ISO 21338:2010
(Main)Water quality — Kinetic determination of the inhibitory effects of sediment, other solids and coloured samples on the light emission of Vibrio fischeri (kinetic luminescent bacteria test)
Water quality — Kinetic determination of the inhibitory effects of sediment, other solids and coloured samples on the light emission of Vibrio fischeri (kinetic luminescent bacteria test)
ISO 21338:2010 specifies the kinetic direct‑contact method for determining the inhibitory effect of suspensions of sediment and other solid samples, and also for problematic turbid or coloured aqueous samples on the light emission of the marine bacterium Vibrio fischeri (NRRL B‑11177). This method is applicable to: a) sediment samples and water suspensions of sediments (fresh water, brackish, and seawater sediments); b) effluents (especially turbid and coloured); c) aqueous extracts (e.g. leachates, eluates, elutriates) of soil, solid waste, and other solid material (especially turbid and coloured).
Qualité de l'eau — Détermination cinétique des effets inhibiteurs des échantillons de sédiment, autres solides et des échantillons colorés sur la luminescence de Vibrio fischeri (essai cinétique de bactéries luminescentes)
Kakovost vode - Določevanje kinetike zaviralnih učinkov sedimenta, drugih trdnih in obarvanih vzorcev na emitirano svetlobo Vibrio fischeri (kinetic luminescent bacteria test)
Ta mednarodni standard opredeljuje kinetično metodo z neposrednim stikom za določevanje zaviralnih učinkov suspenzij sedimenta in drugih trdnih vzorcev ter tudi za problematične motne ali obarvane vodne vzorce na emitirano svetlobo morske bakterije Vibrio fischeri (NRRL B-11177).
Ta metoda se uporablja za:
a) sedimentne vzorce in vodne suspenzije sedimentov (sedimente sladke vode, nepitne vode in morske vode);
b) odplake (zlasti motne in obarvane);
c) vodne ekstrakte (npr. izlužki, eluati in elutriati), zemljine, trdne odpadke in druge trdne materiale(zlasti motne in obarvane).
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INTERNATIONAL ISO
STANDARD 21338
First edition
2010-07-15
Water quality — Kinetic determination of
the inhibitory effects of sediment, other
solids and coloured samples on the light
emission of Vibrio fischeri (kinetic
luminescent bacteria test)
Qualité de l'eau — Détermination cinétique des effets inhibiteurs des
échantillons de sédiment, autres solides et des échantillons colorés sur
la luminescence de Vibrio fischeri (essai cinétique de bactéries
luminescentes)
Reference number
ISO 21338:2010(E)
©
ISO 2010
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ISO 21338:2010(E)
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ii © ISO 2010 – All rights reserved
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ISO 21338:2010(E)
Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Terms and definitions .2
4 Principle.2
5 Interferences .3
6 Reagents and materials .4
7 Apparatus.5
8 Sampling and sample pre-treatment .5
9 Procedure.6
10 Evaluation.7
11 Expression of results.9
12 Criteria of validity .11
13 Test report.11
Annex A (informative) Precision data .13
Annex B (informative) Typical kinetic curves from different samples .17
Annex C (informative) Dilution series .18
Bibliography.20
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ISO 21338:2010(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 21338 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2010 – All rights reserved
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ISO 21338:2010(E)
Introduction
The method specified in this International Standard is a kinetic modification of the luminescent bacteria test
specified in ISO 11348. The kinetic method overcomes the problems arising from intense colour and turbidity
in the samples. There is no need for sedimentation or centrifugation of turbid samples, or for the correction of
colour as described in ISO 11348.
This kinetic method uses luminometers capable of dispensing luminescent bacteria to the samples and
measuring the luminescent intensity over a period of time. In the method, the bacterial suspension is
dispensed and mixed with the sample in the measurement chamber of the luminometer. Several suitable
instruments are commercially available, but only a few of them are capable of cooling the measurement
chamber to (15 ± 1) °C as specified in ISO 11348. However, if the bacterial suspension and test samples are
kept at (15 ± 1) °C in a thermo-block before the measurement and during the whole incubation, the actual
temperature during the contact time is (15 ± 1) °C.
The measurements specified in this International Standard can be carried out using freshly prepared bacteria,
as well as freeze- or liquid-dried bacterial preparations. The various bacterial preparations can deliver different
results, especially in the presence of heavy metals (see ISO 11348). The laboratories responsible for the
results have the opportunity to select the most suitable bacterial preparation based on expert judgement and
information about the samples to be tested.
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INTERNATIONAL STANDARD ISO 21338:2010(E)
Water quality — Kinetic determination of the inhibitory effects
of sediment, other solids and coloured samples on the light
emission of Vibrio fischeri (kinetic luminescent bacteria test)
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This International Standard 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 and to ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this International
Standard be carried out by suitably trained staff.
1 Scope
This International Standard specifies the kinetic direct-contact method for determining the inhibitory effect of
suspensions of sediment and other solid samples, and also for problematic turbid or coloured aqueous
samples on the light emission of the marine bacterium Vibrio fischeri (NRRL B-11177).
This method is applicable to:
a) sediment samples and water suspensions of sediments (fresh water, brackish, and seawater sediments);
b) effluents (especially turbid and coloured);
c) aqueous extracts (e.g. leachates, eluates, elutriates) of soil, solid waste, and other solid material
(especially turbid and coloured).
