Annex B (informative) Multitesting .16
Annex C (informative) Direct seeding of the analysis batches .19
Annex D (informative) Performance data .20
Bibliography .22
ISO 5815-1:2019(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 147, Water quality, Subcommittee SC 5,
Biological methods.
This second edition cancels and replaces the first edition (ISO 5815-1:2003), which has been technically
revised. The main changes compared to the previous edition are as follows:
— change of working range: 1 mg/l instead of 3 mg/l as lower limit;
— changes in test procedure;
— in 5.2, option to check seeding water suitability in advance with a CGA control analysis batch;
— in 5.3.2, phosphate buffer solution pH-value: requirement for preparation of a new solution if the pH
value is out of the range pH 7 and pH 8;
— in 5.5, range for oxygen consumption of seeded dilution water 0,2 mg/l to 1,5 mg/l instead of upper
limit 1,5 mg/l;
— in 5.9, allowable range BOD of the CGA control solution changed to (198 ± 40) mg/l and BOD
5 7
(206 ± 40) mg/l;
— in 6.5, electrochemical probe option to measure the dissolved oxygen concentration added;
— in 8.4, interferences: subclause on presence of peroxides and peroxide compounds added;
— in 9.4, options to determinate the dilutions elaborated;
— in 9.7, control analysis: elaborated description of procedure;
— in 10.3, "approval of results/validity criteria” added;
— Annex A: title changed and “normative” instead of “informative”
— Annex C "Direct seeding of the analysis batches" added;
iv © ISO 2019 – All rights reserved

ISO 5815-1:2019(E)
— new Annex D "Performance data" included.
A list of all parts in the ISO 5815 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
ISO 5815-1:2019(E)
Introduction
The incubation time specified in this document is 5 d or 7 d. The latter corresponds to the practice in
several Nordic countries. Annex A describes an incubation time of (2 + 5) d.
ISO 5815-1 specifies the determination of the biochemical oxygen demand (BOD) of waters with an
expected BOD in the range 1 mg/l to 6 000 mg/l using the dilution method. A lower limit of working
range may result from validation data in the laboratory. For samples with an expected low BOD in the
range of 0,5 mg/l to 6 mg/l ISO 5815-2 provides the option of the determination of the (BOD) of waters
using undiluted samples.
vi © ISO 2019 – All rights reserved

INTERNATIONAL STANDARD ISO 5815-1:2019(E)
Water quality — Determination of biochemical oxygen
demand after n days (BOD ) —
n
Part 1:
Dilution and seeding method with allylthiourea addition
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably qualified staff.
1 Scope
This document specifies the determination of the biochemical oxygen demand of waters by dilution and
seeding with suppression of nitrification after 5 d or 7 d incubation time.
It is applicable to all waters having biochemical oxygen demands usually between 1 mg/l and
6 000 mg/l. It applies particularly to waste waters but also suits for the analysis of natural waters.
For biochemical oxygen demands greater than 6 000 mg/l of oxygen, the method is still applicable, but
special care is needed taking into consideration the representativeness of subsampling for preparation
of the dilution steps. The results obtained are the product of a combination of biochemical and chemical
reactions in presence of living matter which behaves only with occasional reproducibility. The results
do not have the rigorous and unambiguous character of those resulting from, for example, a single,
well-defined, chemical process. Nevertheless, the results provide an indication from which the quality
of waters can be estimated.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 5667-3, Water quality — Preservation and handling of water samples
ISO 5813, Water quality — Determination of dissolved oxygen — Iodometric method
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 6060, Water quality — Determination of the chemical oxygen demand
ISO 8245, Water quality — Guidelines for the determination of total organic carbon (TOC) and dissolved
organic carbon (DOC)
ISO 8467, Water quality — Determination of permanganate index
ISO 10523, Water quality — Determination of pH
ISO 15705, Water quality — Determination of the chemical oxygen demand index (ST-COD) — Small-scale
sealed-tube method
ISO 17289, Water quality — Determination of dissolved oxygen — Optical sensor method
ISO 5815-1:2019(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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/
3.1
biochemical oxygen demand after n days
BOD
n
mass concentration of dissolved oxygen consumed under specified conditions by the biochemical
oxidation of organic and/or inorganic matter in water where n is the incubation time equal to 5 d or 7 d
Note 1 to entry: For the purposes of this document “biochemical oxidation” is taken to mean “biological oxidation”.
