ISO 14380:2011
(Main)Water quality — Determination of the acute toxicity to Thamnocephalus platyurus (Crustacea, Anostraca)
Water quality — Determination of the acute toxicity to Thamnocephalus platyurus (Crustacea, Anostraca)
ISO 14380:2011 specifies a method for the determination of the lethal effects of toxicants to Thamnocephalus platyurus test organisms after 24 h exposure. A second method (rapid test) is described in an annex for the determination of sublethal effects after a very short exposure time (1 h). The methods are applicable to: a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions under the conditions of the test; b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration or centrifugation; c) fresh waters; d) aqueous extracts; e) toxins of blue‑green algae. ISO 14380:2011 is not applicable to the testing of unstable chemicals (hydrolysing, absorbing, etc.) in water unless exposure concentration is measured, nor to the testing of aquatic samples from the estuarine or marine environment.
Qualité de l'eau — Détermination de la toxicité aiguë envers Thamnocephalus platyurus (Crustacea, Anostraca)
Kakovost vode - Določevanje akutne strupenosti z raki Thamnocephalus platyurus (Crustacea, Anostraca)
Ta mednarodni standard določa metodo za določevanje smrtnih učinkov strupenih snovi na preskusne organizme Thamnocephalus platyurus po 24-urni izpostavljenosti. Druga metoda (hitri preskus) za določevanje subletalnih učinkov po zelo kratki izpostavljenosti (1 h) je opisana v dodatku A. Metode se uporabljajo za: a) kemične snovi, ki so topne ali se lahko pod pogoji preskusa ohranijo v stabilni suspenziji ali disperziji; b) obdelane ali neobdelane industrijske ali komunalne odplake, če so primerne po dekantaciji, filtriranju ali centrifugiranju; e) toksine modrozelenih alg. Ta mednarodni standard se ne uporablja za preskušanje nestabilnih kemikalij (hidrolizo, absorpcijo itd.) v vodi, razen če je bila izmerjena koncentracija izpostavljenosti, in preskušanje vodnih vzorcev iz okolja rečnih ustij ali morskega okolja.
General Information
Buy Standard
Standards Content (Sample)
SLOVENSKI STANDARD
SIST ISO 14380:2012
01-junij-2012
.DNRYRVWYRGH'RORþHYDQMHDNXWQHVWUXSHQRVWL]UDNL7KDPQRFHSKDOXVSODW\XUXV
&UXVWDFHD$QRVWUDFD
Water quality - Determination of the acute toxicity to Thamnocephalus platyurus
(Crustacea, Anostraca)
Qualité de l'eau - Détermination de la toxicité aiguë envers Thamnocephalus platyurus
(Crustacea, Anostraca)
Ta slovenski standard je istoveten z: ISO 14380:2011
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 14380:2012 en,fr
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
SIST ISO 14380:2012
---------------------- Page: 2 ----------------------
SIST ISO 14380:2012
INTERNATIONAL ISO
STANDARD 14380
First edition
2011-11-15
Water quality — Determination of the
acute toxicity to Thamnocephalus
platyurus (Crustacea, Anostraca)
Qualité de l’eau — Détermination de la toxicité aiguë envers
Thamnocephalus platyurus (Crustacea, Anostraca)
Reference number
ISO 14380:2011(E)
©
ISO 2011
---------------------- Page: 3 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2011
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2011 – All rights reserved
---------------------- Page: 4 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
Contents Page
Foreword . iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Test environment . 2
6 Reagents, test organisms and media . 2
7 Apparatus and materials . 3
8 Treatment and preparation of samples . 4
8.1 Special precautions . 4
8.2 Preparation of the stock solutions of substances to be tested . 4
9 Procedure . 4
9.1 Hatching of the cysts . 4
9.2 Selection of test concentrations . 5
9.3 Preparation of the test and control solutions . 5
9.4 Introduction of the organisms . 6
9.5 Incubation of the test system . 6
9.6 Measurements . 7
10 Estimation of the LC . 7
50
11 Reference test . 8
12 Validity criteria . 8
13 Test report . 8
Annex A (informative) Rapid test for determination of sublethal effects on Thamnocephalus platyurus
(1 h exposure) .10
Annex B (informative) Culturing of Thamnocephalus platyurus for cyst production .16
Annex C (informative) Precision data.19
Bibliography .20
© ISO 2011 – All rights reserved iii
---------------------- Page: 5 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(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 14380 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2011 – All rights reserved
---------------------- Page: 6 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
Introduction
The evaluation of harmful effects on water quality has for several years involved the performance of biological
tests. Crustaceans are of interest from the ecotoxicological point of view because they are primary consumers
and a major component of the zooplankton in aquatic ecosystems.
The test specified in this International Standard involves determination of the lethal effects on the fresh water
fairy shrimp Thamnocephalus platyurus after 24 h exposure to the toxicant. A rapid test can also be carried out
to determine sublethal effects after a very short exposure time (1 h).
The beavertail fairy shrimp T. platyurus is to date already used extensively in toxicity testing for several reasons:
a) this anostracan crustacean has a sensitivity to chemicals which is quite similar to that of the cladoceran
crustacean Daphnia magna (see References [4][5][6][7]);
b) the assays are performed with neonates hatched from dormant eggs (cysts), which bypasses the need for
culturing or maintaining live stock cultures of test organisms;
c) T. platyurus neonates are substantially smaller than neonates of Daphnia magna, hence the assays require
much smaller test containers, and much less bench space and incubation space;
d) T. platyurus is very sensitive to cyanotoxins produced by algal blooms in eutrophicated waters (see
References [8][9]).
© ISO 2011 – All rights reserved v
---------------------- Page: 7 ----------------------
SIST ISO 14380:2012
---------------------- Page: 8 ----------------------
SIST ISO 14380:2012
INTERNATIONAL STANDARD ISO 14380:2011(E)
Water quality — Determination of the acute toxicity to
Thamnocephalus platyurus (Crustacea, Anostraca)
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This 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 qualified staff.
1 Scope
This International Standard specifies a method for the determination of the lethal effects of toxicants to
Thamnocephalus platyurus test organisms after 24 h exposure. A second method (rapid test) is described in
Annex A for the determination of sublethal effects after a very short exposure time (1 h).
The methods are applicable to:
a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions
under the conditions of the test;
b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration or
centrifugation;
c) fresh waters;
d) aqueous extracts;
e) toxins of blue-green algae.
This International Standard is not applicable to the testing of unstable chemicals (hydrolysing, absorbing, etc.)
in water unless exposure concentration is measured, nor to the testing of aquatic samples from the estuarine
or marine environment.
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, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 10523, Water quality — Determination of pH
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2011 – All rights reserved 1
---------------------- Page: 9 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
3.1
control batch
series of replicates containing control solution
NOTE The role of a control batch in an experimental procedure is to demonstrate the response to the detection
system imposed collectively by compounds of the matrix used in the determination, in the absence of the subject of
interest.
3.2
LC
50
concentration or dilution of the test sample which gives rise to 50 % mortality of the test organisms
3.3
EC
50
concentration or dilution of the sample which gives rise to 50 % effect on the test organisms
3.4
neonate
newly hatched individual
3.5
test batch
series of replicates filled with the same test solution
4 Principle
Freshly hatched T. platyurus larvae are exposed to a range of concentrations of the sample under analysis and
the percentage mortality of the test organisms is determined after 24 h exposure, with subsequent calculation
of the 24 h LC .
50
The test is carried out in one or two stages:
— a “range-finding test” to determine the range of concentrations or dilutions needed for calculation of the
24 h LC ;
50
— a “definitive test” conducted when the data of the range-finding test are not sufficient or adequate for
calculation of the 24 h LC .
50
5 Test environment
The test shall be carried out in the dark, in a temperature-controlled room or incubator at (25 ± 1) °C in the test
containers.
