Water quality — Biochemical and physiological measurements on fish — Part 1: Sampling of fish, handling and preservation of samples

ISO 23893-1:2007 provides guidance on how to sample fish for determination of biochemical and physiological characteristics, such as the composition and enzyme activities of blood, liver, muscle and other tissues in order to asses the health of fish in the field as well as in the laboratory. The biochemical and physiological variables used for this purpose are often called biomarkers. ISO 23893-1:2007 includes recommendations and methods for: obtaining a site‑specific sample of a representative number of fish; sampling fish tissues in the field and in the laboratory; and handling and preservation of samples prior to analysis of biochemical and physiological variables.

Qualité de l'eau — Mesurages biochimiques et physiologiques sur poisson — Partie 1: Échantillonnage des poissons, manipulation et conservation des échantillons

L'ISO 23893-1:2007 fournit des indications sur la manière d'échantillonner des poissons pour déterminer leurs caractéristiques biochimiques et physiologiques, telles que la composition et les activités enzymatiques du sang, du foie, des muscles et d'autres tissus afin d'évaluer l'état de santé des poissons aussi bien sur le terrain qu'en laboratoire. Les variables biochimiques et physiologiques utilisées dans ce but sont souvent appelées marqueurs biologiques. L'ISO 23893-1:2007 inclut des recommandations et des méthodes concernant la manière d'obtenir un échantillon spécifique du site pour un nombre représentatif de poissons, l'échantillonnage des tissus de poissons sur le terrain et en laboratoire, et la manipulation et la conservation des échantillons avant analyse des variables biochimiques et physiologiques.

Kakovost vode - Biokemijske in fiziološke meritve v ribah - 1. del: Vzorčenje rib, ravnanje z vzorci in njihovo konzerviranje

Ta del ISO 23893 podaja vodilo o tem, kako vzorčiti ribe za določevanje biokemijske in fizioloških značilnosti, kot so sestava in dejavnosti encimov v krvi, jetrih, mišicah in drugih tkivih, za vrednotenje zdravja rib tako na terenu kot v laboratoriju. Biokemijske in fiziološke spremenljivke, ki se uporabljajo v ta namen, se pogosto imenujejo biomarkerji. Ta del ISO 23893 vključuje priporočila in metode za: pridobivanje vzorcev reprezentativnega števila rib za določen predel; vzorčenje tkiv rib na terenu in v laboratoriju; ter ravnanje z vzorci in njihovo konzerviranje pred analizo biokemijskih in fizioloških spremenljivk.

General Information

Status
Published
Publication Date
07-Nov-2007
Current Stage
9093 - International Standard confirmed
Completion Date
21-Mar-2022

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INTERNATIONAL ISO
STANDARD 23893-1
First edition
2007-11-15
Water quality — Biochemical and
physiological measurements on fish —
Part 1:
Sampling of fish, handling and
preservation of samples
Qualité de l'eau — Mesurages biochimiques et physiologiques sur
poisson —
Partie 1: Échantillonnage des poissons, manipulation et conservation
des échantillons
Reference number
ISO 23893-1:2007(E)
ISO 2007
---------------------- Page: 1 ----------------------
ISO 23893-1:2007(E)
PDF disclaimer

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shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In

downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat

accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.

Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation

parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In

the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

COPYRIGHT PROTECTED DOCUMENT
© ISO 2007

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,

electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or

ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2007 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 23893-1:2007(E)
Contents Page

Foreword............................................................................................................................................................ iv

Introduction ........................................................................................................................................................ v

1 Scope ..................................................................................................................................................... 1

2 Principle................................................................................................................................................. 1

3 Equipment ............................................................................................................................................. 2

4 Fish sampling........................................................................................................................................ 3

4.1 Statistical aspects................................................................................................................................. 3

4.2 Frequency and season for sampling .................................................................................................. 3

4.3 Selection of sampling sites ................................................................................................................. 3

4.4 Sampling procedures ........................................................................................................................... 4