Note 2 to entry: n is either 5 or 7.
3.2
chemical oxygen demand
COD
mass concentration of oxygen equivalent to the amount of dichromate consumed by dissolved and
suspended matter when a water sample is treated with that oxidant under defined conditions
[SOURCE: ISO 6060:1989, 3]
3.3
total organic carbon
TOC
sum of organically bound carbon present in water, bonded to dissolved or suspended matter, including
cyanate, elemental carbon and thiocyanate
[SOURCE: ISO 8245:1999, 3.3]
3.4
permanganate index (of water)
mass concentration of oxygen equivalent to the amount of permanganate ion consumed when a water
sample is treated with that oxidant under defined conditions
[SOURCE: ISO 8467:1993, 3.1]
3.5
seeding water
water with adapted (aerobic) microorganisms via which the oxidation of the water contents occurs
Note 1 to entry: The seeding water is used for producing the seeded dilution water.
3.6
dilution water
water added to the test sample to prepare a series of defined dilutions
[SOURCE: ISO 20079:2005, 3.7]
3.7
seeded dilution water
dilution water to which a definite amount of seeding water is added
2 © ISO 2019 – All rights reserved

ISO 5815-1:2019(E)
3.8
free chlorine
chlorine present in the form of hypochlorous acid, hypochlorite ion or dissolved elemental chlorine
[SOURCE: ISO 7393-1:1985, 2.1]
3.9
combined chlorine
fraction of total chlorine present in the form of chloramines and organic chloramines
[SOURCE: ISO 7393-1:1985, 2.2]
3.10
nitrification
oxidation of ammonium salts by bacteria where usually the intermediate product is nitrite and the end
product nitrate
[SOURCE: ISO 11733:2004, 3.9]
4 Principle
The BOD , with inhibition of nitrification is determined, using the dilution method. A batch series with
n
different dilutions of a sample is prepared and examined. The dilution water is enriched with oxygen
and seeded with adapted aerobic microorganisms.
The sample is incubated at (20 ± 1) °C for a specified period (n), 5 d or 7 d, in the dark, in a completely
filled and stoppered bottle. The dissolved oxygen concentration is determined before and after
incubation. The mass of consumed oxygen per litre sample is calculated.
5 Reagents
Use only reagents with the degree of purity "for analysis".
5.1 Water, at least grade 3 in accordance with ISO 3696.
The water shall not contain more than 0,01 mg/l of copper, nor chlorine or chloramines.
5.2 Seeding water, which can be obtained in one of the following ways:
a) municipal waste water, decanted or coarsely filtered;
b) surface water containing municipal waste water;
c) settled effluent from a waste water treatment plant;
d) water taken downstream from the discharge of the water to be analysed, or water containing
microorganisms that are adapted to the water to be analysed;
e) commercially available seeding material.
Use seeding water with a COD of about 300 mg/l or a TOC of about 100 mg/l (see 5.5). If the COD or TOC
are higher, adapt to these concentrations with dilution water (5.4) before preparing the seeded dilution
water (5.5) or use a correspondingly changed volume of the seeding waters for seeding the dilution
water (5.4).
If the sample comes from a process that has been subjected to disinfection treatment (chlorination, UV,
ozone or other), use inoculum, even when there is no residual disinfectant present.
For commercially available seeding material consider respective application recommendations.
ISO 5815-1:2019(E)
The selected seeding material can be checked in advance by running the procedure with a control
analysis (9.7) batch only to prove its suitability for the analysis of samples.
5.3 Salt solutions
5.3.1 General
The following solutions can be kept for at least six months in glass bottles in the dark at (5 ± 3) °C.
Discard the solutions at the first signs of precipitation or opaqueness.