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 5667-16:1998, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 11348-1, Water quality — Determination of the inhibitory effect of water samples on the light emission of
Vibrio fischeri (Luminescent bacteria test) — Part 1: Method using freshly prepared bacteria
ISO 11348-2, Water quality — Determination of the inhibitory effect of water samples on the light emission of
Vibrio fischeri (Luminescent bacteria test) — Part 2: Method using liquid-dried bacteria
ISO 11348-3:2007, Water quality — Determination of the inhibitory effect of water samples on the light
emission of Vibrio fischeri (Luminescent bacteria test) — Part 3: Method using freeze-dried bacteria
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ISO 21338:2010(E)
3 Terms and definitions
For the purpose of this document, the following terms and definitions apply.
3.1
contact time
duration of contact between one object or substance and another
NOTE In the test, the contact time is the time available to control or sample for contact with the test bacteria.
3.2
control sample
sample used in a laboratory in order to check or monitor the instrument or measurement performance or to
monitor changes in a sample under investigation
3.3
correction factor
dimensionless multiplier to correct data for known influences affecting their values as measured
NOTE In the test, the correction factor, f , serves to correct the initial luminescence intensity of the sample.
kt
3.4
peak value
maximum signal recorded in response to a stimulus
NOTE In the test, the peak value is the maximum signal which is recorded immediately after all the bacteria are in
contact with the sample.
3.5
reference sample
when the effect or behaviour of a substance is known from previous tests (reference substance) and when this
substance is examined within the framework of a test series as test sample, this is called the reference
sample
NOTE Adapted from ISO 5667-16:1998.
3.6
test sample
test sample is made from the sample by means of various preparatory steps specific to the sample and the
test, e.g. by dissolving, homogenizing, sedimenting, filtering, neutralizing or aeration
[ISO 5667-16:1998]
4 Principle
The inhibition of light emission by cultures of Vibrio fischeri is measured kinetically by following the light
emission of cultures from the very beginning of the assay. This is accomplished by dispensing the luminescent
bacteria suspension into the sample in a cuvette or other suitable vessel (e.g. microtiter plate) already in the
measuring position in the luminometer. The light emission is measured and recorded from the moment of
dispensing of the bacterial suspension to the sample until the maximum value has been reached and not only
at the maximum value of intensity (peak value) which usually occurs within 5 s of mixing, and after a contact
time of 15 min and 30 min or optionally 5 min (Figure 1).
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ISO 21338:2010(E)
Key
t time
y relative light units
1 start measurement
2 inject bacteria
3 record peak value from 0 s to 5 s
4 mix the sample before recording signal at 30 min
Figure 1 — Principal schematic protocol for the kinetic luminescent bacteria test
Vibrio fischeri suspension is dispensed and mixed into the sample in the measurement chamber of the
luminometer.
The test criterion is the decrease of the luminescence at each endpoint compared to the peak value, taking
into account a correction factor, f , which is measured from intensity changes of control samples during the
kt
exposure time. The inhibitory effect of the sample can be determined as the lowest ineffective dilution (LID)
value, or as effective concentration (EC or EC ) values by means of dilution series (e.g. as described in
20 50
Annex C). The LID value is the most concentrated test batch tested at which the inhibition of light emission is
<20 %. For higher levels of inhibition, the dilution effect relationship can be determined graphically or by
statistical analysis. The inhibition by a sample is expressed as the concentrations which results in 20 % and
50 % light reduction compared to the blank (EC and EC ). This value is interpolated within the dilution
20 50
series.
No extra correction procedures for colour and turbidity are needed because these factors remain the same
during the whole measurement. Inhibition at different contact times and different sample concentrations yields
complete kinetic toxicological data about the sample (inhibition, expressed as a percentage vs. concentration
vs. time) and enables assumptions to be made about the nature of the contaminants if compared with existing
data (see Annex B).
5 Interferences
Volatile substances or substances which react with the dilution water or the test suspension, or alter their state
during the test period, may affect the result or impair the reproducibility of the test results.
Since oxygen is required for the bioluminescence (Reference [18]), samples with high oxygen demand (or low
oxygen concentration) may cause deficiency of oxygen and be inhibitory.
Readily biodegradable nutrients in the sample may cause a pollutant-independent reduction in
bioluminescence (Reference [19]).
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ISO 21338:2010(E)
Samples with a pH outside the range 6,0 to 8,5 affect the luminescence of bacteria (References [18][20]).
Adjust the sample when the toxic effect of pH is not of interest.
As the test organism Vibrio fischeri is a marine bacterium, testing salt water samples with the standard
procedure often leads to stimulation effects of bioluminescence, which may mask inhibition effects (see
ISO 11348-3:2007, Annex D).
Salt concentrations in the initial sample exceeding 30 g/l NaCl or contents of other compounds giving equal
osmolarity may lead, together with the salt spiking required by the test, to hyperosmotic effects. The resulting
salt concentration in the test samples should not exceed the osmolarity of a 35 g/l NaCl solution in order to
avoid these effects.
6 Reagents and materials
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled or demineralized water or water of equivalent purity.
6.1 Test bacteria. Use a strain of luminescent bacteria belonging to the species Vibrio fischeri
NRRL B-11177.
The bacterial suspensions used for toxicity measurements are prepared according to the instructions in
ISO 11348. Dilute the bacterial suspension before the assay from the stock suspension to the measuring
concentration (example: ISO 11348-3:2007, variant B).
