ISO/TS 18220:2016
(Main)Water quality — Larval development test with the harpacticoid copepod Nitocra spinipes
Water quality — Larval development test with the harpacticoid copepod Nitocra spinipes
ISO/TS 18220:2016 specifies an early-life stage procedure for determination of the toxic effects of chemicals and water samples on a cold-water brackish water copepod species under semi-static conditions. The biological test variables include survival and development of the early-life stages. The exposure starts with newly hatched ( The benthic living Nitocra complements the planktonic Acartia species in ISO 16778. These organisms represent different life-history strategies as Nitocra is egg-carrying, whereas Acartia is a broadcasting calanoid copepod and thus, different sensitivities of specific life stages. Nitocra is a fresh to brackish water species, which allows testing low salinity waters and is complementary to A. tonsa, which is of marine origin and poorly tolerates low salinities.
Qualité de l'eau — Essai de développement larvaire avec le copépode harpacticoïde Nitocra spinipes
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TECHNICAL ISO/TS
SPECIFICATION 18220
First edition
2016-07-01
Water quality — Larval development
test with the harpacticoid copepod
Nitocra spinipes
Qualité de l’eau — Essai de développement larvaire avec le
copépode harpacticoïde Nitocra spinipes
Reference number
ISO/TS 18220:2016(E)
©
ISO 2016
---------------------- Page: 1 ----------------------
ISO/TS 18220:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 18220:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Terms and definitions . 1
3 Principle . 2
4 Reagents . 2
5 Cultivation . 3
5.1 Test organism . 3
5.2 Algae for feeding . 3
6 Apparatus . 3
7 Procedure. 4
7.1 Production of nauplii to be used in test . 4
7.2 Choice of test concentrations . 4
7.2.1 Hypothesis testing . 5
7.2.2 Regression analysis. 5
7.3 Preparation of solutions to be used in test . 5
7.3.1 Stock solution . 5
7.3.2 Test solutions . 5
7.4 Start of test . 6
7.5 Incubation/exposure . 6
7.6 Maintenance . 6
7.7 Measurements/observations . 6
7.7.1 Mortality . 6
7.7.2 Larval development ratio (LDR). 7
7.7.3 Physical-chemical parameters — oxygen, pH and salinity . 7
7.7.4 Concentration of the test substance . 7
8 Validity criteria . 7
9 Evaluation of results . 8
9.1 Calculation of results . 8
9.2 Expression of results . 8
9.3 Interpretation of results . 8
10 Reproducibility . 8
11 Test report . 8
Annex A (informative) Biology and cultivation of Nitocra spinipes .10
Annex B (informative) Nitocra spinipes larval development ratio .15
Annex C (informative) Calculations .17
Bibliography .19
© ISO 2016 – All rights reserved iii
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ISO/TS 18220:2016(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 18220:2016(E)
Introduction
Harpacticoid copepods are predominantly benthic, occurring widely in marine, brackish and fresh
water ecosystems. They represent important prey items for the benthic larvae of many fish species and
larger invertebrates and constitute an ecologically important energy-transfer link between the organic
phase of the sediment and higher trophic levels.
The euryhaline brackish water harpactoid Nitocra spinipes (Crustacea) is a common component of the
benthic meiofana in shallow coastal waters around the world (see Reference [6]).
© ISO 2016 – All rights reserved v
---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/TS 18220:2016(E)
Water quality — Larval development test with the
harpacticoid copepod Nitocra spinipes
WARNING — Persons using this Technical Specification should be familiar with normal
laboratory practice. This Technical Specification 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 Technical
Specification be carried out by suitably qualified staff.
1 Scope
This Technical Specification specifies an early-life stage procedure for determination of the toxic
effects of chemicals and water samples on a cold-water brackish water copepod species under semi-
static conditions. The biological test variables include survival and development of the early-life stages.
The exposure starts with newly hatched (<24 h) nauplii (larvae) and is continued until emergence of
(c. 50 %) copepodites (juveniles) in the control.