4.5 Handling of samples and analytical procedures ............................................................................... 6

4.6 Background information ...................................................................................................................... 7

5 Quality assurance................................................................................................................................. 7

5.1 General................................................................................................................................................... 7

5.2 Fish sampling........................................................................................................................................ 7

5.3 Tissue sampling.................................................................................................................................... 7

5.4 Biochemical/chemical analysis ........................................................................................................... 7

5.5 Evaluation.............................................................................................................................................. 7

6 Report .................................................................................................................................................... 8

6.1 General................................................................................................................................................... 8

6.2 Data logging, data hosting................................................................................................................... 8

6.3 Evaluation.............................................................................................................................................. 8

Annex A (informative) Summary of variables used as biomarkers in fish ................................................... 9

Annex B (informative) Guide to interpretation of biomarker responses with references......................... 12

Annex C (informative) Suggested report for fish sampling ......................................................................... 15

Annex D (informative) Suggested report for tissue sampling ..................................................................... 16

Bibliography ..................................................................................................................................................... 18

© ISO 2007 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 23893-1:2007(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies

(ISO member bodies). The work of preparing International Standards is normally carried out through ISO

technical committees. Each member body interested in a subject for which a technical committee has been

established has the right to be represented on that committee. International organizations, governmental and

non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the

International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 23893-1 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,

Biological methods.

ISO 23893 consists of the following parts, under the general title Water quality — Biochemical and

physiological measurements on fish:
⎯ Part 1: Sampling of fish, handling and preservation of samples

⎯ Part 2: Determination of ethoxyresorufin-O-deethylase (EROD) [Technical Specification]

iv © ISO 2007 – All rights reserved
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ISO 23893-1:2007(E)
Introduction

Determination of biomarker responses can be used to detect toxicity of known as well as unknown pollutants,

when they occur singly or in combination. Therefore, measurement of biomarkers is a cost-effective way to

assess ecosystem health. In combination with determinations of occurring and suspected pollutants,

determinations of biomarkers can facilitate the interpretation of cause-effect relationships in the environment,

as well as in laboratory toxicity tests. Information on commonly used biomarkers and the interpretation of

biomarker responses is given in Annexes A and B, respectively.

Biomarkers like ethoxyresorufin-O-deethylase (EROD), metallothionein and vitellogenin are used to detect

and quantify sublethal effects of pollutants, especially in fish. However, many of the biochemical and

physiological variables that are used as biomarkers are sensitive not only to disturbances by the pollutants of

concern, but also by the normal biochemical and physiological adjustments made by the fish in response to

seasonal variation, its normal development and sexual maturation. Some variables can also be affected by

general stress to disturbances caused by the handling during fish and fish tissue sampling. Therefore,

standardisation of procedures used for sampling and handling of samples prior to determination of the

biochemical and physiological variables is important.

Sublethal responses at the individual level usually occur before effects are seen at the population and

community level. In the aquatic environment, fish are suitable for detection of physiological effects of

pollutants, because they are exposed both through the water and through their food organisms. Also, the

physiology and biochemistry of fishes is rather similar to that of humans and other vertebrates, making

comparisons with studies on mammals easier than for those with crustaceans and other invertebrates.

This part of ISO 23893 serves as guidance for sampling and a platform for determination of biomarkers in fish,

making it possible to use the measurements to:

describe the state of the environment regarding effects of anthropogenic compounds on the health of fish;

perform time-trend surveillance (monitoring);

provide reference data and material for assessment of effects from point sources;

evaluate and assess environmental threats;
provide background information for environmental measures;
follow up and assess effects of environmental corrective measures;

integrate the biomarker responses with other measurements (e.g. fish abundance, recruitment and

pollutant residues) in order to facilitate the interpretation of environmental status or impact.