6.2 Sodium chloride solution, as diluent.
Dissolve 20 g of sodium chloride (NaCl) in water and make up to 1 l with water.
6.3 Sodium hydroxide solution, e.g. c(NaOH) = 1 mol/l.
6.4 Hydrochloric acid, e.g. c(HCl) = 1 mol/l.
For the adjustment of the pH it may be necessary to use acids or bases of lower or higher concentration.
6.5 Solution for freeze-dried bacteria.
⎯ 20,0 g sodium chloride (NaCl);
⎯ 2,035 g magnesium chloride hexahydrate (MgCl ·6H O);
2 2
⎯ 0,30 g potassium chloride (KCl).
Dissolve in water and make up to 1 l with water. Store the solution in portions in a freezer at −18 °C to −20 °C.
6.6 Reference substances.
Prepare the following three separate reference substance solutions with sodium chloride solution (6.2) as
diluent without adjustment of the pH for the use of freeze-dried bacteria:
a) 6,8 mg/l 3,5-dichlorophenol (DCP, C H OCl );
6 4 2
b) 19,34 mg/l zinc sulfate heptahydrate (ZnSO ·7 H O);
4 2
c) 105,8 mg/l potassium dichromate (K Cr O ).
2 2 7
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ISO 21338:2010(E)
The concentrations in a) to c) are approximately twice the expected EC values for the respective reference
50
substances in ISO 11348-3. For concentrations of reference substance solutions for freshly cultured or
liquid-dried bacteria, see ISO 11348-1 or ISO 11348-2. The volumes required depend on the test set-up.
NOTE 1 It is possible to use commercially available chemical preparations with defined concentrations of ZnSO and
4
K Cr O for the preparation of the solutions of the reference substances.
2 2 7
NOTE 2 For more information about reference substances, see Reference [22].
7 Apparatus
Usual laboratory equipment, and in particular the following.
7.1 Freezer, for the storage of preserved bacteria.
7.2 Incubator or refrigerator, to maintain the solution for freeze-dried bacteria (6.5) at a temperature of
(4 ± 3) °C.
7.3 Thermostatically controlled thermo-block, to maintain the test samples and the Vibrio fischeri
suspension at a temperature of (15 ± 1) °C. Within one test, the temperature deviation should be at most
±0,3 °C.
7.4 Luminometer, equipped with at least one dispenser (minimum injection volume 0,2 ml).
The instrument shall be capable of measuring and recording the luminescence continuously at least for 5 s in
0,2 s intervals or shorter. The injection and recording shall be performed simultaneously.
7.5 Test tubes, cuvettes, test plates or any other suitable test vessels, made of chemically inert material,
appropriate for the selected luminometer.
7.6 pH-meter.
7.7 Chronometer.
[3]
7.8 Piston pipettes, nominal capacity 100 µl to 1 000 µl, ISO 8655-2 .
7.9 Mixer, e.g. vortex mixer, for mixing the samples before the measurements.
7.10 Conductometer.
7.11 Oxygen probe, as specified in ISO 5814.
[1]
7.12 Sieve, nominal size of openings 2 mm, ISO 565 .
8 Sampling and sample pre-treatment
8.1 Sampling
Collect the samples in chemically inert, clean containers as specified in ISO 5667-16. Fill the containers
completely and seal them. Cool the samples on ice, or in a refrigerator or a cooling box at 2 °C to 5 °C and
test them as soon as possible after collection. Where necessary, store samples at a temperature of 2 °C to
5 °C in the dark for no longer than 48 h. If the samples have to be frozen store them at a temperature of
−18 °C or below in the dark for no longer than 2 months. Prepare and measure frozen samples immediately
after thawing in a water bath. For long-term storage, the samples may be freeze-dried and stored in the dark
at room temperature. Do not use chemicals to preserve the samples. Perform the necessary pH adjustment
and salt addition just before testing.
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ISO 21338:2010(E)
8.2 Sample preparation
Sieve the samples to remove any coarse material (e.g. plant roots and particles larger than 2 mm).
Suspend sediment samples in water (e.g. 20 % mass fraction) by vigorous shaking (vortexing) in order to
obtain homogeneous suspensions (Reference [22]). Also, mass fractions higher than 20 % can be measured
if the suspensions stay homogeneous during the whole measurement.
Measure the oxygen concentration in all samples and sample water suspensions. An oxygen concentration
>3 mg/l is required for the test. If the oxygen concentration of the undiluted sample is less than 3 mg/l, use
appropriate methods to oxygenate the sample, e.g. aeration or stirring.
Measure the pH of all samples and sample water suspensions. If the pH is between 6,0 and 8,5, no
adjustment is necessary. The adjustment of the pH value, however, may alter the nature of the sample. On
the other hand, the pH of the sample and the pH of the test batch can differ because of the buffer capacity of
the test medium. It may be necessary to carry out tests on both the pH-adjusted and the non-pH-adjusted
samples.
If necessary, adjust the pH of the samples by adding either hydrochloric acid (6.4) or sodium hydroxide
solution (6.3). Depending on the purpose of the test, the pH may be adjusted to 7,0 ± 0,2 or to the upper
(8,5 ± 0,2) or lower (6,0 ± 0,2) limits. Choose the concentration of the hydrochloric acid or the sodium
hydroxide solution to restrict the volume added to not more than 5 % of total volume.