Maintain the atmosphere free from toxic dusts or vapours. The use of control solutions is a double check that
the test is performed in an atmosphere free from toxic dusts and vapours.
6 Reagents, test organisms and media
Use only reagents of recognized analytical grade, unless otherwise specified.
6.1 Test organisms. The test organisms are neonates of the beavertail fairy shrimp T. platyurus, which are
hatched from dormant eggs (cysts) of this crustacean.
Cysts of T. platyurus are obtained from laboratory cultures of the crustacean as described in Annex B or can
1)
be purchased from a specialized company .
1) MicroBioTests Inc., Mariakerke, Belgium, is an example of a supplier able to provide suitable Thamnocephalus platyurus
cysts commercially. This information is given for the convenience of the users of this document and does not constitute an
endorsement by ISO of this supplier.
2 © ISO 2011 – All rights reserved
---------------------- Page: 10 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
6.2 Pure water, conductivity below 10 µS/cm.
6.3 Test medium, prepared by dissolving the following mineral substances in 1 l of pure water (6.2):
NaHCO 96 mg
3
CaSO⋅2H O 60 mg
4 2
MgSO 60 mg
4
KCl 4 mg
This test medium corresponds to a synthetic water of moderate hardness, i.e. containing CaCO at concentrations
3
of 80 mg/l to 100 mg/l (see Reference [13]). Thus prepared, the medium has a pH of 7,6 ± 0,3.
When stored in a refrigerator at (4 ± 2) °C in the dark, the solution can be used for several months.
Aerate the test medium until the dissolved oxygen concentration has reached the air saturation value and until
the pH has stabilized. If necessary, adjust the pH to 7,6 ± 0,3 using sodium hydroxide or hydrochloric acid
solutions. The concentration of the acid or base required shall be selected so that the volume to be admixed is
as small as possible. Bring the temperature of the test medium up to (25 ± 1) °C prior to use.
6.4 Hatching medium. An eightfold dilution of the test medium (6.3) with pure water (6.2).
6.5 Reference substance. Potassium dichromate (K Cr O ) is the recommended reference chemical.
2 2 7
7 Apparatus and materials
Usual laboratory apparatus and glassware and in particular the following.
7.1 Temperature-controlled room or chamber.
7.2 Hatching Petri dishes, small Petri dishes, diameter 5 cm, in glass or in inert plastic material.
7.3 Test containers. Disposable microplates made from chemically inert material, comprising wells with
a capacity >1 ml. For example, 24 (4 × 6) well microplates with a well diameter of approximately 16 mm are
suitable.
7.4 Pipette for sampling the test organisms, with a sufficient diameter for capturing the animals while
allowing sampling of only a small volume of medium.
Micropipettes of inert plastic material with a bulb at the end are very suitable for the operations.
7.5 Stereomicroscope with incident (bottom) illumination, with a magnification of at least eight times and,
if possible, a continuous magnification.
7.6 Light source, providing a range of light intensity in the hatching Petri dish of 3 000 lx to 4 000 lx.
7.7 Sample collecting bottles, as specified in ISO 5667-16.
© ISO 2011 – All rights reserved 3
---------------------- Page: 11 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
8 Treatment and preparation of samples
8.1 Special precautions
Special precautions are required for sampling, transportation, storage and treatment of water, effluent, or
aqueous extract samples to be tested.
Sampling, transportation and storage of the samples should be performed as specified in ISO 5667-16.
Carry out the toxicity test as soon as possible, ideally within 12 h of collection. If this time interval cannot be
met, cool the sample to 0 °C to 5 °C and test the sample within 24 h. If it is not possible to perform the test
within 72 h, the sample may be frozen and maintained deep-frozen (below −18 °C) for testing within 2 months
of collection, provided that characteristics are known to be unaffected by freezing. At the time of testing,
homogenize the sample to be analysed by shaking manually, and, if necessary, allow to settle for 2 h in a
container, and sample by drawing off (using a pipette) the required quantity of supernatant, maintaining the end
of the pipette in the centre of the section of the test tube and halfway between the surface of the deposited
substances and the surface of the liquid.
If the raw sample of the decanted supernatant is likely to interfere with the test (due to the presence of residual
suspended matter, protozoa, microorganisms, etc.), filter or centrifuge the raw or decanted sample.
The sample obtained by either of these methods is the sample submitted to testing.
Measure the pH (as specified in ISO 10523) and the dissolved oxygen concentration (as specified in ISO 5814)
and record these values in the test report.
If the aim of the test is to assess the acute toxicity without considering the pH effects, the test may also be
carried out after adjustment of the pH value to 7,6 ± 0,3 with hydrochloric acid or sodium hydroxide solutions.
Proceed, if appropriate, as indicated above, for the separation of the suspended matter formed following the
adjustment of the pH. Mention any pH adjustment in the test report.
8.2 Preparation of the stock solutions of substances to be tested
Prepare the stock solution of the substance to be tested by dissolving a known quantity of substance in a
specified volume of test medium (6.3) at the time of use. However, if the stock solution of the substance is
stable under certain conditions, it may be prepared in advance and stored under these conditions.
For substances sparingly soluble in the test medium, refer to the specifications given in ISO 5667-16.
9 Procedure
9.1 Hatching of the cysts
9.1.1 General
T. platyurus cysts shall be hatched under the conditions specified in 9.1.2 to 9.1.4.
9.1.2 Preparation of hatching medium
Prepare 20 ml hatching medium (6.4) by adding 17,5 ml pure water (6.2) to 2,5 ml test medium (6.3) in a small
glass container.
9.1.3 Prehydration of the cysts
Transfer approximately 10 mg to 15 mg dry cysts into a 1 ml tube in glass or in inert plastic material. The
amount depends on the hatchability of the cysts and should be sufficient to provide enough nauplii to perform a
complete toxicity test (i.e. >180 nauplii). Fill the tube with hatching medium. Close the tube and shake it several
times during a 30 min period to hydrate the cysts.
4 © ISO 2011 – All rights reserved
---------------------- Page: 12 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
9.1.4 Transfer of the prehydrated cysts into the hatching Petri dish
Empty the contents of the tube with prehydrated cysts into a Petri dish (7.2). Make sure that most of the cysts
are transferred by rinsing the tube with hatching medium.
Add 10 ml hatching medium to the Petri dish and swirl gently to distribute the cysts evenly.
Cover the hatching Petri dish and incubate at (25 ± 1) °C for 20 h to 22 h under continuous illumination (3 000 lx
−2 −1
to 4 000 lx, corresponding to 40 to 55 µmol m s ).
9.2 Selection of test concentrations
The test should comprise at least five concentrations of the sample to be tested. The dilutions shall be selected
within a geometric series with a separation factor which depends on the nature of the sample to be analysed
(chemical substances, effluents, waters or extracts) and of the type of assay (range finding or definitive).
For the range-finding test with chemical substances, the separation factor for the serial dilutions is usually 10
(one order of magnitude difference between two successive dilutions). A suitable concentration range is best
determined by carrying out a preliminary range-finding test covering several orders of magnitude of difference
in test concentration. Replication of test concentrations is not a requirement in the preliminary test.
For effluents, water or extracts a 1+1 dilution factor is normally applied (i.e. dilution of the previous concentration
by half).
Dilutions series for the definitive test on chemical substances are prepared with a separation factor not
exceeding 3,2.
The test is carried out with three replicates for each dilution plus a control (i.e. the test medium without sample)
also in three replicates.
When using a solvent to dissolve or disperse chemical substances, the test is carried out with three replicates
for the control (the test medium without sample) plus three replicates for the control with solvent.
NOTE The latter application requires the use of a second microplate at the highest concentration of solvent.
9.3 Preparation of the test and control solutions
Prepare the test solutions by mixing the appropriate volumes of the sample to be tested (Clause 8 and 9.2) or
of its initial dilution, with test medium (6.3).