© ISO 2007 – All rights reserved v
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INTERNATIONAL STANDARD ISO 23893-1:2007(E)
Water quality — Biochemical and physiological measurements
on fish —
Part 1:
Sampling of fish, handling and preservation of samples
1 Scope

This part of ISO 23893 provides guidance on how to sample fish for determination of biochemical and

physiological characteristics, such as the composition and enzyme activities of blood, liver, muscle and other

tissues in order to asses the health of fish in the field as well as in the laboratory. The biochemical and

physiological variables used for this purpose are often called biomarkers. This part of ISO 23893 includes

recommendations and methods for:
obtaining a site-specific sample of a representative number of fish;
sampling fish tissues in the field and in the laboratory; and

handling and preservation of samples prior to analysis of biochemical and physiological variables.

2 Principle

Fish of a suitable species, age (size), and sex are sampled at selected sites at a suitable time of the year in

order to reduce variability due to biological, geographical, and seasonal influences. Standardised sampling

and measurement procedures, and qualified staff are used for collection of samples, transport, storage, and

analysis. By these means, the results from time series of comparable data can be used to detect changes in

the environment that are caused by anthropogenic compounds.

Necessary permits for fish and fish tissue sampling shall be obtained to comply with national legislation. This

may include permits from the (land) owner of the fishing rights, regional environmental and fishery authorities,

and ethical (animal rights) authorities.

The health of fish can be assessed by determination of biochemical, physiological, histological, and

pathological methods. The subcellular and cellular variables are often called biomarkers. The primary toxic

effect of pollutants, which occurs at the subcellular level, results in a biochemical or physiological change. This

reaction is usually fast, and it can progress further and cause disturbances at higher levels of biological

organisation within the organism, resulting in changes at the cellular and tissue (organ) level (histological

changes). These can lead to disturbances of reproduction and growth, and can eventually cause death of the

organism. Monitoring of fish health can, therefore, serve as an early warning system for anthropogenic

disturbance. Through a combination with other measurements (integrated monitoring), it may be possible to

correlate biomarker responses with for instance pollutant residues, distance from point sources, and

ecological variables such as reproductive recruitment, which are known to be sensitive to pollutants.

In principle, this method can be applied to all species of fish from all types of environments (fresh, salt,

brackish, cold, and warm water) and in shallow as well as reasonably deep water habitats. However, it is

usually advantageous to restrict these methods to certain species of fish, which can be used as indicator

species for fish health. These species shall be stationary, readily available (catchable in most locations) and

reasonably resistant to handling stress. Their biology and physiology should be well known in order to make

the interpretation of data easier. Examples of such species are the perch (Perca fluviatilis) and the eelpout

(viviparous blenny, Zoarces viviparus), which are used for monitoring along the Swedish coast.

© ISO 2007 – All rights reserved 1
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ISO 23893-1:2007(E)

Preferably the fish species used in the field should be suitable for keeping in the laboratory for toxicological

studies to investigate and confirm cause-effect relationships detected or suspected to take place in the field.

Procedures for organ and tissue sampling are essentially identical in both field and laboratory studies.

Procedures for collection of fish for field studies and for collection of organs are, therefore, described in

separate sections.
3 Equipment
3.1 Fish sampling equipment
3.1.1 Fishing boat, suitable for the area.
3.1.2 Clothing for outdoor work.
3.1.3 Lifejacket, of suitable size and buoyancy for each crew member.

3.1.4 Gill nets, made from textile or nylon fibres and of specified and suitable size for catching the desired

species and size and their gentle release into the fish chest used for storage.

3.1.5 Other equipment for fish capture, e.g. electroshocker and fyke nets, shall be described in enough

detail to allow interpretation and repeated sampling.

3.1.6 Global positioning system (GPS) instrument, for exact location of sampling sites.

3.1.7 Nautical map, for marking of sampling sites.
3.1.8 Knife and pair of scissors, for gentle removal of fish from gill nets.

3.1.9 Fish chest, made from wood or other inert material for storage of fish before tissue sampling.

3.1.10 Instruments for measurement of physical and chemical characteristics of water, e.g. thermometer,

pH meter, conductivity meter.