Add 20 g of sodium chloride per litre to the sample or to the neutralized sample.
For salt water samples, ISO 11348-3:2007, Annex D gives further information.
9 Procedure
9.1 Initial preparations
9.1.1 Preparation of test solutions
Prepare the reference samples according to 6.6. Test each batch of bacteria after delivery with all three
reference substances. Test at least one of the three reference substances with each vial of stock suspension
reconstituted.
Prepare the samples according to 8.2.
Prepare, in the first set of test tubes (7.5), the sample dilution series, the reference sample (6.6) and the
control (6.2) required.
Serially dilute the sample. Make the dilutions in separate tubes and transfer them to the measurement
cuvettes or plates. Normally, in this test, equal volumes of test suspension and sample (or diluted sample) are
mixed, giving tested dilution levels within a series of D W 2.
The minimum dilution which can be tested is 1,00 in 1,25, prepared by mixing 4 volumes of sample with
1 volume of test suspension (e.g. 800 µl sample plus 200 µl test suspension). The corresponding D value is 1.
For this D value, an extra control batch is needed which is made up by adding 200 µl of the test suspension to
800 µl sodium chloride solution (see Annex C).
Maintain the test tubes containing the sodium chloride solution (6.2) for controls, the reference samples (6.6),
the samples (8.2) and the samples of the dilution series (Annex C) at (15 ± 1) °C.
Choose test conditions which ensure that the maximum temperature deviation in the thermo-block within one
test is no more than ±0,3 °C.
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ISO 21338:2010(E)
9.1.2 Preparation of test suspensions
Prepare the luminescent bacterial stock suspension for the test according to the instructions for freshly
cultured (ISO 11348-1), liquid-dried (ISO 11348-2) or freeze-dried (ISO 11348-3) bacteria test.
Prepare the test suspension used in the kinetic test as specified, for example, in ISO 11348-3:2007, 8.3.3,
Variant B, where the test suspension is prepared outside the test tubes. Dilute reconstituted bacterial
suspension with solution (6.5) to form the test suspension before starting the test.
6
Store the luminescent bacteria test suspension for the measurement (cell concentration about 2 × 10 cells/ml)
at (4 ± 3) °C. The suspension is ready to use after a minimum of 30 min in a refrigerator and can be used for
testing purposes as long as the validity criteria stated in Clause 12 are met.
Before initiating the measurement, store the test suspension at (15 ± 1) °C for at least 30 min.
9.2 Test procedure
Carry out duplicate or triplicate determinations per dilution level at a test temperature of (15 ± 1) °C.
Set the dispensing volume from 200 µl to 500 µl (equal to the sample volume) and prime the dispenser with
the Vibrio fischeri bacterial test suspension.
Mix (7.9) the samples well and pipette 200 µl to 500 µl of each well-homogenized sample into the luminometer
cuvettes or microtiter plate wells depending on the instrument used. If the sample contains large particles,
cutting the sharp point of the pipette tip can facilitate the pipetting. Wider pipette tips are also commercially
available.
Load the sample cuvette (or microtiter plate) into the luminometer and start the run by dispensing Vibrio
fischeri bacterial suspension into the cuvette. Determine and record the maximum luminescence value of the
test suspension during the first 5 s of contact (at least 5 measurements/s). The maximum luminescence
intensity is the peak-value, I . Immediately after the measurement, put the cuvette back into the incubator and
p
measure the next cuvette. Repeat this process for each sample and control. When a microtiter plate reader is
used, run the controls and test sample dilution series immediately before transferring the plate to the incubator.
Store the cuvettes at (15 ± 1) °C during the incubation.
Determine and record the luminescence intensity after, optionally 5 min, I , and again after 15 min, I , and
5 15
30 min, I , as required. Mix the samples by hand before each measurement. Repeat this procedure for every
30
control and sample.
Intervals of 30 s between the samples have been found adequate to allow sufficient time for the readings to be
made and the change of the cuvettes.
10 Evaluation
10.1 Inhibitory effect on luminescent bacteria
Calculate the correction factor, f , for the contact time (5 min, 15 min or 30 min) from the measured
kt
luminescence intensity using Equation (1). This factor serves to correct the initial values, I , of all test samples
p
before they can be used as reference values for the determination of the water-dependent decrease of
luminescence.
I
kt
f = (1)
kt
I
p
where
I is the luminescence intensity in the control sample after the contact time of 5 min, 15 min or 30 min,
kt
in relative luminescence units;
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ISO 21338:2010(E)
I is the maximum luminescence intensity (the peak value), in relative luminescence units, of the control
p
test suspension immediately after dispensing the bacteria into the sample.
Calculate the mean correction factor f . Find the deviation of the individual replicates from the mean,
kt
expressed as a percentage to one significant figure, using Formula (2):
⎡⎤
ff±
()
k t, i
kt
⎢⎥
×100 (2)
⎢⎥
f
kt
⎢⎥
⎣⎦
where f is either of the two individual values of the correction factor.
kt,i
Calculate the corrected value of I for test sample cuvettes (corrected peak intensity value), I , using
p ct
Equation (3):
I =⋅If (3)
cpt
kt
where
f is the mean of f ;
kt kt,i
I is the maximum luminescence intensity (the peak value), in relative luminescence units, of the test
p
suspension immediately after dispensing the bacteria into the sample.
