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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Status
Published
Publication Date
15-Jun-2016
Current Stage
6060 - International Standard published
Start Date
27-Apr-2016
Completion Date
16-Jun-2016
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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)
© ISO 2016 – All rights reserved 1
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ISO/TS 18220:2016(E)
2.6
effect concentration
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

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.
© ISO 2016 – All rights reserved 3
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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

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

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

alternative; see Reference [4]).
4 © ISO 2016 – All rights reserved
---------------------- Page: 9 ----------------------
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

(optimally the only one without effects), and the highest concentration is greater than EC , and that

sufficient concentrations are used to define the EC with appropriate level of confidence.

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

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

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.
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]).
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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
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

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

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

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

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

(optimally the only one without effects), and the highest concentration is greater than EC , and that

sufficient concentrations are used to define the EC with appropriate level of confidence.

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

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

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.
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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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