3.1.11 Equipment for determination of water depth, an echo-sounder or a calibrated line can be used to

determine the depth.
3.2 Tissue sampling equipment

3.2.1 Jetty, with easy access to fish chest and within 100 m of the field laboratory.

3.2.2 Landing net, suitable for the fish species and size.

3.2.3 Field laboratory, boathouse, garage or mobile laboratory supplied with electricity.

3.2.4 Stick (baton), for stunning the fish prior to sampling of blood.

3.2.5 Anaesthetic, to anaesthetise the fish (details on usage are given in 4.4.3).

3.2.6 Dissection equipment: forceps, scissors, scalpel, syringes, needles.
3.2.7 Ruler, for determination of body length.

3.2.8 Balance, for determination of body mass and tissue (liver, gonad, spleen) mass.

3.2.9 Centrifuge and tubes, for blood plasma.
3.2.10 Microscope slides, for preparation of blood smears.
2 © ISO 2007 – All rights reserved
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ISO 23893-1:2007(E)

3.2.11 Sample containers, of suitable sizes for tissue samples (e.g. of plastic with snap locks).

3.2.12 Marking pen, waterproof and freezeproof.

3.2.13 Vacuum flask with liquid nitrogen, for rapid freezing and temporary storage of samples.

3.2.14 Container with solid carbon dioxide, for transport of deep-frozen tissue samples between field

laboratory and analytical laboratory.
3.3 Biomarker determination equipment for the field laboratory
3.3.1 Haematocrit tubes and centrifuge, if required.
3.3.2 Blood glucose meter, if required.
3.3.3 Haemoglobin meter, if required.
4 Fish sampling
4.1 Statistical aspects

Feral fish, like other wild animals, are affected by a number of natural factors besides those caused by

anthropogenic load. Important natural factors for fish are climate, hydrology, oxygen and salinity (abiotic

factors), as well as age, size, sex, maturation, nutritional status, parasites and diseases (biotic factors). All

these factors can contribute to the overall variability of the measured response variables. In order to detect

temporal changes in trend monitoring and geographical variation in mapping of potential disturbance, all the

abiotic and biotic factors mentioned above shall be reduced in importance as much as possible.

4.2 Frequency and season for sampling

Fish should be sampled once a year during the autumn period in order to avoid the effects of rapid changes in

physiological conditions due to the reproduction season. During the autumn, most species of fish are not

reproducing, and the conditions to get enough fish by stationary gear like gill nets (3.1.4) and fyke nets (3.1.5)

are still good because the fish are still active. More frequent sampling at other times of the year does generally

not provide any new information in trend monitoring.

In Sweden, perch for fish-health monitoring is sampled by gill nets in September, and eelpout by fyke nets in

November. The most suitable period differs between countries and regions due to differences in climate. Often

only sexually mature fish of one sex (e.g. females for perch and eelpout, and males for chub and zebrafish)

within a certain size interval are used for each species in order to minimise the influence of sex and size.

4.3 Selection of sampling sites

In fish-health monitoring, it is of utmost importance to have as much detailed information as possible about the

anthropogenic load on sites to be used as reference locations. These sites should be monitored regularly,

preferably each year, in order to detect any large-scale impact from diffuse sources of pollution.

Fish-health monitoring can also be applied on a local scale. The locations of the sampling sites should then be

determined by the objectives, which are usually related to the location of point sources of pollution. A suitable

number of sites should be placed in a gradient from the local discharge point, or at sites which should be

protected from disturbances. A reference site with a biotope, which is as similar as possible to the recipient,

should also be selected.