Calculate the inhibitory effect of a test sample after the contact time (5 min, 15 min or 30 min), H , expressed
t
as a percentage, using Equation (4):
()II−
ctt
H=×100 (4)
t
I
ct
where
I is the corrected peak intensity value given by Equation (3);
ct
I is the luminescence intensity of the test sample after the contact time (5 min, 15 min or 30 min), in
t
relative luminescence units.
Calculate the mean of the inhibitory effect H for each dilution level, expressed as a percentage.
t
Calculate the arithmetic difference, expressed in per cent points to one significant figure:
HH(%) − (%)
t
ti,
where
H is either of the two individual values of the inhibitory effects of at least one test sample;
t,i
H is the respective mean value.
t
10.2 Determination of EC values
Calculate the concentration-effect relationship for each exposure time using suitable standard linear or
non-linear regression analysis (see Reference [19]).
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ISO 21338:2010(E)
For evaluation of the relationship between concentration and effect using a linear regression technique,
Γ , for each dilution
calculate the gamma value (ratio of light lost to the amount of light remaining at time t),
t
level of the test sample after the contact time (5 min, 15 min or 30 min) using Equation (5):
Ht
Γ = (5)
t
(100 − H )
t
where H is the mean of the values of H obtained by Equation (4).
t
t
NOTE When a certain test concentration gives 0 % or 100 % inhibition of bioluminescence, the gamma value cannot
be calculated. Therefore, usually
...
SLOVENSKI STANDARD
SIST ISO 21338:2011
01-junij-2011
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Water quality - Kinetic determination of the inhibitory effects of sediment, other solids and
coloured samples on the light emission of Vibrio fischeri (kinetic luminescent bacteria
test)
Qualité de l'eau - Détermination cinétique des effets inhibiteurs des échantillons de
sédiment, autres solides et des échantillons colorés sur la luminescence de Vibrio
fischeri (essai cinétique de bactéries luminescentes)
Ta slovenski standard je istoveten z: ISO 21338:2010
ICS:
13.060.60 Preiskava fizikalnih lastnosti Examination of physical
vode properties of water
SIST ISO 21338:2011 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST ISO 21338:2011
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SIST ISO 21338:2011
INTERNATIONAL ISO
STANDARD 21338
First edition
2010-07-15
Water quality — Kinetic determination of
the inhibitory effects of sediment, other
solids and coloured samples on the light
emission of Vibrio fischeri (kinetic
luminescent bacteria test)
Qualité de l'eau — Détermination cinétique des effets inhibiteurs des
échantillons de sédiment, autres solides et des échantillons colorés sur
la luminescence de Vibrio fischeri (essai cinétique de bactéries
luminescentes)
Reference number
ISO 21338:2010(E)
©
ISO 2010
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SIST ISO 21338:2011
ISO 21338:2010(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
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Contents Page
Foreword .iv
Introduction.v
1 Scope.1
2 Normative references.1
3 Terms and definitions .2
4 Principle.2
5 Interferences .3
6 Reagents and materials .4
7 Apparatus.5
8 Sampling and sample pre-treatment .5
9 Procedure.6
10 Evaluation.7
11 Expression of results.9
12 Criteria of validity .11
13 Test report.11
Annex A (informative) Precision data .13
Annex B (informative) Typical kinetic curves from different samples .17
Annex C (informative) Dilution series .18
Bibliography.20
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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 21338 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
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Introduction
The method specified in this International Standard is a kinetic modification of the luminescent bacteria test
specified in ISO 11348. The kinetic method overcomes the problems arising from intense colour and turbidity
in the samples. There is no need for sedimentation or centrifugation of turbid samples, or for the correction of
colour as described in ISO 11348.
This kinetic method uses luminometers capable of dispensing luminescent bacteria to the samples and
measuring the luminescent intensity over a period of time. In the method, the bacterial suspension is
dispensed and mixed with the sample in the measurement chamber of the luminometer. Several suitable
instruments are commercially available, but only a few of them are capable of cooling the measurement
chamber to (15 ± 1) °C as specified in ISO 11348. However, if the bacterial suspension and test samples are
kept at (15 ± 1) °C in a thermo-block before the measurement and during the whole incubation, the actual
temperature during the contact time is (15 ± 1) °C.
The measurements specified in this International Standard can be carried out using freshly prepared bacteria,
as well as freeze- or liquid-dried bacterial preparations. The various bacterial preparations can deliver different
results, especially in the presence of heavy metals (see ISO 11348). The laboratories responsible for the
results have the opportunity to select the most suitable bacterial preparation based on expert judgement and
information about the samples to be tested.
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INTERNATIONAL STANDARD ISO 21338:2010(E)
Water quality — Kinetic determination of the inhibitory effects
of sediment, other solids and coloured samples on the light
emission of Vibrio fischeri (kinetic luminescent bacteria test)
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This International Standard 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 and to ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this International
Standard be carried out by suitably trained staff.
1 Scope
This International Standard specifies the kinetic direct-contact method for determining the inhibitory effect of
suspensions of sediment and other solid samples, and also for problematic turbid or coloured aqueous
samples on the light emission of the marine bacterium Vibrio fischeri (NRRL B-11177).
This method is applicable to:
a) sediment samples and water suspensions of sediments (fresh water, brackish, and seawater sediments);
b) effluents (especially turbid and coloured);
c) aqueous extracts (e.g. leachates, eluates, elutriates) of soil, solid waste, and other solid material
(especially turbid and coloured).