Control and test solutions can be prepared in 10 ml containers (e.g. tubes in glass or in inert plastic material).
The containers shall be labelled as: control, C1, C2, C3, C4 and C5, in sequence of the highest to the lowest
test concentration.
Distribute the test and control solutions in the microplate in a volume of 1 ml per well and according to the
spatial distribution of the solutions in the wells as shown in Figure 1.
The microplate of 24 wells has six columns (1 to 6) and four rows (A to D).
The four wells in the left column (C6) are filled with the control batch (3.1).
Those of the other columns are filled with the toxicants (test batches 3.5) as follows: the four wells in column 2
are filled with the lowest toxicant dilution (C5), those of column 3 with the second lowest toxicant dilution (C4),
etc.
The wells in rows A, B and C are for the three replicates of the control batch columns and the test batch
columns respectively.
The wells in row D are “rinsing wells” intended to avoid dilution of the toxicant in the test wells during the
transfer of the organisms from the hatching Petri dish to the microplate.
© ISO 2011 – All rights reserved 5
---------------------- Page: 13 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
9.4 Introduction of the organisms
Put the hatching Petri dish on the glass stage of the stereomicroscope (7.5) and collect a number of actively
swimming T. platyurus larvae with the pipette (7.4), taking care to suck up as little hatching medium as possible
during this operation.
Transfer at least 35 test organisms into well D of column 1 (rinsing well) of the microplate and repeat this
operation for the other (rinsing) wells of row D.
Put the microplate on the glass stage of the stereomicroscope and transfer 10 neonates from the (rinsing) well
in column 1 (control batch) into the three wells of this column.
Key
1 test medium
2 test containers, 10 ml
3 test wells
4 rinsing wells
Figure 1 — Filling of the microplate with control and test solutions
Repeat this operation for the five other columns, going from left to right, i.e. starting with column 2 (lowest test
concentration) to column 5 (highest test concentration).
The pipette should be rinsed with test medium after the organisms have been transferred from the rinsing cup
to the three test cups in each individual column.
On completion of the transfers, cover the microplate with a sheet of e.g. polyethylene and the microplate cover.
9.5 Incubation of the test system
Incubate the microplate at (25 ± 1) °C in the dark for 24 h.
6 © ISO 2011 – All rights reserved
---------------------- Page: 14 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
9.6 Measurements
Take the cover and the sheet from the microplate and put the microplate on the glass stage of the
stereomicroscope.
Check all the wells of rows A, B and C of the six columns, and record the number of dead larvae in each well.
NOTE The larvae are considered dead if they do not show any movement during 10 s of observation.
Insert the numbers into the data report template (see Table 1).
On completion of the count, collect the contents of the three wells (A, B, C) of the control column in a suitable
container and measure the pH as specified in ISO 10523, and the oxygen concentration as specified in
ISO 5814.
Transfer the contents of the three wells into a separate container and carry out the mixing of the solution with
great care, in order to avoid adding oxygen from the air which could bias the oxygen measurement.
Perform the same operation and measurements for the three test wells of the most concentrated test
concentration (column C1).
Explanation to Table 1:
Test dilution series
C5: (lowest test concentration)
C4:
C3:
C2:
C1: (highest test concentration)
Table 1 — Data report template (24 h exposure time)
Column
Row
Control C5 C4 C3 C2 C1
A
B
C
Total /30 /30 /30 /30 /30 /30
Mortality, %
If testing chemicals, analytical confirmation is strongly recommended in order to verify test substance
concentrations.
10 Estimation of the LC
50
Calculate the mean percentage mortality in the control and in each test concentration.
Determine the 24 h LC (plus, if deemed necessary, other effect percentages, e.g. LC or LC ) by an
50 10 90
[3]
appropriate statistical method (see ISO/TS 20281 and Reference [18]), e.g. moving average or probit,
depending on the mortality values in the dilution series. Other models may be used depending on the shape of
[3]
the dose-response curve, as the objective is to obtain the best fit to the data (see ISO/TS 20281 ).
© ISO 2011 – All rights reserved 7
---------------------- Page: 15 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
11 Reference test
Periodically determine the 24 h LC of potassium dichromate (6.5) in order to verify the sensitivity of the test
50
organisms and the conformity to the test procedure.
The following dilution series of potassium dichromate shall be prepared with test medium for the reference test.
C1: 0,32 mg/l
C2: 0,18 mg/l
C3: 0,10 mg/l
C4: 0,056 mg/l
C5: 0,032 mg/l
According to an international interlaboratory comparison (Annex C) with 23 participants, after having excluded
one outlier (Mandel’s �-statistic), the mean LC value is a K Cr O concentration of 0,100 mg/l (95 % confidence
50 2 2 7
limits: 0,086 to 0,113), with a repeatability standard deviation � (within-laboratory variability) of 0,010 (a � of
� �,�
9,74 %), and a reproducibility standard deviation � (between-laboratory variability) of 0,024 (a � of 23,74 %).
� �,�
Therefore, according to the data of this extensive international interlaboratory comparison, the results of a test
with the reference chemical should be in the K Cr O concentration range 0,052 mg/l to 0,148 mg/l (calculated
2 2 7
as the mean LC 24 h ± 2 �).
50 �
12 Validity criteria
The test is considered valid if the following conditions are met:
a) the percentage mortality in the controls is not higher than 10 %;
b) the dissolved oxygen concentration at the end of the test (measured as indicated in 9.6) is ≥2 mg/l.
13 Test report
This test report shall contain at least the following information:
a) the test method used, together with a reference to this International Standard (ISO 14380:2011);
b) all information required for the complete identification of the sample or of the substrate under test;
c) the methods of preparation of the samples:
1) for effluents, waters and aqueous extracts, the method and the storage time of the samples, the pH
and the dissolved oxygen concentration of the initial sample, if need be, the conditions in which the
decantation, filtration or centrifugation of the sample and a possible adjustment of the pH were carried
out,
2) for chemicals, the method of preparation of the stock and test solutions;
d) all biological, chemical, and physical information relative to the test specified in this International Standard;
e) all information relative to the test organism, and, if need be, the origin and number of the batch of T.
platyurus cysts used;
f) all information relative to the test (sample concentrations, pH of the test and control solutions, etc.);
g) the test results in accordance with Clause 10 and the method with which they were calculated;
h) the results obtained from the reference test (Clause 11) as well as the date of the reference test;
8 © ISO 2011 – All rights reserved
---------------------- Page: 16 ----------------------
SIST ISO 14380:2012
ISO 14380:2011(E)
i) data to prove that the validity criteria (Clause 12) are met;
j) all operating details not specified in this International Standard, or regarded as optional, together with
details of any incident that may have influenced the
...
INTERNATIONAL ISO
STANDARD 14380
First edition
2011-11-15
Water quality — Determination of the
acute toxicity to Thamnocephalus
platyurus (Crustacea, Anostraca)
Qualité de l’eau — Détermination de la toxicité aiguë envers
Thamnocephalus platyurus (Crustacea, Anostraca)
Reference number
ISO 14380:2011(E)
©
ISO 2011
---------------------- Page: 1 ----------------------
ISO 14380:2011(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2011
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2011 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 14380:2011(E)
Contents Page
Foreword . iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Test environment . 2
6 Reagents, test organisms and media . 2
7 Apparatus and materials . 3
8 Treatment and preparation of samples . 4
8.1 Special precautions . 4
8.2 Preparation of the stock solutions of substances to be tested . 4
9 Procedure . 4
9.1 Hatching of the cysts . 4
9.2 Selection of test concentrations . 5
9.3 Preparation of the test and control solutions . 5
9.4 Introduction of the organisms . 6
9.5 Incubation of the test system . 6
9.6 Measurements . 7
10 Estimation of the LC . 7
50
11 Reference test . 8
12 Validity criteria . 8
13 Test report . 8
Annex A (informative) Rapid test for determination of sublethal effects on Thamnocephalus platyurus
(1 h exposure) .10
Annex B (informative) Culturing of Thamnocephalus platyurus for cyst production .16
Annex C (informative) Precision data.19
Bibliography .20
© ISO 2011 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 14380:2011(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 14380 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2011 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 14380:2011(E)
Introduction
The evaluation of harmful effects on water quality has for several years involved the performance of biological
tests. Crustaceans are of interest from the ecotoxicological point of view because they are primary consumers
and a major component of the zooplankton in aquatic ecosystems.