5.3.2 Phosphate-buffer solution
Dissolve 8,50 g of potassium dihydrogen phosphate (KH PO ), 21,75 g of dipotassium hydrogen
2 4
phosphate (K HPO ), 33,4 g of disodium hydrogen phosphate-heptahydrate (Na HPO ⋅7H O) and 1,70 g
2 4 2 4 2
of ammonium chloride (NH Cl), in about 500 ml of water (5.1). Dilute with water (5.1) to 1 000 ml and
mix. Measure the pH value. If the pH value is outside the range pH 7 to pH 8, prepare a new solution.
5.3.3 Magnesium sulfate heptahydrate solution, ρ = 22,5 g/l.
Dissolve 22,5 g of magnesium sulfate heptahydrate (MgSO ⋅7H O) in water (5.1). Dilute with water (5.1)
4 2
to 1 000 ml and mix.
5.3.4 Calcium chloride solution, ρ = 27,5 g/l.
Dissolve 27,5 g of anhydrous calcium chloride (CaCl ) (or an equivalent amount, if the hydrate is used
(for example 36,4 g CaCl ⋅2H O) in water (5.1), dilute with water (5.1) to 1 000 ml and mix.
2 2
5.3.5 Iron (III)-chloride-hexahydrate solution, ρ = 0,25 g/l.
Dissolve 0,25 g of iron (III)-chloride-hexahydrate (FeCl ⋅6H O), in water (5.1). Dilute with water (5.1) to
3 2
1 000 ml and mix.
5.4 Dilution water
Determine the total volume of dilution water required for the actual test. Pour about half the required
volume of water (5.1) into the feed vessel (6.3) for the dilution water and add 1 ml of each of the salt
solutions (5.3.2, 5.3.3, 5.3.4 and 5.3.5) for each litre of the total volume. Then fill to the required total
volume with water (5.1) and mix by stirring, aeration or shaking. Bring the dilution water obtained in
this way to a temperature of (20 ± 2) °C, keep at this temperature and aerate slightly by mixing. If, for
example, specially adapted seeding water or seeding material is necessary, the procedure according to
Annex C can be followed.
EXAMPLE If 20 l of dilution water are required, prepare 10 l of water (5.1). Stirring continuously, add 20 ml
of each of the salt solutions individually and fill up with water (5.1) to 20 l.
5.5 Seeded dilution water
The preparation of a seeded dilution water is needed when the test solutions are prepared according to
9.3 The mass concentration of oxygen consumed over 5 d (or 7 d) at (20 ± 1) °C by the seeded dilution
water with the addition of allylthiourea (ATU) solution to inhibit nitrification [blank value (see 9.5)],
shall be between 0,2 mg/l and 1,5 mg/l.
The volume increase of the dilution water by seeding water should be as low as possible.
4 © ISO 2019 – All rights reserved

ISO 5815-1:2019(E)
The amount of the seeding water (5.2) needed to attain a hypothetic COD of 0,6 mg/l to 3,0 mg/l,
corresponding to the aimed oxygen consumption in the blank values (9.5), is calculated with
Formula (1):
COD ⋅V
target dilutionwater
V = (1)
seedingwater
COD
seedingwater
where
V is the volume of the seeding water (5.2) to be added to the dilution water (5.4) in
seeding water
litres, l;
COD is the hypothetic COD (0,6 mg/l O to 3 mg/l O ) in the seeded dilution water (5.5)
target 2 2
in milligrams per litre of oxygen, mg/l O ;
COD is the COD of the seeding water (5.2) in milligrams per litre of oxygen, mg/l O ;
seeding water 2
V is the calculated amount of the dilution water to be seeded (5.4) in litres, l.
dilution water
For direct seeding of test batches or automated systems which use a direct seeding, see instructions in
Annex C.
Add the seeding water (5.2) to the dilution water (5.4) and mix by stirring or shaking. Determine the
oxygen content as specified in ISO 5813, ISO 17289 or ISO 5814. Aerate the seeded dilution water up to
an oxygen content of preferably a minimum 8 mg/l. The water shall not be supersaturated with oxygen
by aeration: let it stand about 1 h in an unstopped container before use. Keep the seeded dilution water
at (20 ± 2) °C. The so prepared seeded dilution water can be used immediately for the preparation of
the analysis batches.