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 5667-16:1998, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 11348-1, Water quality — Determination of the inhibitory effect of water samples on the light emission of
Vibrio fischeri (Luminescent bacteria test) — Part 1: Method using freshly prepared bacteria
ISO 11348-2, Water quality — Determination of the inhibitory effect of water samples on the light emission of
Vibrio fischeri (Luminescent bacteria test) — Part 2: Method using liquid-dried bacteria
ISO 11348-3:2007, Water quality — Determination of the inhibitory effect of water samples on the light
emission of Vibrio fischeri (Luminescent bacteria test) — Part 3: Method using freeze-dried bacteria
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3 Terms and definitions
For the purpose of this document, the following terms and definitions apply.
3.1
contact time
duration of contact between one object or substance and another
NOTE In the test, the contact time is the time available to control or sample for contact with the test bacteria.
3.2
control sample
sample used in a laboratory in order to check or monitor the instrument or measurement performance or to
monitor changes in a sample under investigation
3.3
correction factor
dimensionless multiplier to correct data for known influences affecting their values as measured
NOTE In the test, the correction factor, f , serves to correct the initial luminescence intensity of the sample.
kt
3.4
peak value
maximum signal recorded in response to a stimulus
NOTE In the test, the peak value is the maximum signal which is recorded immediately after all the bacteria are in
contact with the sample.
3.5
reference sample
when the effect or behaviour of a substance is known from previous tests (reference substance) and when this
substance is examined within the framework of a test series as test sample, this is called the reference
sample
NOTE Adapted from ISO 5667-16:1998.
3.6
test sample
test sample is made from the sample by means of various preparatory steps specific to the sample and the
test, e.g. by dissolving, homogenizing, sedimenting, filtering, neutralizing or aeration
[ISO 5667-16:1998]
4 Principle
The inhibition of light emission by cultures of Vibrio fischeri is measured kinetically by following the light
emission of cultures from the very beginning of the assay. This is accomplished by dispensing the luminescent
bacteria suspension into the sample in a cuvette or other suitable vessel (e.g. microtiter plate) already in the
measuring position in the luminometer. The light emission is measured and recorded from the moment of
dispensing of the bacterial suspension to the sample until the maximum value has been reached and not only
at the maximum value of intensity (peak value) which usually occurs within 5 s of mixing, and after a contact
time of 15 min and 30 min or optionally 5 min (Figure 1).
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Key
t time
y relative light units
1 start measurement
2 inject bacteria
3 record peak value from 0 s to 5 s
4 mix the sample before recording signal at 30 min
Figure 1 — Principal schematic protocol for the kinetic luminescent bacteria test
Vibrio fischeri suspension is dispensed and mixed into the sample in the measurement chamber of the
luminometer.
The test criterion is the decrease of the luminescence at each endpoint compared to the peak value, taking
into account a correction factor, f , which is measured from intensity changes of control samples during the
kt
exposure time. The inhibitory effect of the sample can be determined as the lowest ineffective dilution (LID)
value, or as effective concentration (EC or EC ) values by means of dilution series (e.g. as described in
20 50
Annex C). The LID value is the most concentrated test batch tested at which the inhibition of light emission is
<20 %. For higher levels of inhibition, the dilution effect relationship can be determined graphically or by
statistical analysis. The inhibition by a sample is expressed as the concentrations which results in 20 % and
50 % light reduction compared to the blank (EC and EC ). This value is interpolated within the dilution
20 50
series.
No extra correction procedures for colour and turbidity are needed because these factors remain the same
during the whole measurement. Inhibition at different contact times and different sample concentrations yields
complete kinetic toxicological data about the sample (inhibition, expressed as a percentage vs. concentration
vs. time) and enables assumptions to be made about the nature of the contaminants if compared with existing
data (see Annex B).
5 Interferences
Volatile substances or substances which react with the dilution water or the test suspension, or alter their state
during the test period, may affect the result or impair the reproducibility of the test results.
Since oxygen is required for the bioluminescence (Reference [18]), samples with high oxygen demand (or low
oxygen concentration) may cause deficiency of oxygen and be inhibitory.
Readily biodegradable nutrients in the sample may cause a pollutant-independent reduction in
bioluminescence (Reference [19]).
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Samples with a pH outside the range 6,0 to 8,5 affect the luminescence of bacteria (References [18][20]).
Adjust the sample when the toxic effect of pH is not of interest.
As the test organism Vibrio fischeri is a marine bacterium, testing salt water samples with the standard
procedure often leads to stimulation effects of bioluminescence, which may mask inhibition effects (see
ISO 11348-3:2007, Annex D).
Salt concentrations in the initial sample exceeding 30 g/l NaCl or contents of other compounds giving equal
osmolarity may lead, together with the salt spiking required by the test, to hyperosmotic effects. The resulting
salt concentration in the test samples should not exceed the osmolarity of a 35 g/l NaCl solution in order to
avoid these effects.
6 Reagents and materials
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled or demineralized water or water of equivalent purity.
6.1 Test bacteria. Use a strain of luminescent bacteria belonging to the species Vibrio fischeri
NRRL B-11177.
The bacterial suspensions used for toxicity measurements are prepared according to the instructions in
ISO 11348. Dilute the bacterial suspension before the assay from the stock suspension to the measuring
concentration (example: ISO 11348-3:2007, variant B).