The benthic living Nitocra complements the planktonic Acartia species in ISO 16778. These organisms
represent different life-history strategies as Nitocra is egg-carrying, whereas Acartia is a broadcasting
calanoid copepod and thus, different sensitivities of specific life stages. Nitocra is a fresh to brackish
water species, which allows testing low salinity waters and is complementary to A. tonsa, which is of
marine origin and poorly tolerates low salinities.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
nauplii
larvae
2.2
copepodites
juveniles
2.3
larval development ratio
LDR
ratio of copepodites (2.2) to the total number of surviving early-life stages (nauplii + copepodites) at the
end of the test
2.4
lowest observed effect concentration
LOEC
lowest concentration within the experimental range at which a significant effect is observed
2.5
no observed effect concentration
NOEC
tested concentration just below the LOEC (2.4)
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ISO/TS 18220:2016(E)
2.6
effect concentration
EC
x
calculated concentration from which an effect of x % is expected
2.7
mortality
calculated on dead and missing animals at the end of the test divided by animals at start
2.8
confidence interval
A x %
interval of values within which the measured or calculated value is likely to be present with a
probability of x %
2.9
salinity
S
dimensionless value of which, for the purpose of checking water quality, may be regarded as an estimate
of the concentration, in grams per kilogram, of dissolved salts in sea water
Note 1 to entry: It is defined algorithmically, in terms of the ratio (K15) of the electrical conductivity of the
sample, at 15 °C and 1 atm, to that of defined potassium chloride solution (32,436 6 g/kg of sample) at the same
temperature and pressure.
3 Principle
The test is an early-life stage test, in which the organisms are exposed to various concentrations of a test
substance or water sample under semi-static test conditions from the first naupliar stage (N-1) to the
first copepodite stages (C-1, C-2, etc.). Survival and development of early-life stages [larval development
ratio (LDR)], are the investigated test variables. The exposure starts with newly hatched (<24 h) nauplii
(larvae) and is continued until the emergence of (approximately 50 %) copepodites (juveniles) in the
control. The total test duration is about 6 d to 7 d, which is sufficient time to investigate the development
from N-1 to 50 % copepodites in the control. The naupliar (larval) and copepodite (juvenile) stages are
morphologically distinct, and therefore, the transition from the last naupliar to the first copepodite
stage is easily observed.
The outcome of the test is either the no observed effect concentration (NOEC) and the lowest observed
effect concentration (LOEC) values or the effect concentrations with a certain degree (x %) of inhibition
(EC ) (e.g. EC and EC ).
x 50 10
4 Reagents
4.1 Test organism
The species to be used is the brackish water harpacticoid copepod Nitocra spinipes Boeck.
Newly hatched (less than 24 h of age) nauplii should be collected from a healthy stock (i.e. showing
no signs of stress, such as high mortality, poor fecundity, etc.). The stock animals shall be maintained
in culture conditions (light, temperature, medium and feeding) similar to those to be used in the test
(culturing method for N. spinipes is described in Annex A).
2 © ISO 2016 – All rights reserved
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ISO/TS 18220:2016(E)
4.2 Water, deionized or of equivalent purity, to prepare artificial sea water or to dilute natural sea water.
4.2.1 Artificial sea water
An example of artificial sea water suitable for cultivation and testing is included in Annex A. Any
artificial sea water with a known composition in which the copepods show suitable long-term survival,
normal behaviour, development and fecundity may be used as culture and dilution medium (4.3).
4.2.2 Natural sea water
Natural sea water shall be collected from an unpolluted location. Any natural sea water with a known
composition in which the copepods show suitable long-term survival, normal behaviour, development
and fecundity may be used as culture and dilution medium.
Suspended particles shall be <10 mg/l and can be stored cold for approximately 6 months before
preparation of culture or dilution medium.
4.3 Medium
4.3.1 Culture medium
Culture medium is used for cultivating Nitocra spinipes and is prepared from either natural or artificial
sea water (4.2). Natural sea water shall be filtered (30 µm) and heated to 80 °C to kill undesired
organisms and then conditioned (24 h) to culture temperature and oxygen saturation. The culture
medium can be stored cold for several weeks.
4.3.2 Dilution medium
Dilution medium is used for diluting water samples or dissolving chemicals and is prepared from
culture medium that has first been filtered (GF/C-1,2 µm) before use. Salinity of the dilution medium
should be the same as the culture medium. Salinities between 3 ‰ and 25 ‰ can be used. The dilution
medium shall have a dissolved oxygen concentration above 70 % of the air saturation value and a pH
of 7,5 ± 1,0 before being used to prepare the test solutions. If the physical conditions or the salinity of
the medium to be used in the test differ more than 5 °C or 10 % from those used for routine culturing,
it is good practice to include an adequate cultivating acclimation period at the same salinity (±2 ‰) of
2 weeks to 3 weeks to avoid stressing of the larvae.
5 Cultivation
5.1 Test organism
See Annex A for detailed information.
5.2 Algae for feeding
See Annex A for detailed information.
6 Apparatus
All equipment, which will come in contact with the test medium, shall be made of glass or chemically
inert material.