Throw away the residue of the dilution water at the end of the working day, unless the laboratory
experience reveals via the complied-with control analysis (9.7) with the control solution (5.9), and the
blank value determination (9.5) that the water is acceptable for a longer time.
5.6 Hydrochloric acid (HCl) or sulfuric acid (H SO ) solution, for example c(HCl) ≈ 0,5 mol/l or
2 4
c(H SO ) ≈ 0,25 mol/l.
2 4
5.7 Sodium hydroxide (NaOH) solution, for example c(NaOH) = 0,5 mol/l, ρ ≈ 20 g/l.
5.8 Sodium sulfite (Na SO ) solution, for example ρ(Na SO ) = 50 g/l.
2 3 2 3
5.9 Glucose-glutamic acid (GGA), control solution.
Dry about 200 mg to 300 mg of anhydrous D-glucose (C H O ) and 200 mg to 300 mg of anhydrous
6 12 6
L-glutamic acid (C H NO ) at (105 ± 5) °C for 1 h. Weigh (150 ± 1) mg of each substance, dissolve in
5 9 4
water (5.1), dilute with water to 1 000 ml, and mix. The theoretical oxygen demand of this solution is
307 mg/l of oxygen for BOD (the empirical BOD is (198 ± 40) mg/l of oxygen and the BOD (based on
5 5 7
conversion factor BOD /BOD = 1,04 from Table D.3 and previous empirical BOD ) is (206 ± 40) mg/l of
7 5 5
oxygen).
Prepare the solution immediately before use and discard any remaining solution at the end of the
working day. The solution may also be frozen in small amounts. The frozen solution can be kept for a
maximum of three months. Use the thawed solution immediately after thawing.
5.10 Allylthiourea (ATU) solution, ρ = 1,0 g/l.
Dissolve 200 mg of allylthiourea (C H N S) in water (5.1), dilute with water (5.1) to 200 ml and mix.
4 8 2
Store the solution at (5 ± 3) °C. The solution is stable for at least two weeks.
ISO 5815-1:2019(E)
WARNING — The reagent is toxic and shall therefore be handled according to the safety data sheet.
The nitrification inhibition is not attained in all cases by addition of 2 ml of the ATU-solution (ρ = 1,0 g/l)
per litre of analysis batch. The addition of a significantly higher volume than 2 ml of this ATU-solution
can disturb the titration according to ISO 5813 (see 9.6.1).
6 Apparatus
Usual laboratory equipment, and in particular the following.
6.1 General
Plastic and glass vessels shall be carefully cleaned and, in particular, made free of absorbed toxic and
biodegradable compounds and shall be protected from contamination.
6.2 Incubation bottles, BOD-bottles (Karlsruhe type) with a content between 100 ml and 300 ml
or conical shoulder bottles with stoppers and a suitable funnel, or other suitable, bubble-free closing
bottles. For the use of automatic systems, it is important to use incubation bottles with a definite volume,
as the incubation bottles serve as dilution vessels.
6.3 Feed vessel for the seeded and non-seeded dilution water, made from glass or plastic.
Take measures to ensure that the vessel is kept clean and free from microorganism growths, and
protected from light.
6.4 Tempering cabinet, room or incubator, capable of being maintained at (20 ± 1) °C and darkened.
6.5 Equipment for determining dissolved oxygen concentration, as specified in ISO 5813
(iodometric method), or ISO 5814 (electrochemical probe method) using an oxygen probe or ISO 17289
(optical sensor method) using optical oxygen measurement.
6.6 Cooling and freezing device, for transport and storage of the samples.
6.7 Dilution vessel, mixing vessel preferably made from glass, for example volumetric flask or
graduated measuring cylinder, with sufficient volume capacity for the dilution batch and the possibility
of a thorough mixing.
6.8 Aeration equipment, bottle of compressed air or a compressor. The air quality shall be such that
the aeration does not lead to any contamination, especially by the addition of organic matter, oxidizing or
reducing materials, or metals. If contamination is suspected, filter and wash the air.
6.9 pH-measuring equipment, which fulfils the requirements for the determination of pH, as
specified in ISO 10523.
6.10 Stirrer, to ensure that the sample is homogeneous for the extraction of partial samples and no air
is taken in.