6.2 Sodium chloride solution, as diluent.
Dissolve 20 g of sodium chloride (NaCl) in water and make up to 1 l with water.
6.3 Sodium hydroxide solution, e.g. c(NaOH) = 1 mol/l.
6.4 Hydrochloric acid, e.g. c(HCl) = 1 mol/l.
For the adjustment of the pH it may be necessary to use acids or bases of lower or higher concentration.
6.5 Solution for freeze-dried bacteria.
⎯ 20,0 g sodium chloride (NaCl);
⎯ 2,035 g magnesium chloride hexahydrate (MgCl ·6H O);
2 2
⎯ 0,30 g potassium chloride (KCl).
Dissolve in water and make up to 1 l with water. Store the solution in portions in a freezer at −18 °C to −20 °C.
6.6 Reference substances.
Prepare the following three separate reference substance solutions with sodium chloride solution (6.2) as
diluent without adjustment of the pH for the use of freeze-dried bacteria:
a) 6,8 mg/l 3,5-dichlorophenol (DCP, C H OCl );
6 4 2
b) 19,34 mg/l zinc sulfate heptahydrate (ZnSO ·7 H O);
4 2
c) 105,8 mg/l potassium dichromate (K Cr O ).
2 2 7
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The concentrations in a) to c) are approximately twice the expected EC values for the respective reference
50
substances in ISO 11348-3. For concentrations of reference substance solutions for freshly cultured or
liquid-dried bacteria, see ISO 11348-1 or ISO 11348-2. The volumes required depend on the test set-up.
NOTE 1 It is possible to use commercially available chemical preparations with defined concentrations of ZnSO and
4
K Cr O for the preparation of the solutions of the reference substances.
2 2 7
NOTE 2 For more information about reference substances, see Reference [22].
7 Apparatus
Usual laboratory equipment, and in particular the following.
7.1 Freezer, for the storage of preserved bacteria.
7.2 Incubator or refrigerator, to maintain the solution for freeze-dried bacteria (6.5) at a temperature of
(4 ± 3) °C.
7.3 Thermostatically controlled thermo-block, to maintain the test samples and the Vibrio fischeri
suspension at a temperature of (15 ± 1) °C. Within one test, the temperature deviation should be at most
±0,3 °C.
7.4 Luminometer, equipped with at least one dispenser (minimum injection volume 0,2 ml).
The instrument shall be capable of measuring and recording the luminescence continuously at least for 5 s in
0,2 s intervals or shorter. The injection and recording shall be performed simultaneously.
7.5 Test tubes, cuvettes, test plates or any other suitable test vessels, made of chemically inert material,
appropriate for the selected luminometer.
7.6 pH-meter.
7.7 Chronometer.
[3]
7.8 Piston pipettes, nominal capacity 100 µl to 1 000 µl, ISO 8655-2 .
7.9 Mixer, e.g. vortex mixer, for mixing the samples before the measurements.
7.10 Conductometer.
7.11 Oxygen probe, as specified in ISO 5814.
[1]
7.12 Sieve, nominal size of openings 2 mm, ISO 565 .
8 Sampling and sample pre-treatment
8.1 Sampling
Collect the samples in chemically inert, clean containers as specified in ISO 5667-16. Fill the containers
completely and seal them. Cool the samples on ice, or in a refrigerator or a cooling box at 2 °C to 5 °C and
test them as soon as possible after collection. Where necessary, store samples at a temperature of 2 °C to
5 °C in the dark for no longer than 48 h. If the samples have to be frozen store them at a temperature of
−18 °C or below in the dark for no longer than 2 months. Prepare and measure frozen samples immediately
after thawing in a water bath. For long-term storage, the samples may be freeze-dried and stored in the dark
at room temperature. Do not use chemicals to preserve the samples. Perform the necessary pH adjustment
and salt addition just before testing.
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8.2 Sample preparation
Sieve the samples to remove any coarse material (e.g. plant roots and particles larger than 2 mm).
Suspend sediment samples in water (e.g. 20 % mass fraction) by vigorous shaking (vortexing) in order to
obtain homogeneous suspensions (Reference [22]). Also, mass fractions higher than 20 % can be measured
if the suspensions stay homogeneous during the whole measurement.
Measure the oxygen concentration in all samples and sample water suspensions. An oxygen concentration
>3 mg/l is required for the test. If the oxygen concentration of the undiluted sample is less than 3 mg/l, use
appropriate methods to oxygenate the sample, e.g. aeration or stirring.
Measure the pH of all samples and sample water suspensions. If the pH is between 6,0 and 8,5, no
adjustment is necessary. The adjustment of the pH value, however, may alter the nature of the sample. On
the other hand, the pH of the sample and the pH of the test batch can differ because of the buffer capacity of
the test medium. It may be necessary to carry out tests on both the pH-adjusted and the non-pH-adjusted
samples.
If necessary, adjust the pH of the samples by adding either hydrochloric acid (6.4) or sodium hydroxide
solution (6.3). Depending on the purpose of the test, the pH may be adjusted to 7,0 ± 0,2 or to the upper
(8,5 ± 0,2) or lower (6,0 ± 0,2) limits. Choose the concentration of the hydrochloric acid or the sodium
hydroxide solution to restrict the volume added to not more than 5 % of total volume.
Add 20 g of sodium chloride per litre to the sample or to the neutralized sample.