6.1 Glass vessels, approximately 150 ml, diameter 8 cm, and height 4,5 cm, for Nitocra spinipes
cultivation.
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ISO/TS 18220:2016(E)
6.2 Test vessels, approximately 15 ml, diameter 2,5 cm, height 4 cm, with flat bottom.
6.3 pH meter.
6.4 Oxygen meter.
6.5 Conductivity meter.
6.6 Wide pipettes, for sampling animals, preferably salinized to prevent copepods from adhering to
pipette walls.
6.7 Temperature-control cabinet or room, (22 ± 1) °C.
6.8 Low-magnifying stereo microscope.
6.9 Inverted microscope.
6.10 Apparatus for membrane filtration.
6.11 Filters, 1,2 µm and 30 µm.
7 Procedure
7.1 Production of nauplii to be used in test
Nauplii aged less than 24 h are used for initiating a test. To produce a sufficient number of nauplii, the
procedure presented below should be followed.
The day before the test starts, approximately 300 females with well-developed egg sacs are sampled
by pipette under a low-magnifying stereomicroscope and randomly transferred (approximately 60 in
each) to six glass “hatch” vessels containing 100 ml dilution medium. The isolated female copepods are
5
fed with a suspension of Rhodomonas salina to a concentration of 2,5 × 10 cells/ml.
7.2 Choice of test concentrations
The range of the test concentrations should preferably not include any concentrations that have a
significant effect on survival since the main objective of the test is to measure sublethal effects (i.e.
development).
Prior knowledge of the toxicity of the test substance, i.e. from an acute test (see Reference [2]) or from
range-finding studies, should help in selecting appropriate test concentrations. As a rule of thumb, the
highest concentration in the early-life stage test should be set at 10 % to 20 % of the acute 96 h-LC to
50
avoid significant effect on survival.
At least five different concentrations should be tested in a geometric series with a factor between
concentrations not exceeding 3,3. Justification should be provided if fewer than five concentrations
are used. Substances should not be tested above their solubility limits in dilution medium. A dilution-
medium control shall be included, and also, if relevant, a solvent-control containing the same
concentration of solvent as the test series should be run additionally.
The number of replicates generally depends on the statistical design (hypothesis testing or regression
analysis). When planning the test, it should be taken into consideration if the aim is to achieve a
NOEC/LOEC (by use of Chi-square) or an EC value (by use of regression analysis or none parametric
x
alternative; see Reference [4]).
4 © ISO 2016 – All rights reserved
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ISO/TS 18220:2016(E)
The number of replicates in control(s) and each test concentration should not be lower than 8. For
regression analysis, the number of replicates may be lowered (≥4) but such a statistical design usually
requires more test concentrations as compared to a hypothesis testing design.
In setting the range of concentrations, the following should be borne in mind.
7.2.1 Hypothesis testing
If the aim is to obtain the NOEC, the lowest test concentration shall be low enough so that the biological
endpoint at that concentration is not significantly different from that of the control. If this is not the
case, the test will have to be repeated with a decreased lowest concentration.
If the aim is to obtain the LOEC, the highest concentration shall be high enough to cause a statistically
significant effect when compared to the control on the biological endpoint. If this is not the case, the
test will have to be repeated with an increased highest concentration.
7.2.2 Regression analysis
If EC for effects on development is estimated, it is optimal that the lowest concentration has no effects
x
(optimally the only one without effects), and the highest concentration is greater than EC , and that
50
sufficient concentrations are used to define the EC with appropriate level of confidence.
x
The range of test concentrations should preferably not include any concentrations that have a significant
effect on survival since the main objective of the test is to measure sublethal effects (i.e. development).
7.3 Preparation of solutions to be used in test
7.3.1 Stock solution
The stock solution should preferably be prepared by dissolving the substance in dilution medium. The
preferred options for preparing stock solutions are physical methods, such as stirring and sonication.
[1]
NOTE See ISO 5667-16.
When preparing the stock solution, the pH should be measured to assure it is in the valid range (6 to
9). The pH adjustment of the stock solution shall not change the concentration to any significant extent
or lead to chemical reaction or precipitation of the test substance. HCl and NaOH are preferred for pH
adjustments and preferably used in small volumes.