The test specified in this International Standard involves determination of the lethal effects on the fresh water
fairy shrimp Thamnocephalus platyurus after 24 h exposure to the toxicant. A rapid test can also be carried out
to determine sublethal effects after a very short exposure time (1 h).
The beavertail fairy shrimp T. platyurus is to date already used extensively in toxicity testing for several reasons:
a) this anostracan crustacean has a sensitivity to chemicals which is quite similar to that of the cladoceran
crustacean Daphnia magna (see References [4][5][6][7]);
b) the assays are performed with neonates hatched from dormant eggs (cysts), which bypasses the need for
culturing or maintaining live stock cultures of test organisms;
c) T. platyurus neonates are substantially smaller than neonates of Daphnia magna, hence the assays require
much smaller test containers, and much less bench space and incubation space;
d) T. platyurus is very sensitive to cyanotoxins produced by algal blooms in eutrophicated waters (see
References [8][9]).
© ISO 2011 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 14380:2011(E)
Water quality — Determination of the acute toxicity to
Thamnocephalus platyurus (Crustacea, Anostraca)
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This 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 qualified staff.
1 Scope
This International Standard specifies a method for the determination of the lethal effects of toxicants to
Thamnocephalus platyurus test organisms after 24 h exposure. A second method (rapid test) is described in
Annex A for the determination of sublethal effects after a very short exposure time (1 h).
The methods are applicable to:
a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions
under the conditions of the test;
b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration or
centrifugation;
c) fresh waters;
d) aqueous extracts;
e) toxins of blue-green algae.
This International Standard is not applicable to the testing of unstable chemicals (hydrolysing, absorbing, etc.)
in water unless exposure concentration is measured, nor to the testing of aquatic samples from the estuarine
or marine environment.
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, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 10523, Water quality — Determination of pH
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2011 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 14380:2011(E)
3.1
control batch
series of replicates containing control solution
NOTE The role of a control batch in an experimental procedure is to demonstrate the response to the detection
system imposed collectively by compounds of the matrix used in the determination, in the absence of the subject of
interest.
3.2
LC
50
concentration or dilution of the test sample which gives rise to 50 % mortality of the test organisms
3.3
EC
50
concentration or dilution of the sample which gives rise to 50 % effect on the test organisms
3.4
neonate
newly hatched individual
3.5
test batch
series of replicates filled with the same test solution
4 Principle
Freshly hatched T. platyurus larvae are exposed to a range of concentrations of the sample under analysis and
the percentage mortality of the test organisms is determined after 24 h exposure, with subsequent calculation
of the 24 h LC .
50
The test is carried out in one or two stages:
— a “range-finding test” to determine the range of concentrations or dilutions needed for calculation of the
24 h LC ;
50
— a “definitive test” conducted when the data of the range-finding test are not sufficient or adequate for
calculation of the 24 h LC .
50
5 Test environment
The test shall be carried out in the dark, in a temperature-controlled room or incubator at (25 ± 1) °C in the test
containers.
Maintain the atmosphere free from toxic dusts or vapours. The use of control solutions is a double check that
the test is performed in an atmosphere free from toxic dusts and vapours.
6 Reagents, test organisms and media
Use only reagents of recognized analytical grade, unless otherwise specified.
6.1 Test organisms. The test organisms are neonates of the beavertail fairy shrimp T. platyurus, which are
hatched from dormant eggs (cysts) of this crustacean.
Cysts of T. platyurus are obtained from laboratory cultures of the crustacean as described in Annex B or can
1)
be purchased from a specialized company .
1) MicroBioTests Inc., Mariakerke, Belgium, is an example of a supplier able to provide suitable Thamnocephalus platyurus
cysts commercially. This information is given for the convenience of the users of this document and does not constitute an
endorsement by ISO of this supplier.
2 © ISO 2011 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 14380:2011(E)
6.2 Pure water, conductivity below 10 µS/cm.
6.3 Test medium, prepared by dissolving the following mineral substances in 1 l of pure water (6.2):
NaHCO 96 mg
3
CaSO⋅2H O 60 mg
4 2
MgSO 60 mg
4
KCl 4 mg
This test medium corresponds to a synthetic water of moderate hardness, i.e. containing CaCO at concentrations
3
of 80 mg/l to 100 mg/l (see Reference [13]). Thus prepared, the medium has a pH of 7,6 ± 0,3.
When stored in a refrigerator at (4 ± 2) °C in the dark, the solution can be used for several months.
Aerate the test medium until the dissolved oxygen concentration has reached the air saturation value and until
the pH has stabilized. If necessary, adjust the pH to 7,6 ± 0,3 using sodium hydroxide or hydrochloric acid
solutions. The concentration of the acid or base required shall be selected so that the volume to be admixed is
as small as possible. Bring the temperature of the test medium up to (25 ± 1) °C prior to use.
6.4 Hatching medium. An eightfold dilution of the test medium (6.3) with pure water (6.2).
6.5 Reference substance. Potassium dichromate (K Cr O ) is the recommended reference chemical.
2 2 7
7 Apparatus and materials
Usual laboratory apparatus and glassware and in particular the following.
7.1 Temperature-controlled room or chamber.
7.2 Hatching Petri dishes, small Petri dishes, diameter 5 cm, in glass or in inert plastic material.
7.3 Test containers. Disposable microplates made from chemically inert material, comprising wells with
a capacity >1 ml. For example, 24 (4 × 6) well microplates with a well diameter of approximately 16 mm are
suitable.
7.4 Pipette for sampling the test organisms, with a sufficient diameter for capturing the animals while
allowing sampling of only a small volume of medium.
Micropipettes of inert plastic material with a bulb at the end are very suitable for the operations.
7.5 Stereomicroscope with incident (bottom) illumination, with a magnification of at least eight times and,
if possible, a continuous magnification.
7.6 Light source, providing a range of light intensity in the hatching Petri dish of 3 000 lx to 4 000 lx.
7.7 Sample collecting bottles, as specified in ISO 5667-16.
© ISO 2011 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 14380:2011(E)
8 Treatment and preparation of samples
8.1 Special precautions
Special precautions are required for sampling, transportation, storage and treatment of water, effluent, or
aqueous extract samples to be tested.
Sampling, transportation and storage of the samples should be performed as specified in ISO 5667-16.
Carry out the toxicity test as soon as possible, ideally within 12 h of collection. If this time interval cannot be
met, cool the sample to 0 °C to 5 °C and test the sample within 24 h. If it is not possible to perform the test
within 72 h, the sample may be frozen and maintained deep-frozen (below −18 °C) for testing within 2 months
of collection, provided that characteristics are known to be unaffected by freezing. At the time of testing,
homogenize the sample to be analysed by shaking manually, and, if necessary, allow to settle for 2 h in a
container, and sample by drawing off (using a pipette) the required quantity of supernatant, maintaining the end
of the pipette in the centre of the section of the test tube and halfway between the surface of the deposited
substances and the surface of the liquid.
If the raw sample of the decanted supernatant is likely to interfere with the test (due to the presence of residual
suspended matter, protozoa, microorganisms, etc.), filter or centrifuge the raw or decanted sample.
The sample obtained by either of these methods is the sample submitted to testing.
Measure the pH (as specified in ISO 10523) and the dissolved oxygen concentration (as specified in ISO 5814)
and record these values in the test report.