For salt water samples, ISO 11348-3:2007, Annex D gives further information.
9 Procedure
9.1 Initial preparations
9.1.1 Preparation of test solutions
Prepare the reference samples according to 6.6. Test each batch of bacteria after delivery with all three
reference substances. Test at least one of the three reference substances with each vial of stock suspension
reconstituted.
Prepare the samples according to 8.2.
Prepare, in the first set of test tubes (7.5), the sample dilution series, the reference sample (6.6) and the
control (6.2) required.
Serially dilute the sample. Make the dilutions in separate tubes and transfer them to the measurement
cuvettes or plates. Normally, in this test, equal volumes of test suspension and sample (or diluted sample) are
mixed, giving tested dilution levels within a series of D W 2.
The minimum dilution which can be tested is 1,00 in 1,25, prepared by mixing 4 volumes of sample with
1 volume of test suspension (e.g. 800 µl sample plus 200 µl test suspension). The corresponding D value is 1.
For this D value, an extra control batch is needed which is made up by adding 200 µl of the test suspension to
800 µl sodium chloride solution (see Annex C).
Maintain the test tubes containing the sodium chloride solution (6.2) for controls, the reference samples (6.6),
the samples (8.2) and the samples of the dilution series (Annex C) at (15 ± 1) °C.
Choose test conditions which ensure that the maximum temperature deviation in the thermo-block within one
test is no more than ±0,3 °C.
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9.1.2 Preparation of test suspensions
Prepare the luminescent bacterial stock suspension for the test according to the instructions for freshly
cultured (ISO 11348-1), liquid-dried (ISO 11348-2) or freeze-dried (ISO 11348-3) bacteria test.
Prepare the test suspension used in the kinetic test as specified, for example, in ISO 11348-3:2007, 8.3.3,
Variant B, where the test suspension is prepared outside the test tubes. Dilute reconstituted bacterial
suspension with solution (6.5) to form the test suspension before starting the test.
6
Store the luminescent bacteria test suspension for the measurement (cell concentration about 2 × 10 cells/ml)
at (4 ± 3) °C. The suspension is ready to use after a minimum of 30 min in a refrigerator and can be used for
testing purposes as long as the validity criteria stated in Clause 12 are met.
Before initiating the measurement, store the test suspension at (15 ± 1) °C for at least 30 min.
9.2 Test procedure
Carry out duplicate or triplicate determinations per dilution level at a test temperature of (15 ± 1) °C.
Set the dispensing volume from 200 µl to 500 µl (equal to the sample volume) and prime the dispenser with
the Vibrio fischeri bacterial test suspension.
Mix (7.9) the samples well and pipette 200 µl to 500 µl of each well-homogenized sample into the luminometer
cuvettes or microtiter plate wells depending on the instrument used. If the sample contains large particles,
cutting the sharp point of the pipette tip can facilitate the pipetting. Wider pipette tips are also commercially
available.
Load the sample cuvette (or microtiter plate) into the luminometer and start the run by dispensing Vibrio
fischeri bacterial suspension into the cuvette. Determine and record the maximum luminescence value of the
test suspension during the first 5 s of contact (at least 5 measurements/s). The maximum luminescence
intensity is the peak-value, I . Immediately after the measurement, put the cuvette back into the incubator and
p
measure the next cuvette. Repeat this process for each sample and control. When a microtiter plate reader is
used, run the controls and test sample dilution series immediately before transferring the plate to the incubator.
Store the cuvettes at (15 ± 1) °C during the incubation.
Determine and record the luminescence intensity after, optionally 5 min, I , and again after 15 min, I , and
5 15
30 min, I , as required. Mix the samples by hand before each measurement. Repeat this procedure for every
30
control and sample.
Intervals of 30 s between the samples have been found adequate to allow sufficient time for the readings to be
made and the change of the cuvettes.
10 Evaluation
10.1 Inhibitory effect on luminescent bacteria
Calculate the correction factor, f , for the contact time (5 min, 15 min or 30 min) from the measured
kt
luminescence intensity using Equation (1). This factor serves to correct the initial values, I , of all test samples
p
before they can be used as reference values for the determination of the water-dependent decrease of
luminescence.
I
kt
f = (1)
kt
I
p
where
I is the luminescence intensity in the control sample after the contact time of 5 min, 15 min or 30 min,
kt
in relative luminescence units;
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I is the maximum luminescence intensity (the peak value), in relative luminescence units, of the control
p
test suspension immediately after dispensing the bacteria into the sample.
Calculate the mean correction factor f . Find the deviation of the individual replicates from the mean,
kt
expressed as a percentage to one significant figure, using Formula (2):
⎡⎤
ff±
()
k t, i
kt
⎢⎥
×100 (2)
⎢⎥
f
kt
⎢⎥
⎣⎦
where f is either of the two individual values of the correction factor.
kt,i
Calculate the corrected value of I for test sample cuvettes (corrected peak intensity value), I , using
p ct
Equation (3):
I =⋅If (3)
cpt
kt
where
f is the mean of f ;
kt kt,i
I is the maximum luminescence intensity (the peak value), in relative luminescence units, of the test
p
suspension immediately after dispensing the bacteria into the sample.
Calculate the inhibitory effect of a test sample after the contact time (5 min, 15 min or 30 min), H , expressed
t
as a percentage, using Equation (4):
()II−
ctt
H=×100 (4)
t
I
ct
where
I
...
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