The use of organic solvents may be required in some cases in order to produce a suitable concentrated
stock solution of so-called “difficult substances” as described in Reference [7], but every effort should
be made to avoid the use of such carrier solvents. Solvents are used to produce a stock solution that
can be dosed accurately into water; the recommended maximum solvent concentration in the final
test medium is 0,01 ml/l and should preferably be the same in all test vessels. If a higher solvent
concentration is used, it should be documented that it has no effects on the test variables investigated
in the test. Solvents may be essential in handling some substances; for example, for preparing stock
solutions of hydrolytically unstable or highly viscous substances. When necessary, use of solvents of
low toxicity at low concentrations is recommended to aid preparation of test solutions. Examples of
suitable solvents are presented in References [7] and [8]. Care should be taken when using readily
degradable agents (e.g. acetone) as these can cause problems with bacterial growth in the test vessels.
7.3.2 Test solutions
Test solutions are prepared by dilution of stock solution using dilution medium.
To be able to allocate newborn nauplii among control(s) and test concentrations in a random fashion
and avoiding cross-contamination, 5 ml of dilution media is initially added to each test vessel to be used
in the test. When newborn nauplii have been transferred to each test vessel (see below), either 5 ml
dilution media (control) or test solution is added to the test vessels. Hence, each test solution to be used
© ISO 2016 – All rights reserved 5
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ISO/TS 18220:2016(E)
at start of the test shall have a concentration that is two times the intended final test concentrations. At
test solution renewals (see below), the concentrations of the test solutions should, however, be the same
as the chosen test concentrations.
7.4 Start of test
Experiments are preferably to be started on Wednesdays or Fridays to avoid LDR readings and feeding
at weekends.
Day 0
Nauplii hatched within 24 h (see 7.1) are collected by pipette, transferred in a volume of 5 µl/nauplius
to 5 ml dilution medium/test vessel and distributed evenly (so that all vessels contain nauplia from all
the six hatch vessels) among the vessels. The optimal number of nauplii per test vessel is 10 and should
be between 8 and 12.
Test solutions (5 ml/test vessel) are added to test vessels and dilution medium (5 ml/control vessel) is
added to the control vessels as described in 7.3.2.
The vessels should be labelled according to treatment and individual identity.
Test medium levels should be marked on the test vessels to allow compensation for evaporation during
the test with distilled/deionized water.
Make a final check of the number of nauplii in each vessel and register on the data sheet. Feed the nauplii
5
in each test vessel with a suspension of R. salina to a concentration of 2,5 × 10 cells/ml.
7.5 Incubation/exposure
The vessels can preferably be placed in darkness. A photoperiod of 16:8 h light:dark may also be used
-1 -2
but at a low light intensity (5 μmol to 10 μmol × s × m ).
The time needed to complete (at 22 °C and ~6 ‰ salinity) the larval development test is 6 d to 7 d. At
lower temperatures and higher salinities, the development may be slower, and thus, testing at these
conditions may last longer. From day 5 and onward, the development needs to be investigated daily.
7.6 Maintenance
Day 2 or 3 and 5: Compensate for evaporation with deionized water. With a pipette, carefully transfer
7 ml from each test vessel into an additional beaker in order to control that no animals are entrapped
in the pipette. Collect the extracted water from the control, lowest and highest test concentrations,
respectively, and measure oxygen, salinity and pH immediately. Make notation of the parameter values
on the data sheet. Add 7 ml of fresh dilution media to the control test vessels and the test solutions
to treatment test vessels. Feed the copepods in each test vessel with a suspension of R. salina to a
5
concentration of 2,5 × 10 cells/ml.
Day 6 or 7: Count the number of nauplii and copepodites in the control. If the ratio of copepodites to
the total number of surviving early-life stages (nauplii + copepodites) is within 60 % ± 20 % (preferably
close to 50 %) the test is terminated and all vessels are counted. If the ratio is lower than 40 % on day 7,
the maintenance procedures described above for day 2 or 3 and 5 are repeated and the exposure period
is prolonged by another 24 h (or more, if necessary). To eliminate errors from long reading times, during
which animals may continue to develop, readings should be made across treatments.
7.7 Measurements/observations
7.7.1 Mortality
Dead and missing animals at the end of the test are registered and are all considered “dead” when
calculating the LC or NOEC/LOEC.
x
6 © ISO 2016 – All rights reserved
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ISO/TS 18220:2016(E)
7.7.2 Larval development ratio (LDR)
The LDR is expressed as the ratio of copepodites to the total number of surviving early-life stages
(nauplii + copepodites) at the end of the test. Mortality of the animals should be presented along with
the LDR.
Observations made during the test should be recorded on data sheets, examples of which are provided
in Annex B.
7.7.3 Physical-chemical parameters — oxygen, pH and salinity
During the test, dissolved oxygen, salinity and pH should be measured in the control and all test
concentrations each time test medium is renewed.