If the aim of the test is to assess the acute toxicity without considering the pH effects, the test may also be
carried out after adjustment of the pH value to 7,6 ± 0,3 with hydrochloric acid or sodium hydroxide solutions.
Proceed, if appropriate, as indicated above, for the separation of the suspended matter formed following the
adjustment of the pH. Mention any pH adjustment in the test report.
8.2 Preparation of the stock solutions of substances to be tested
Prepare the stock solution of the substance to be tested by dissolving a known quantity of substance in a
specified volume of test medium (6.3) at the time of use. However, if the stock solution of the substance is
stable under certain conditions, it may be prepared in advance and stored under these conditions.
For substances sparingly soluble in the test medium, refer to the specifications given in ISO 5667-16.
9 Procedure
9.1 Hatching of the cysts
9.1.1 General
T. platyurus cysts shall be hatched under the conditions specified in 9.1.2 to 9.1.4.
9.1.2 Preparation of hatching medium
Prepare 20 ml hatching medium (6.4) by adding 17,5 ml pure water (6.2) to 2,5 ml test medium (6.3) in a small
glass container.
9.1.3 Prehydration of the cysts
Transfer approximately 10 mg to 15 mg dry cysts into a 1 ml tube in glass or in inert plastic material. The
amount depends on the hatchability of the cysts and should be sufficient to provide enough nauplii to perform a
complete toxicity test (i.e. >180 nauplii). Fill the tube with hatching medium. Close the tube and shake it several
times during a 30 min period to hydrate the cysts.
4 © ISO 2011 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 14380:2011(E)
9.1.4 Transfer of the prehydrated cysts into the hatching Petri dish
Empty the contents of the tube with prehydrated cysts into a Petri dish (7.2). Make sure that most of the cysts
are transferred by rinsing the tube with hatching medium.
Add 10 ml hatching medium to the Petri dish and swirl gently to distribute the cysts evenly.
Cover the hatching Petri dish and incubate at (25 ± 1) °C for 20 h to 22 h under continuous illumination (3 000 lx
−2 −1
to 4 000 lx, corresponding to 40 to 55 µmol m s ).
9.2 Selection of test concentrations
The test should comprise at least five concentrations of the sample to be tested. The dilutions shall be selected
within a geometric series with a separation factor which depends on the nature of the sample to be analysed
(chemical substances, effluents, waters or extracts) and of the type of assay (range finding or definitive).
For the range-finding test with chemical substances, the separation factor for the serial dilutions is usually 10
(one order of magnitude difference between two successive dilutions). A suitable concentration range is best
determined by carrying out a preliminary range-finding test covering several orders of magnitude of difference
in test concentration. Replication of test concentrations is not a requirement in the preliminary test.
For effluents, water or extracts a 1+1 dilution factor is normally applied (i.e. dilution of the previous concentration
by half).
Dilutions series for the definitive test on chemical substances are prepared with a separation factor not
exceeding 3,2.
The test is carried out with three replicates for each dilution plus a control (i.e. the test medium without sample)
also in three replicates.
When using a solvent to dissolve or disperse chemical substances, the test is carried out with three replicates
for the control (the test medium without sample) plus three replicates for the control with solvent.
NOTE The latter application requires the use of a second microplate at the highest concentration of solvent.
9.3 Preparation of the test and control solutions
Prepare the test solutions by mixing the appropriate volumes of the sample to be tested (Clause 8 and 9.2) or
of its initial dilution, with test medium (6.3).
Control and test solutions can be prepared in 10 ml containers (e.g. tubes in glass or in inert plastic material).
The containers shall be labelled as: control, C1, C2, C3, C4 and C5, in sequence of the highest to the lowest
test concentration.
Distribute the test and control solutions in the microplate in a volume of 1 ml per well and according to the
spatial distribution of the solutions in the wells as shown in Figure 1.
The microplate of 24 wells has six columns (1 to 6) and four rows (A to D).
The four wells in the left column (C6) are filled with the control batch (3.1).
Those of the other columns are filled with the toxicants (test batches 3.5) as follows: the four wells in column 2
are filled with the lowest toxicant dilution (C5), those of column 3 with the second lowest toxicant dilution (C4),
etc.
The wells in rows A, B and C are for the three replicates of the control batch columns and the test batch
columns respectively.
The wells in row D are “rinsing wells” intended to avoid dilution of the toxicant in the test wells during the
transfer of the organisms from the hatching Petri dish to the microplate.
© ISO 2011 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 14380:2011(E)
9.4 Introduction of the organisms
Put the hatching Petri dish on the glass stage of the stereomicroscope (7.5) and collect a number of actively
swimming T. platyurus larvae with the pipette (7.4), taking care to suck up as little hatching medium as possible
during this operation.
Transfer at least 35 test organisms into well D of column 1 (rinsing well) of the microplate and repeat this
operation for the other (rinsing) wells of row D.
Put the microplate on the glass stage of the stereomicroscope and transfer 10 neonates from the (rinsing) well
in column 1 (control batch) into the three wells of this column.
Key
1 test medium
2 test containers, 10 ml
3 test wells
4 rinsing wells
Figure 1 — Filling of the microplate with control and test solutions
Repeat this operation for the five other columns, going from left to right, i.e. starting with column 2 (lowest test
concentration) to column 5 (highest test concentration).
The pipette should be rinsed with test medium after the organisms have been transferred from the rinsing cup
to the three test cups in each individual column.
On completion of the transfers, cover the microplate with a sheet of e.g. polyethylene and the microplate cover.
9.5 Incubation of the test system
Incubate the microplate at (25 ± 1) °C in the dark for 24 h.
6 © ISO 2011 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 14380:2011(E)
9.6 Measurements
Take the cover and the sheet from the microplate and put the microplate on the glass stage of the
stereomicroscope.
Check all the wells of rows A, B and C of the six columns, and record the number of dead larvae in each well.
NOTE The larvae are considered dead if they do not show any movement during 10 s of observation.
Insert the numbers into the data report template (see Table 1).
On completion of the count, collect the contents of the three wells (A, B, C) of the control column in a suitable
container and measure the pH as specified in ISO 10523, and the oxygen concentration as specified in
ISO 5814.
Transfer the contents of the three wells into a separate container and carry out the mixing of the solution with
great care, in order to avoid adding oxygen from the air which could bias the oxygen measurement.
Perform the same operation and measurements for the three test wells of the most concentrated test
concentration (column C1).
Explanation to Table 1:
Test dilution series
C5: (lowest test concentration)
C4:
C3:
C2:
C1: (highest test concentration)
Table 1 — Data report template (24 h exposure time)
Column
Row
Control C5 C4 C3 C2 C1
A
B
C
Total /30 /30 /30 /30 /30 /30
Mortality, %
If testing chemicals, analytical confirmation is strongly recommended in order to verify test substance
concentrations.
10 Estimation of the LC
50
Calculate the mean percentage mortality in the control and in each test concentration.
Determine the 24 h LC (plus, if deemed necessary, other effect percentages, e.g. LC or LC ) by an
50 10 90
[3]
appropriate statistical method (see ISO/TS 20281 and Reference [18]), e.g. moving average or probit,
depending on the mortality values in the dilution series. Other models may be used depending on the shape of
[3]
the dose-response curve, as the objective is to obtain the best fit to the data (see ISO/TS 20281 ).
© ISO 2011 – All rights reserved 7
---------------------- Page: 12 ----------------------
ISO 14380:2011(E)
11 Reference test
Periodically determine the 24 h LC of potassium dichromate (6.5) in order to verify the sensitivity of the test
50
organisms and the conformity to the test procedure.
The following dilution series of potassium dichromate shall be prepared with test medium for the reference test.