6.11 Glass fibre filter GF 6.
7 Sampling and preservation
Closable vessels made from glass or plastic are suitable for sampling. The volume should be large
enough to ensure that a proper dilution series can be derived. Fill the sampling vessels completely, close
6 © ISO 2019 – All rights reserved

ISO 5815-1:2019(E)
them and cool to (5 ± 3) °C as soon as possible. Store the samples in the dark to prevent algal growth
and cool at (5 ± 3) °C until processing at latest the day after the date of sampling.
If samples cannot be analysed in between the day after sampling freeze the samples as soon as possible
after sampling and store samples at ≤−18 °C for up to one month, or up to six months if BOD is >50 mg/l,
in a suitable vessel in the dark or in dark coloured bottles as specified in ISO 5667-3. Make sure
that the bottles do not cause excessive blank values (9.5). Since completely filled sample containers
are transported, the volume reduction up to a volume which allows expansion caused by freezing
(prevention of breakage) takes place in the laboratory after homogenization of the sample.
Thaw the sample at maximum (20 ± 2) °C, but not for more than 16 h, as otherwise the onset of bacterial
processes can falsify the results. The sample should therefore be frozen in portions, each of which does
not exceed a volume of one litre. The thawing of the samples through any means of heating equipment,
for example microwave or heating plate in which (a part of) the sample reaches a temperature of more
than 22 °C is not allowed and leads to false results. The temperature of a heated water bath shall not
exceed 22 °C. Complete thawing of a sample before use is essential, as the freezing process may lead
to the concentration of certain components in the inner part of the sample which freezes last. A once
thawed sample shall not be frozen again.
8 Interferences
8.1 General
Substances that are toxic to microorganisms, for example bactericides, free chlorine etc., inhibit the
biochemical oxidation and lead to reduced findings. Increased results can occur due to the presence of
nitrifying microorganisms. The presence of algae can lead to an overestimation of BOD and disturb the
determination.
8.2 Presence of free and/or combined chlorine
Remove free and/or combined chlorine in the sample by adding the required volume of sodium sulfite
solution (5.8). Take care to avoid adding an excess: perform the determination of the free and combined
[3] [4]
chlorine concentration as specified in ISO 7393-1 or ISO 7393-2 and calculate the volume of the
sodium sulfite solution according to Formula (2).
17, 8××V ρ
p 2
V = (2)
ρ
where
V is the volume of the sodium sulfite solution (5.8) in millilitres, ml;
V is the sample volume to be treated in millilitres, ml;
p
ρ is the measured free chlorine in the sample in grams per litre, g/l;
ρ is the concentration of sodium sulfite solution in grams per litre, g/l.
8.3 Presence of algae
In the case of samples containing algae, consider filtration after sampling in the field or directly
after arrival at the laboratory to avoid producing unusually high results. Use glass fibre filters (6.11).
Filtering can change BOD results radically and it shall only be performed if deemed necessary in the
evaluation of the quality of the water or if the analysis from an algae-free sample is required. Indicate
any filtration in the test report (see Clause 11).
ISO 5815-1:2019(E)
8.4 Presence of peroxides and peroxide compounds
NOTE Peroxides and peroxide compounds can be found in waste water of the chip industry or large
laundries, etc.
Peroxides and peroxide compounds create reduced findings; hence peroxides shall be eliminated from
samples to be examined.
The following methods can be applied for indication if peroxides are suspected:
— Proof with iodide starch paper. Iodide and filter paper containing starch is coloured blue in the
presence of peroxide, but it reacts with chlorine as well. Peroxide-test strips are commercially
available for this method.
— Proof by using oxygen content measurement (9.6.2). An oversaturation of the dissolved oxygen
concentration in the sample can indicate peroxides and peroxide compounds.
If peroxides have been detected, various methods of destroying peroxides can be applied:
— Peroxides can be expelled by shaking or stirring the sample intensively in an open vessel. The
presence of peroxides shall be tested using peroxide test strips or measurement of dissolved oxygen
concentration from time to time, but not for more than a 2 h time period. The expulsion of the
peroxides is finished when the dissolved oxygen concentration no longer decreases within a period
of 30 min or the test strips no longer indicate any peroxides.