As a minimum, these measurements shall be made in the control, lowest and the highest test
concentration.
7.7.4 Concentration of the test substance
It is recommended that, as a minimum, the highest and lowest test concentrations are analysed when
freshly prepared — at the start of the test and immediately prior to renewals and at the end of the test.
It is recommended that results be based on measured concentrations.
NOTE If there is evidence that the concentration of the substance tested has been satisfactorily maintained
within ±20 % of the nominal concentration throughout the test, then results can be based on nominal or
measured initial values.
For tests where the concentration of the test substance is not expected to remain within ±20 % of the
nominal concentration, it is necessar
...
TECHNICAL ISO/TS
SPECIFICATION 18220
First edition
Water quality — Larval development
test with the harpacticoid copepod
Nitocra spinipes
Qualité de l’eau — Essai de développement larvaire avec le copépode
harpacticoïde Nitocra spinipes
PROOF/ÉPREUVE
Reference number
ISO/TS 18220:2016(E)
©
ISO 2016
---------------------- Page: 1 ----------------------
ISO/TS 18220:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 18220:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Terms and definitions . 1
3 Principle . 2
4 Reagents . 2
5 Cultivation . 3
5.1 Test organism . 3
5.2 Algae for feeding . 3
6 Apparatus . 3
7 Procedure. 4
7.1 Production of nauplii to be used in test . 4
7.2 Choice of test concentrations . 4
7.2.1 Hypothesis testing . 5
7.2.2 Regression analysis. 5
7.3 Preparation of solutions to be used in test . 5
7.3.1 Stock solution . 5
7.3.2 Test solutions . 5
7.4 Start of test . 6
7.5 Incubation/exposure . 6
7.6 Maintenance . 6
7.7 Measurements/observations . 6
7.7.1 Mortality . 6
7.7.2 Larval development ratio (LDR). 7
7.7.3 Physical-chemical parameters — oxygen, pH and salinity . 7
7.7.4 Concentration of the test substance . 7
8 Validity criteria . 7
9 Evaluation of results . 8
9.1 Calculation of results . 8
9.2 Expression of results . 8
9.3 Interpretation of results . 8
10 Reproducibility . 8
11 Test report . 8
Annex A (informative) Biology and cultivation of Nitocra spinipes .10
Annex B (informative) Nitocra spinipes larval development ratio .15
Annex C (informative) Calculations .17
Bibliography .19
© ISO 2016 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO/TS 18220:2016(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 147, Water quality, Subcommittee SC 5, Biological
methods.
iv PROOF/ÉPREUVE © ISO 2016 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 18220:2016(E)
Introduction
Harpacticoid copepods are predominantly benthic, occurring widely in marine, brackish and fresh
water ecosystems. They represent important prey items for the benthic larvae of many fish species and
larger invertebrates and constitute an ecologically important energy-transfer link between the organic
phase of the sediment and higher trophic levels.
The euryhaline brackish water harpactoid Nitocra spinipes (Crustacea) is a common component of the
benthic meiofana in shallow coastal waters around the world (see Reference [6]).
© ISO 2016 – All rights reserved PROOF/ÉPREUVE v
---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/TS 18220:2016(E)
Water quality — Larval development test with the
harpacticoid copepod Nitocra spinipes
WARNING — Persons using this Technical Specification should be familiar with normal
laboratory practice. This Technical Specification 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 Technical
Specification be carried out by suitably qualified staff.
1 Scope
This Technical Specification specifies an early-life stage procedure for determination of the toxic
effects of chemicals and water samples on a cold-water brackish water copepod species under semi-
static conditions. The biological test variables include survival and development of the early-life stages.
The exposure starts with newly hatched (<24 h) nauplii (larvae) and is continued until emergence of
(c. 50 %) copepodites (juveniles) in the control.