C1: 0,32 mg/l
C2: 0,18 mg/l
C3: 0,10 mg/l
C4: 0,056 mg/l
C5: 0,032 mg/l
According to an international interlaboratory comparison (Annex C) with 23 participants, after having excluded
one outlier (Mandel’s �-statistic), the mean LC value is a K Cr O concentration of 0,100 mg/l (95 % confidence
50 2 2 7
limits: 0,086 to 0,113), with a repeatability standard deviation � (within-laboratory variability) of 0,010 (a � of
� �,�
9,74 %), and a reproducibility standard deviation � (between-laboratory variability) of 0,024 (a � of 23,74 %).
� �,�
Therefore, according to the data of this extensive international interlaboratory comparison, the results of a test
with the reference chemical should be in the K Cr O concentration range 0,052 mg/l to 0,148 mg/l (calculated
2 2 7
as the mean LC 24 h ± 2 �).
50 �
12 Validity criteria
The test is considered valid if the following conditions are met:
a) the percentage mortality in the controls is not higher than 10 %;
b) the dissolved oxygen concentration at the end of the test (measured as indicated in 9.6) is ≥2 mg/l.
13 Test report
This test report shall contain at least the following information:
a) the test method used, together with a reference to this International Standard (ISO 14380:2011);
b) all information required for the complete identification of the sample or of the substrate under test;
c) the methods of preparation of the samples:
1) for effluents, waters and aqueous extracts, the method and the storage time of the samples, the pH
and the dissolved oxygen concentration of the initial sample, if need be, the conditions in which the
decantation, filtration or centrifugation of the sample and a possible adjustment of the pH were carried
out,
2) for chemicals, the method of preparation of the stock and test solutions;
d) all biological, chemical, and physical information relative to the test specified in this International Standard;
e) all information relative to the test organism, and, if need be, the origin and number of the batch of T.
platyurus cysts used;
f) all information relative to the test (sample concentrations, pH of the test and control solutions, etc.);
g) the test results in accordance with Clause 10 and the method with which they were calculated;
h) the results obtained from the reference test (Clause 11) as well as the date of the reference test;
8 © ISO 2011 – All rights reserved
---------------------- Page: 13 ----------------------
ISO 14380:2011(E)
i) data to prove that the validity criteria (Clause 12) are met;
j) all operating details not specified in this International Standard, or regarded as optional, together with
details of any incident that may have influenced the results;
k) name and address of the testing laboratory, the persons carrying out the test, and the person approving
the report.
© ISO 2011 – All rights reserved 9
---------------------- Page: 14 ----------------------
ISO 14380:2011(E)
Annex A
(informative)
Rapid test for determination of sublethal effects on
Thamnocephalus platyurus (1 h exposure)
A.1 General
This International Standard can be applied to testing of pure chemicals as well as to effluents, waste waters
and other environmental aqueous samples.
The test procedure is based on the determination of the uptake of coloured microspheres by the test organisms
in a non-toxic medium (control) as compared to the uptake (or absence of uptake) in the test solution under
analysis.
Even after a very short exposure time (1 h), stressed crustaceans already either cease to take up the particles
or ingest them at a much lower rate.
Comparative studies on chemicals and cyanotoxins on the sensitivity of the sublethal effect criterion after 1 h
exposure versus the mortality criterion after 24 h exposure revealed a positive correlation between the 1 h
EC and the 24 h LC (see References [13][14]).
50 50
T
...
SLOVENSKI STANDARD
oSIST ISO 14380:2012
01-februar-2012
.DNRYRVWYRGH'RORþHYDQMHDNXWQHVWUXSHQRVWL]UDNL7KDPQRFHSKDOXVSODW\XUXV
&UXVWDFHD$QRVWUDFD
Water quality - Determination of the acute toxicity to Thamnocephalus platyurus
(Crustacea, Anostraca)
Qualité de l'eau - Détermination de la toxicité aiguë envers Thamnocephalus platyurus
(Crustacea, Anostraca)
Ta slovenski standard je istoveten z: ISO 14380:2011
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
oSIST ISO 14380:2012 en,fr
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST ISO 14380:2012
---------------------- Page: 2 ----------------------
oSIST ISO 14380:2012
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 14380
ISO/TC 147/SC 5
Water quality — Determination of the
Secretariat: DIN
acute toxicity to Thamnocephalus
Voting begins on:
platyurus (Crustacea, Anostraca)
2011-09-01
Voting terminates on:
Qualité de l’eau — Détermination de la toxicité aiguë envers
2011-11-01
Thamnocephalus platyurus (Crustacea, Anostraca)
Please see the administrative notes on page iii
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR
TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
ISO/FDIS 14380:2011(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN
DARDS TO WHICH REFERENCE MAY BE MADE IN
©
NATIONAL REGULATIONS. ISO 2011
---------------------- Page: 3 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
Copyright notice
This ISO document is a Draft International Standard and is copyrightprotected by ISO. Except as permitted
under the applicable laws of the user’s country, neither this ISO draft nor any extract from it may be reproduced,
stored in a retrieval system or transmitted in any form or by any means, electronic, photocopying, recording
or otherwise, without prior written permission being secured.
Requests for permission to reproduce should be addressed to either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
Email copyright@iso.org
Web www.iso.org
Reproduction may be subject to royalty payments or a licensing agreement.
Violators may be prosecuted.
ii © ISO 2011 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
In accordance with the provisions of Council Resolution 15/1993, this document is circulated in the
English language only.
© ISO 2011 – All rights reserved iii
---------------------- Page: 5 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
Contents Page
Foreword . v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Test environment . 2
6 Reagents, test organisms and media . 2
7 Apparatus and materials . 3
8 Treatment and preparation of samples . 3
8.1 Special precautions . 3
8.2 Preparation of the stock solutions of substances to be tested . 4
9 Procedure . 4
9.1 Hatching of the cysts . 4
9.2 Selection of test concentrations . 5
9.3 Preparation of the test and control solutions . 5
9.4 Introduction of the organisms . 5
9.5 Incubation of the test system . 6
9.6 Measurements . 6
10 Estimation of the LC
50 .7
11 Reference test . 7
12 Validity criteria . 8
13 Test report . 8
Annex A (informative) Rapid test for determination of sublethal effects on Thamnocephalus platyurus
(1 h exposure) . 9
Annex B (informative) Culturing of Thamnocephalus platyurus for cyst production .15
Annex C (informative) Precision data.18
Bibliography .19
iv © ISO 2011 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(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
nongovernmental, 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 14380 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
© ISO 2011 – All rights reserved v
---------------------- Page: 7 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
Introduction
The evaluation of harmful effects on water quality has for several years involved the performance of biological
tests. Crustaceans are of interest from the ecotoxicological point of view because they are primary consumers
and a major component of the zooplankton in aquatic ecosystems.
The test specified in this International Standard involves determination of the lethal effects on the fresh water
fairy shrimp Thamnocephalus platyurus after 24 h exposure to the toxicant. A rapid test can also be carried out
to determine sublethal effects after a very short exposure time (1 h).
The beavertail fairy shrimp T. platyurus is to date already used extensively in toxicity testing for several reasons:
a) this anostracan crustacean has a sensitivity to chemicals which is quite similar to that of the cladoceran
crustacean Daphnia magna (see References [4][5][6][7]);
b) the assays are performed with neonates hatched from dormant eggs (cysts), which bypasses the need for
culturing or maintaining live stock cultures of test organisms;
c) T. platyurus neonates are substantially smaller than neonates of Daphnia magna, hence the assays require
much smaller test containers, and much less bench space and incubation space;
d) T. platyurus is very sensitive to cyanotoxins produced by algal blooms in eutrophicated waters (see
References [8][9]).
vi © ISO 2011 – All rights reserved
---------------------- Page: 8 ----------------------
oSIST ISO 14380:2012
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 14380:2011(E)
Water quality — Determination of the acute toxicity to
Thamnocephalus platyurus (Crustacea, Anostraca)
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This 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 qualified staff.