— Some peroxide compounds cannot be eliminated with these methods. The peroxide compounds can
then be destroyed with a sodium sulfite solution (5.8). Use an aliquot of the sample to determine the
volume of sodium sulfite solution (5.8) needed to destroy the peroxides. Avoid an excess and test
the spontaneous oxygen consumption. The test for complete destruction can be carried out with
peroxide test strips. Based on these results calculate the amount of sodium sulfite solution needed
to remove peroxides and peroxide compounds in the sample volume required for the test. Add the
equivalent to the sample aliquot that is used for the BOD determination.
It shall be noted that this treatment of the sample can change it, hence document the sample treatment
for the elimination of peroxides in the test report.
9 Procedure
9.1 General
If the presence of substances that are toxic to microorganisms is suspected, Annex B with the testing of
several different dilutions of the sample should be applied.
The number of bottles to be prepared depends on the technique to measure the dissolved oxygen
content and the number of replicates desired.
The direct seeding of the analysis batches is described in Annex C.
9.2 Pretreatment
Pre-treatment is carried out for samples within at latest the day after sampling or for frozen samples
after complete thawing of the sample.
9.2.1 Neutralization of the sample
Neutralize the sample or the diluted samples (for example in automatic systems) with hydrochloric
acid (5.6) or sodium hydroxide solution (5.7) if the pH-value is not between 6 and 8. Choose the
concentration of the hydrochloric acid (5.6) or the sodium hydroxide (5.7) solutions to restrict the
8 © ISO 2019 – All rights reserved

ISO 5815-1:2019(E)
volume added to not more than 5 % of total volume. Indicate potential precipitation resulting hereby in
the test report.
NOTE If the whole sample meets pH requirements, so will any dilution.
9.2.2 Homogenization
For sub-sampling the fresh or thawed sample shall be thoroughly mixed before distribution. An even
distribution of all soluble and particulate components should be ensured (for example by gentle
agitation or vigorous shaking). If the sample contains large particles, which complicate the withdrawal
of partial samples of equal quality, homogenization by crushing the particles with for example a
dispersing device is recommended.
9.3 Preparation of test solutions
Temper the sample to a temperature of (20 ± 2) °C. Assure homogeneity of the sample during preparation
of the dilutions, for example by gentle agitation or vigorous shaking either manually or with a stirrer
without the inclusion of air until all analysis batches are produced.
Carry out the test with preferably two replicates per dilution. Depending on the purpose of testing
and quality assurance requirements for selected samples a testing of only one replicate per dilution is
acceptable. Report the number of dilutions (see 9.4) and the number of replicates per dilution tested for
the sample in the test report [Clause 11 d)].
The following description of the procedure takes into account at least two replicates per dilution and
several dilutions.
Calculate the volumes of sample and seeded dilution water needed to prepare the test solutions for
the selected number of dilutions and replicates considering 9.4 for the selection of dilutions. If the
iodometric method (9.6.1) is used, a second series of bottles with replicates is needed to determine the
concentration of dissolved oxygen at time zero.
Place a known volume of the sample (or pretreated sample) each in the dilution vessels (6.7), add 2 ml
of allylthiourea solution (5.10) per litre of diluted sample and fill to the mark with seeded dilution
water (5.5). If the dilution factor to be used is greater than 100, carry out serial dilutions in two or
more steps. When automatic analysis systems are applied, add a definite volume of the sample (or the
pre-treated sample) into the incubation bottle. The addition of 2 ml allylthiourea solution (5.10) per
litre batch and the seeded dilution water (5.5) occur fully automatically through the system.
9.4 Calculation of dilutions
9.4.1 Empirical determination of the dilutions
As the correct dilution degree of the sample which gives a measurable BOD in at least one of the
n
dilutions cannot be precisely attained, several different dilutions varying according to the dilution
factor (as reciprocal value of sample volume to total volume of test batch) and including the dilution
corresponding to the expected BOD (see Table 1 and Annex B) are prepared. Various options for
n
the determination of dilutions factors and dilution series are described in 9.4.2 to 9.4.3. If sufficient
information on the oxygen consumption of the sample is available (for example known, reproducible,
stable composition of the
...