The benthic living Nitocra complements the planktonic Acartia species in ISO 16778. These organisms
represent different life-history strategies as Nitocra is egg-carrying, whereas Acartia is a broadcasting
calanoid copepod and thus, different sensitivities of specific life stages. Nitocra is a fresh to brackish
water species, which allows testing low salinity waters and is complementary to A. tonsa, which is of
marine origin and poorly tolerates low salinities.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
nauplii
larvae
2.2
copepodites
juveniles
2.3
larval development ratio
LDR
ratio of copepodites (2.2) to the total number of surviving early-life stages (nauplii + copepodites) at the
end of the test
2.4
lowest observed effect concentration
LOEC
lowest concentration within the experimental range at which a significant effect is observed
2.5
no observed effect concentration
NOEC
tested concentration just below the LOEC (2.4)
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2.6
effect concentration
EC
x
calculated concentration from which an effect of x % is expected
2.7
mortality
calculated on dead and missing animals at the end of the test divided by animals at start
2.8
confidence interval
A x %
interval of values within which the measured or calculated value is likely to be present with a
probability of x %
2.9
salinity
S
dimensionless value of which, for the purpose of checking water quality, may be regarded as an estimate
of the concentration, in grams per kilogram, of dissolved salts in sea water
Note 1 to entry: It is defined algorithmically, in terms of the ratio (K15) of the electrical conductivity of the
sample, at 15 °C and 1 atm, to that of defined potassium chloride solution (32,436 6 g/kg of sample) at the same
temperature and pressure.
3 Principle
The test is an early-life stage test, in which the organisms are exposed to various concentrations of a test
substance or water sample under semi-static test conditions from the first naupliar stage (N-1) to the
first copepodite stages (C-1, C-2, etc.). Survival and development of early-life stages [larval development
ratio (LDR)], are the investigated test variables. The exposure starts with newly hatched (<24 h) nauplii
(larvae) and is continued until the emergence of (approximately 50 %) copepodites (juveniles) in the
control. The total test duration is about 6 d to 7 d, which is sufficient time to investigate the development
from N-1 to 50 % copepodites in the control. The naupliar (larval) and copepodite (juvenile) stages are
morphologically distinct, and therefore, the transition from the last naupliar to the first copepodite
stage is easily observed.
The outcome of the test is either the no observed effect concentration (NOEC) and the lowest observed
effect concentration (LOEC) values or the effect concentrations with a certain degree (x %) of inhibition
(EC ) (e.g. EC and EC ).
x 50 10
4 Reagents
4.1 Test organism
The species to be used is the brackish water harpacticoid copepod Nitocra spinipes Boeck.
Newly hatched (less than 24 h of age) nauplii should be collected from a healthy stock (i.e. showing
no signs of stress, such as high mortality, poor fecundity, etc.). The stock animals shall be maintained
in culture conditions (light, temperature, medium and feeding) similar to those to be used in the test
(culturing method for N. spinipes is described in Annex A).
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4.2 Water, deionized or of equivalent purity, to prepare artificial sea water or to dilute natural sea water.
4.2.1 Artificial sea water
An example of artificial sea water suitable for cultivation and testing is included in Annex A. Any
artificial sea water with a known composition in which the copepods show suitable long-term survival,
normal behaviour, development and fecundity may be used as culture and dilution medium (4.3).
4.2.2 Natural sea water
Natural sea water shall be collected from an unpolluted location. Any natural sea water with a known
composition in which the copepods show suitable long-term survival, normal behaviour, development
and fecundity may be used as culture and dilution medium.
Suspended particles shall be <10 mg/l and can be stored cold for approximately 6 months before
preparation of culture or dilution medium.
4.3 Medium
4.3.1 Culture medium
Culture medium is used for cultivating Nitocra spinipes and is prepared from either natural or artificial
sea water (4.2). Natural sea water shall be filtered (30 µm) and heated to 80 °C to kill undesired
organisms and then conditioned (24 h) to culture temperature and oxygen saturation. The culture
medium can be stored cold for several weeks.
4.3.2 Dilution medium
Dilution medium is used for diluting water samples or dissolving chemicals and is prepared from
culture medium that has first been filtered (GF/C-1,2 µm) before use. Salinity of the dilution medium
should be the same as the culture medium. Salinities between 3 ‰ and 25 ‰ can be used. The dilution
medium shall have a dissolved oxygen concentration above 70 % of the air saturation value and a pH
of 7,5 ± 1,0 before being used to prepare the test solutions. If the physical conditions or the salinity of
the medium to be used in the test differ more than 5 °C or 10 % from those used for routine culturing,
it is good practice to include an adequate cultivating acclimation period at the same salinity (±2 ‰) of
2 weeks to 3 weeks to avoid stressing of the larvae.
5 Cultivation
5.1 Test organism
See Annex A for detailed information.
5.2 Algae for feeding
See Annex A for detailed information.
6 Apparatus
All equipment, which will come in contact with the test medium, shall be made of glass or chemically
inert material.
6.1 Glass vessels, approximately 150 ml, diameter 8 cm, and height 4,5 cm, for Nitocra spinipes
cultivation.
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6.2 Test vessels, approximately 15 ml, diameter 2,5 cm, height 4 cm, with flat bottom.