1 Scope
This International Standard specifies a method for the determination of the lethal effects of toxicants to
Thamnocephalus platyurus test organisms after 24 h exposure. A second method (rapid test) is described in
Annex A for the determination of sublethal effects after a very short exposure time (1 h).
The methods are applicable to:
a) chemical substances which are soluble or which can be maintained as stable suspensions or dispersions
under the conditions of the test;
b) industrial or sewage effluents, treated or untreated, if appropriate after decantation, filtration or
centrifugation;
c) fresh waters;
d) aqueous extracts;
e) toxins of bluegreen algae.
This International Standard is not applicable to the testing of unstable chemicals (hydrolysing, absorbing, etc.)
in water unless exposure concentration is measured, nor to the testing of aquatic samples from the estuarine
or marine environment.
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 566716, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 10523, Water quality — Determination of pH
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
© ISO 2011 – All rights reserved 1
---------------------- Page: 9 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
3.1
control batch
series of replicates containing control solution
NOTE The role of a control batch in an experimental procedure is to demonstrate the response to the detection
system imposed collectively by compounds of the matrix used in the determination, in the absence of the subject of interest.
3.2
LC
50
concentration or dilution of the test sample which gives rise to 50 % mortality of the test organisms
3.3
EC
50
concentration or dilution of the sample which gives rise to 50 % effect on the test organisms
3.4
neonate
newly hatched individual
3.5
test batch
series of replicates filled with the same test solution
4 Principle
Freshly hatched T. platyurus larvae are exposed to a range of concentrations of the sample under analysis and
the percentage mortality of the test organisms is determined after 24 h exposure, with subsequent calculation
of the 24 h LC .
50
The test is carried out in one or two stages:
— a “rangefinding test” to determine the range of concentrations or dilutions needed for calculation of
the 24 h LC ;
50
— a “definitive test” conducted when the data of the rangefinding test are not sufficient or adequate for
calculation of the 24 h LC .
50
5 Test environment
The test shall be carried out in the dark, in a temperaturecontrolled room or incubator at (25 ± 1) °C in the
test containers.
Maintain the atmosphere free from toxic dusts or vapours. The use of control solutions is a double check that
the test is performed in an atmosphere free from toxic dusts and vapours.
6 Reagents, test organisms and media
Use only reagents of recognized analytical grade, unless otherwise specified.
6.1 Test organisms. The test organisms are neonates of the beavertail fairy shrimp T. platyurus, which are
hatched from dormant eggs (cysts) of this crustacean.
Cysts of T. platyurus are obtained from laboratory cultures of the crustacean as described in Annex B or can
1)
be purchased from a specialized company .
1) MicroBioTests Inc., Mariakerke, Belgium, is an example of a supplier able to provide suitable Thamnocephalus platyurus
cysts commercially. This information is given for the convenience of the users of this document and does not constitute an
endorsement by ISO of this supplier.
2 © ISO 2011 – All rights reserved
---------------------- Page: 10 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
6.2 Pure water, conductivity below 10 µS/cm.
6.3 Test medium, prepared by dissolving the following mineral substances in 1 l of pure water (6.2):
NaHCO 96 mg
3
CaSO⋅2H O 60 mg
4 2
MgSO 60 mg
4
KCl 4 mg
This test medium corresponds to a synthetic water of moderate hardness, i.e. containing CaCO at concentrations
3
of 80 mg/l to 100 mg/l (see Reference [13]). Thus prepared, the medium has a pH of 7,6 ± 0,3.
When stored in a refrigerator at (4 ± 2) °C in the dark, the solution can be used for several months.
Aerate the test medium until the dissolved oxygen concentration has reached the air saturation value and until
the pH has stabilized. If necessary, adjust the pH to 7,6 ± 0,3 using sodium hydroxide or hydrochloric acid
solutions. The concentration of the acid or base required shall be selected so that the volume to be admixed is
as small as possible. Bring the temperature of the test medium up to (25 ± 1) °C prior to use.
6.4 Hatching medium. An eightfold dilution of the test medium (6.3) with pure water (6.2).
6.5 Reference substance. Potassium dichromate (K Cr O ) is the recommended reference chemical.
2 2 7
7 Apparatus and materials
Usual laboratory apparatus and glassware and in particular the following.
7.1 Temperature-controlled room or chamber.
7.2 Hatching Petri dishes, small Petri dishes, diameter 5 cm, in glass or in inert plastic material.
7.3 Test containers. Disposable microplates made from chemically inert material, comprising wells with a
capacity >1 ml. For example, 24 (4 × 6) well microplates with a well diameter of approximately 16 mm are suitable.
7.4 Pipette for sampling the test organisms, with a sufficient diameter for capturing the animals while
allowing sampling of only a small volume of medium.
Micropipettes of inert plastic material with a bulb at the end are very suitable for the operations.
7.5 Stereomicroscope with incident (bottom) illumination, with a magnification of at least eight times and,
if possible, a continuous magnification.
7.6 Light source, providing a range of light intensity in the hatching Petri dish of 3 000 lx to 4 000 lx.
7.7 Sample collecting bottles, as specified in ISO 566716.
8 Treatment and preparation of samples
8.1 Special precautions
Special precautions are required for sampling, transportation, storage and treatment of water, effluent, or
aqueous extract samples to be tested.
© ISO 2011 – All rights reserved 3
---------------------- Page: 11 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
Sampling, transportation and storage of the samples should be performed as specified in ISO 566716.
Carry out the toxicity test as soon as possible, ideally within 12 h of collection. If this time interval cannot be
met, cool the sample to 0 °C to 5 °C and test the sample within 24 h. If it is not possible to perform the test
within 72 h, the sample may be frozen and maintained deepfrozen (below −18 °C) for testing within 2 months
of collection, provided that characteristics are known to be unaffected by freezing. At the time of testing,
homogenize the sample to be analysed by shaking manually, and, if necessary, allow to settle for 2 h in a
container, and sample by drawing off (using a pipette) the required quantity of supernatant, maintaining the end
of the pipette in the centre of the section of the test tube and halfway between the surface of the deposited
substances and the surface of the liquid.
If the raw sample of the decanted supernatant is likely to interfere with the test (due to the presence of residual
suspended matter, protozoa, microorganisms, etc.), filter or centrifuge the raw or decanted sample.
The sample obtained by either of these methods is the sample submitted to testing.
Measure the pH (as specified in ISO 10523) and the dissolved oxygen concentration (as specified in ISO 5814)
and record these values in the test report.
If the aim of the test is to assess the acute toxicity without considering the pH effects, the test may also be
carried out after adjustment of the pH value to 7,6 ± 0,3 with hydrochloric acid or sodium hydroxide solutions.
Proceed, if appropriate, as indicated above, for the separation of the suspended matter formed following the
adjustment of the pH. Mention any pH adjustment in the test report.
8.2 Preparation of the stock solutions of substances to be tested
Prepare the stock solution of the substance to be tested by dissolving a known quantity of substance in a
specified volume of test medium (6.3) at the time of use. However, if the stock solution of the substance is
stable under certain conditions, it may be prepared in advance and stored under these conditions.
For substances sparingly soluble in the test medium, refer to the specifications given in ISO 566716.
9 Procedure
9.1 Hatching of the cysts
9.1.1 General
T. platyurus cysts shall be hatched under the conditions specified in 9.1.2 to 9.1.4.
9.1.2 Preparation of hatching medium
Prepare 20 ml hatching medium (6.4) by adding 17,5 ml pure water (6.2) to 2,5 ml test medium (6.3) in a small
glass container.
9.1.3 Prehydration of the cysts
Transfer approximately 10 mg to 15 mg dry cysts into a 1 ml tube in glass or in inert plastic material. The
amount depends on the hatchability of the cysts and should be sufficient to provide enough nauplii to perform a
complete toxicity test (i.e. >180 nauplii). Fill the tube with hatching medium. Close the tube and shake it several
times during a 30 min period to hydrate the cysts.