Another aspect to be considered in the selection of sampling sites is availability of fish and reasonably easy

access to the sampling site, or at least to the site where the fish is to be killed for taking the samples [the fish

chest site (3.1.9)].
© ISO 2007 – All rights reserved 3
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ISO 23893-1:2007(E)
4.4 Sampling procedures
4.4.1 General

The number of fish should be sufficient in order to detect a predetermined change in the response variable

within a certain number of years. An experienced statistician can give advice on this. It should also be

considered that an additional number of fish to be sampled does not necessarily add much to the total cost of

the monitoring programme. For example for perch and eelpout, 25 females each, with a total length of 20 cm

to 30 cm, are sampled at each station in the Swedish monitoring programme. This number fulfils the statistical

need for determination of differences between stations for all the monitoring response variables used in that

programme. These are presented in Annexes A and B. If more stations are used, as in mapping of

disturbance from a point source, a lower number (10 to 20) should be used at each station. By these means,

more sites can be included at the same overall cost. The sex of the fish shall be determined and recorded,

and a sufficient number of the sex to be used shall be sampled. For most variables, females are the preferred

sex, but in some studies males should be used (e.g. for determination of vitellogenin in blood plasma).

4.4.2 Fish sampling

Fish can be caught by several methods (reviewed in Reference [3]) like angling and electric fishing gear

(Reference [1]), if they are killed immediately on site, sampled directly and samples are handled appropriately.

However, in most long-term monitoring programmes, adult fish are captured by gill nets (3.1.4), traps or fyke

nets (3.1.5) in order to get a sufficient sample of fish of suitable size and sex.

In order to avoid unnecessary stress on the fish when they are caught and killed for tissue sampling, they

should first be brought to a fish chest (3.1.9) and kept there for 2 days to 4 days before they are killed. This

stabilises stress-sensitive response variables like blood glucose, blood lactate and haematocrit.

In the field, fishes should preferably be caught by gill nets or fyke nets and kept alive through frequent

sampling carried out with the fishing equipment. However, other fishing techniques may also be used to

collect fish. Reference to the method used or a detailed description shall be given in a report in such cases.

The intention is that the fish are sampled from predetermined sampling sites by suitable fishing gear, e.g. gill

nets for perch and fyke nets for eelpout. Gill nets shall be made from suitable material that facilitates the

removal of fish with a minimum of damage. The mesh shall be adjusted to the species and size of the fish to

be used in the study. For perch of 20 cm to 30 cm body length, a mesh size of 30 mm to 33 mm is suitable.

The gill nets used for sampling of fish for population studies, as described in Reference [2], are multi-mesh

gill nets, and these are not the same as the nets used in this part of ISO 23893. Ordinary fyke nets can be

used to catch eelpout.

The nets should be set 3 days to 5 days before the fish tissues are to be sampled, in order to keep the fish in

the fish chest for 2 days to 4 days prior to sampling the tissues. The gill nets shall be sampled frequently, and

at least every 12 h, in order to collect as many live fishes as possible. They should be set at sunset and

collected during sunrise. This also means that the laboratory staff sampling the tissues shall maintain contact

with the local fishermen involved in the fishery to check that enough fish of suitable size is available before

they arrive.
An example of a sampling form is given in Annex C.
4.4.3 Fish tissue sampling

The fish shall be collected from the fish chest (3.1.9) one by one by a landing net (3.2.2), taking care to disturb

the remaining fish as little as possible. Tissue sampling shall be performed in a locality that is less than 100 m

from the fish chest. The sampling locality (3.2.3) shall have electricity and adequate lighting, and be

reasonably comfortable, so that the staff can operate safely and under suitable working conditions. A

boathouse, a garage or a caravan is a suitable locality.
4 © ISO 2007 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 23893-1:2007(E)

After its capture, the fish is either stunned by a blow on the back of the head with a wooden rod or a rubber

baton (3.2.4) or anaesthetised with a suitable anaesthetic (3.2.5) such as MS-222 (tricaine methanesulfonate

or ethyl 3-aminobenzoate methanesulfonate) . Then the samples should be taken in the following order:

blood, bile, liver, spleen, muscle, gonads, and other tissues (see Figure 1).
Key
1 brain
2 gills
3 muscle
4 backbone
5 blood
6 spleen
7 liver
8 kidney

Figure 1 — Sampling of blood from caudal blood vessels using a heparinised syringe

If some tissues are not needed then just proceed to the next item.