6.3 pH meter.
6.4 Oxygen meter.
6.5 Conductivity meter.
6.6 Wide pipettes, for sampling animals, preferably salinized to prevent copepods from adhering to
pipette walls.
6.7 Temperature-control cabinet or room, (22 ± 1) °C.
6.8 Low-magnifying stereo microscope.
6.9 Inverted microscope.
6.10 Apparatus for membrane filtration.
6.11 Filters, 1,2 µm and 30 µm.
7 Procedure
7.1 Production of nauplii to be used in test
Nauplii aged less than 24 h are used for initiating a test. To produce a sufficient number of nauplii, the
procedure presented below should be followed.
The day before the test starts, approximately 300 females with well-developed egg sacs are sampled
by pipette under a low-magnifying stereomicroscope and randomly transferred (approximately 60 in
each) to six glass “hatch” vessels containing 100 ml dilution medium. The isolated female copepods are
5
fed with a suspension of Rhodomonas salina to a concentration of 2,5 × 10 cells/ml.
7.2 Choice of test concentrations
The range of the test concentrations should preferably not include any concentrations that have a
significant effect on survival since the main objective of the test is to measure sublethal effects (i.e.
development).
Prior knowledge of the toxicity of the test substance, i.e. from an acute test (see Reference [2]) or from
range-finding studies, should help in selecting appropriate test concentrations. As a rule of thumb, the
highest concentration in the early-life stage test should be set at 10 % to 20 % of the acute 96 h-LC to
50
avoid significant effect on survival.
At least five different concentrations should be tested in a geometric series with a factor between
concentrations not exceeding 3,3. Justification should be provided if fewer than five concentrations
are used. Substances should not be tested above their solubility limits in dilution medium. A dilution-
medium control shall be included, and also, if relevant, a solvent-control containing the same
concentration of solvent as the test series should be run additionally.
The number of replicates generally depends on the statistical design (hypothesis testing or regression
analysis). When planning the test, it should be taken into consideration if the aim is to achieve a
NOEC/LOEC (by use of Chi-square) or an EC value (by use of regression analysis or none parametric
x
alternative; see Reference [4]).
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The number of replicates in control(s) and each test concentration should not be lower than 8. For
regression analysis, the number of replicates may be lowered (≥4) but such a statistical design usually
requires more test concentrations as compared to a hypothesis testing design.
In setting the range of concentrations, the following should be borne in mind.
7.2.1 Hypothesis testing
If the aim is to obtain the NOEC, the lowest test concentration shall be low enough so that the biological
endpoint at that concentration is not significantly different from that of the control. If this is not the
case, the test will have to be repeated with a decreased lowest concentration.
If the aim is to obtain the LOEC, the highest concentration shall be high enough to cause a statistically
significant effect when compared to the control on the biological endpoint. If this is not the case, the
test will have to be repeated with an increased highest concentration.
7.2.2 Regression analysis
If EC for effects on development is estimated, it is optimal that the lowest concentration has no effects
x
(optimally the only one without effects), and the highest concentration is greater than EC , and that
50
sufficient concentrations are used to define the EC with appropriate level of confidence.
x
The range of test concentrations should preferably not include any concentrations that have a significant
effect on survival since the main objective of the test is to measure sublethal effects (i.e. development).
7.3 Preparation of solutions to be used in test
7.3.1 Stock solution
The stock solution should preferably be prepared by dissolving the substance in dilution medium. The
preferred options for preparing stock solutions are physical methods, such as stirring and sonication.
[1]
NOTE See ISO 5667-16.
When preparing the stock solution, the pH should be measured to assure it is in the valid range (6 to
9). The pH adjustment of the stock solution shall not change the concentration to any significant extent
or lead to chemical reaction or precipitation of the test substance. HCl and NaOH are preferred for pH
adjustments and preferably used in small volumes.
The use of organic solvents may be required in some cases in order to produce a suitable concentrated
stock solution of so-called “difficult substances” as described in Reference [7], but every effort should
be made to avoid the use of such carrier solvents. Solvents are used to produce a stock solution that
can be dosed accurately into water; the recommended maximum solvent concentration in the final
test medium is 0,01 ml/l and should preferably be the same in all test vessels. If a higher solvent
concentration is used, it should be documented that it has no effects on the test variables investigated
in the test. Solvents may be essential in handling some substances; for example, for preparing stock
solutions of hydrolytically unstable or highly viscous substances. When necessary, use of solvents of
low toxicity at low concentrations is recommended to aid preparation of test solutions. Examples of
suitable solvents are presented in References [7] and [8]. Care should be taken when using readily
degradable agents (e.g. acetone) as these can cause problems with bacterial growth in the test vessels.