9.1.4 Transfer of the prehydrated cysts into the hatching Petri dish
Empty the contents of the tube with prehydrated cysts into a Petri dish (7.2). Make sure that most of the cysts
are transferred by rinsing the tube with hatching medium.
Add 10 ml hatching medium to the Petri dish and swirl gently to distribute the cysts evenly.
4 © ISO 2011 – All rights reserved
---------------------- Page: 12 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
Cover the hatching Petri dish and incubate at (25 ± 1) °C for 20 h to 22 h under continuous illumination (3 000 lx
−2 −1
to 4 000 lx, corresponding to 40 to 55 µmol m s ).
9.2 Selection of test concentrations
The test should comprise at least five concentrations of the sample to be tested. The dilutions shall be selected
within a geometric series with a separation factor which depends on the nature of the sample to be analysed
(chemical substances, effluents, waters or extracts) and of the type of assay (range finding or definitive).
For the rangefinding test with chemical substances, the separation factor for the serial dilutions is usually 10
(one order of magnitude difference between two successive dilutions). A suitable concentration range is best
determined by carrying out a preliminary rangefinding test covering several orders of magnitude of difference
in test concentration. Replication of test concentrations is not a requirement in the preliminary test.
For effluents, water or extracts a 1+1 dilution factor is normally applied (i.e. dilution of the previous
concentration by half).
Dilutions series for the definitive test on chemical substances are prepared with a separation factor not
exceeding 3,2.
The test is carried out with three replicates for each dilution plus a control (i.e. the test medium without sample)
also in three replicates.
When using a solvent to dissolve or disperse chemical substances, the test is carried out with three replicates
for the control (the test medium without sample) plus three replicates for the control with solvent.
NOTE The latter application requires the use of a second microplate at the highest concentration of solvent.
9.3 Preparation of the test and control solutions
Prepare the test solutions by mixing the appropriate volumes of the sample to be tested (Clause 8 and 9.2) or
of its initial dilution, with test medium (6.3).
Control and test solutions can be prepared in 10 ml containers (e.g. tubes in glass or in inert plastic material).
The containers shall be labelled as: control, C1, C2, C3, C4 and C5, in sequence of the highest to the lowest
test concentration.
Distribute the test and control solutions in the microplate in a volume of 1 ml per well and according to the
spatial distribution of the solutions in the wells as shown in Figure 1.
The microplate of 24 wells has six columns (1 to 6) and four rows (A to D).
The four wells in the left column (C6) are filled with the control batch (3.1).
Those of the other columns are filled with the toxicants (test batches 3.5) as follows: the four wells in column 2
are filled with the lowest toxicant dilution (C5), those of column 3 with the second lowest toxicant dilution (C4), etc.
The wells in rows A, B and C are for the three replicates of the control batch columns and the test batch
columns respectively.
The wells in row D are “rinsing wells” intended to avoid dilution of the toxicant in the test wells during the
transfer of the organisms from the hatching Petri dish to the microplate.
9.4 Introduction of the organisms
Put the hatching Petri dish on the glass stage of the stereomicroscope (7.5) and collect a number of actively
swimming T. platyurus larvae with the pipette (7.4), taking care to suck up as little hatching medium as possible
during this operation.
Transfer at least 35 test organisms into well D of column 1 (rinsing well) of the microplate and repeat this
operation for the other (rinsing) wells of row D.
© ISO 2011 – All rights reserved 5
---------------------- Page: 13 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
Put the microplate on the glass stage of the stereomicroscope and transfer 10 neonates from the (rinsing) well
in column 1 (control batch) into the three wells of this column.
Key
1 test medium
2 test containers, 10 ml
3 test wells
4 rinsing wells
Figure 1 — Filling of the microplate with control and test solutions
Repeat this operation for the five other columns, going from left to right, i.e. starting with column 2 (lowest test
concentration) to column 5 (highest test concentration).
The pipette should be rinsed with test medium after the organisms have been transferred from the rinsing cup
to the three test cups in each individual column.
On completion of the transfers, cover the microplate with a sheet of e.g. polyethylene and the microplate cover.
9.5 Incubation of the test system
Incubate the microplate at (25 ± 1) °C in the dark for 24 h.
9.6 Measurements
Take the cover and the sheet from the microplate and put the microplate on the glass stage of the
stereomicroscope.
Check all the wells of rows A, B and C of the six columns, and record the number of dead larvae in each well.
NOTE The larvae are considered dead if they do not show any movement during 10 s of observation.
6 © ISO 2011 – All rights reserved
---------------------- Page: 14 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
Insert the numbers into the data report template (see Table 1).
On completion of the count, collect the contents of the three wells (A, B, C) of the control column in a suitable
container and measure the pH as specified in ISO 10523, and the oxygen concentration as specified in ISO 5814.
Transfer the contents of the three wells into a separate container and carry out the mixing of the solution with
great care, in order to avoid adding oxygen from the air which could bias the oxygen measurement.
Perform the same operation and measurements for the three test wells of the most concentrated test
concentration (column C1).
Explanation to Table 1:
Test dilution series
C5: (lowest test concentration)
C4:
C3:
C2:
C1: (highest test concentration)
Table 1 — Data report template (24 h exposure time)
Column
Row
Control C5 C4 C3 C2 C1
A
B
C
Total /30 /30 /30 /30 /30 /30
Mortality, %
If testing chemicals, analytical confirmation is strongly recommended in order to verify test substance
concentrations.
10 Estimation of the LC
50
Calculate the mean percentage mortality in the control and in each test concentration.
Determine the 24 h LC (plus, if deemed necessary, other effect percentages, e.g. LC or LC ) by an
50 10 90
[3]
appropriate statistical method (see ISO/TS 20281 and Reference [18]), e.g. moving average or probit,
depending on the mortality values in the dilution series. Other models may be used depending on the shape of
[3]
the doseresponse curve, as the objective is to obtain the best fit to the data (see ISO/TS 20281 ).
11 Reference test
Periodically determine the 24 h LC of potassium dichromate (6.5) in order to verify the sensitivity of the test
50
organisms and the conformity to the test procedure.
The following dilution series of potassium dichromate shall be prepared with test medium for the reference test.
C1: 0,32 mg/l
C2: 0,18 mg/l
© ISO 2011 – All rights reserved 7
---------------------- Page: 15 ----------------------
oSIST ISO 14380:2012
ISO/FDIS 14380:2011(E)
C3: 0,10 mg/l
C4: 0,056 mg/l
C5: 0,032 mg/l
According to an international interlaboratory comparison (Annex C) with 23 participants, after having excluded
one outlier (Mandel’s hstatistic), the mean LC value is a K Cr O concentration of 0,100 mg/l (95 % confidence
50 2 2 7
limits: 0,086 to 0,113), with a repeatability standard deviation s (withinlaboratory variability) of 0,010 (a C of
r V,r
9,74 %), and a reproducibility standard deviation s (betweenlaboratory variability) of 0,024 (a C of 23,74 %).
R V,R
Therefore, according to the data of this extensive international interlaboratory comparison, the results of a test
with the reference chemical should be in the K Cr O concentration range 0,052 mg/l to 0,148 mg/l (calculated
2 2 7
as the mean LC 24 h ± 2 s ).
50 R
12 Validity criteria
The test is considered valid if the following conditions are met:
a) the percentage mortality in the controls is not higher than 10 %;
b) the dissolved oxygen concentration at the end of the test (measured as indicated in 9.6) is ≤2 mg/l.
13 Test report
This test report shall contain at least the following information:
a) the test method used, together with a reference to this International Standard (ISO 14380:2011);
b) all information required for the complete identification of the sample or of the substrate under test;
c) the methods of preparation of the samples:
1) for effluents, waters and aqueous extracts, the method and the storage time of the samples, the pH and the
dissolved oxygen concentratio
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.