7.3.2 Test solutions
Test solutions are prepared by dilution of stock solution using dilution medium.
To be able to allocate newborn nauplii among control(s) and test concentrations in a random fashion
and avoiding cross-contamination, 5 ml of dilution media is initially added to each test vessel to be used
in the test. When newborn nauplii have been transferred to each test vessel (see below), either 5 ml
dilution media (control) or test solution is added to the test vessels. Hence, each test solution to be used
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at start of the test shall have a concentration that is two times the intended final test concentrations. At
test solution renewals (see below), the concentrations of the test solutions should, however, be the same
as the chosen test concentrations.
7.4 Start of test
Experiments are preferably to be started on Wednesdays or Fridays to avoid LDR readings and feeding
at weekends.
Day 0
Nauplii hatched within 24 h (see 7.1) are collected by pipette, transferred in a volume of 5 µl/nauplius
to 5 ml dilution medium/test vessel and distributed evenly (so that all vessels contain nauplia from all
the six hatch vessels) among the vessels. The optimal number of nauplii per test vessel is 10 and should
be between 8 and 12.
Test solutions (5 ml/test vessel) are added to test vessels and dilution medium (5 ml/control vessel) is
added to the control vessels as described in 7.3.2.
The vessels should be labelled according to treatment and individual identity.
Test medium levels should be marked on the test vessels to allow compensation for evaporation during
the test with distilled/deionized water.
Make a final check of the number of nauplii in each vessel and register on the data sheet. Feed the nauplii
5
in each test vessel with a suspension of R. salina to a concentration of 2,5 × 10 cells/ml.
7.5 Incubation/exposure
The vessels can preferably be placed in darkness. A photoperiod of 16:8 h light:dark may also be used
-1 -2
but at a low light intensity (5 μmol to 10 μmol × s × m ).
The time needed to complete (at 22 °C and ~6 ‰ salinity) the larval development test is 6 d to 7 d. At
lower temperatures and higher salinities, the development may be slower, and thus, testing at these
conditions may last longer. From day 5 and onward, the development needs to be investigated daily.
7.6 Maintenance
Day 2 or 3 and 5: Compensate for evaporation with deionized water. With a pipette, carefully transfer
7 ml from each test vessel into an additional beaker in order to control that no animals are entrapped
in the pipette. Collect the extracted water from the control, lowest and highest test concentrations,
respectively, and measure oxygen, salinity and pH immediately. Make notation of the parameter values
on the data sheet. Add 7 ml of fresh dilution media to the control test vessels and the test solutions
to treatment test vessels. Feed the copepods in each test vessel with a suspension of R. salina to a
5
concentration of 2,5 × 10 cells/ml.
Day 6 or 7: Count the number of nauplii and copepodites in the control. If the ratio of copepodites to
the total number of surviving early-life stages (nauplii + copepodites) is within 60 % ± 20 % (preferably
close to 50 %) the test is terminated and all vessels are counted. If the ratio is lower than 40 % on day 7,
the maintenance procedures described above for day 2 or 3 and 5 are repeated and the exposure period
is prolonged by another 24 h (or more, if necessary). To eliminate errors from long reading times, during
which animals may continue to develop, readings should be made across treatments.
7.7 Measurements/observations
7.7.1 Mortality
Dead and missing animals at the end of the test are registered and are all considered “dead” when
calculating the LC or NOEC/LOEC.
x
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7.7.2 Larval development ratio (LDR)
The LDR is expressed as the ratio of copepodites to the total number of surviving early-life stages
(nauplii + copepodites) at the end of the test. Mortality of the animals should be presented along with
the LDR.
Observations made during the test should be recorded on data sheets, examples of which are provided
in Annex B.
7.7.3 Physical-chemical parameters — oxygen, pH and salinity
During the test, dissolved oxygen, salinity and pH should be measured in the control and all test
concentrations each time test medium is renewed.
As a minimum, these measurements shall be made in the control, lowest and the highest test
concentration.
7.7.4 Concentration of the test substance
It is recommended that, as a minimum, the highest and lowest test concentrations are analysed when
freshly prepared — at the start of the test and immediately prior to renewals and at the end of the test.
It is recommended that results be based on measured concentrations.
NOTE If there is evidence that the concentration of the substance tested has been satisfactorily maintained
within ±20 % of the nominal concentration throughout the test, then results can be based on nominal or
measured initial values.
For tests where the c
...
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