1) The body mass is determined to the nearest gram and the total body length is determined to the nearest

millimeter.

2) Blood is taken by a heparinised syringe from the caudal vessels (see Figure 1).

3) The fish is decapitated.
4) The body cavity is cut open, taking care not to damage the gall bladder.

1) MS-222 is an example of a suitable product available commercially. This information is given for the convenience of

users of this part of ISO 23893, and does not constitute an endorsement of this product by ISO.

Tricaine methanesulfonate is probably the most widely used fish anaesthetic, even if it is rather expensive. A dilution of

1:1 000 is lethal in 5 min to 10 min. Other commonly used anaesthetics for fish are quinaldine (2-methylquinoline), for

which a dilution of 1:20 000 is lethal in 5 min to 10 min, and benzocaine (ethyl 4-aminobenzoate) which can be used in the

field by dissolving 0,2 g in 5 ml acetone (to facilitate solubility) and adding it to 8 l of water (Reference [11]). It should be

noted that the use of anaesthetics may affect certain biomarkers.
© ISO 2007 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 23893-1:2007(E)
5) Bile is sampled using a syringe (no heparin needed).
6) The liver is removed and weighed to the nearest 0,1 g.
7) The gonads are removed and weighed to the nearest 0,1 g.
8) The spleen is removed and weighed to the nearest 0,1 g.
9) The intestinal tract is removed and weighed to determine somatic mass.
10) A piece of muscle tissue is removed from a predetermined place.

11) Otoliths or other tissues (opercular bone, scales) suitable for determination of age are taken.

12) Samples of tissues for histological examination are put into vessels with preservative.

13) The remaining part is sometimes stored frozen for later chemical analysis or for additional biochemical or

physiological analysis.

All tissue samples taken for biochemical measurements (liver and muscle) shall be processed further

immediately by taking subsamples for different analyses. The subsamples shall have been taken from the

same part of the organ for each variable to be analysed. This is crucial because enzyme activities may differ

within an organ (like the liver). The anatomy of the liver may differ between species of fish. This may require

standardisation within national and regional monitoring programmes in order to reduce variations due to

micro-anatomical variation. It is especially important for the liver, which is often used for determination of

several biochemical variables and also for histological examination.

Blood samples shall be processed directly to determine haematocrit by centrifugation, haemoglobin by

spectrophotometry, and to produce blood plasma by centrifugation. Blood smears for differential counts of

blood cells shall be prepared directly. Determination of blood glucose and lactate may be determined on site,

or samples of blood for glucose and lactate may be subsampled and processed further in an analytical

laboratory.

Samples of blood plasma, bile and other tissues shall be placed in labelled containers (3.2.11) and frozen

directly in liquid nitrogen or in contact with solid carbon dioxide. Labelling of containers shall be tested to be

sure that they withstand the freezing and processing of samples. Samples for histological examination and

determination of fish age shall be treated as required by the methods that are used.

The entire procedure, from taking the fish from the fish chest to processing of all samples, should not exceed

10 min. This requires some training of the team, which normally consists of two, three or four persons, when

all tissues mentioned above are to be sampled. When fewer tissues are to be sampled, the number of staff

may be reduced.

Examples of variables for monitoring of fish health mentioned above are described in more detail in Annex A

and an example of a sampling form is given in Annex D.
4.5 Handling of samples and analytical procedures

All samples, which are not determined on site, and which are to be used for biochemical and chemical

analysis, shall be kept frozen below −70 °C until analysed. If samples are to be stored for longer periods

(decades, centuries), then they shall be sent to a certified tissue bank. Samples to be used for histological

analyses can be stored in a bank at a lower cost.

If available, standard analytical procedures shall be used, and the sampling and handling of samples should

refer to this part of ISO 23893 and be supplemented with details pertaining to the specific samples.

Raw data for all inf
...

SLOVENSKI STANDARD
SIST ISO 23893-1:2010
01-september-2010

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UDYQDQMH]Y]RUFLLQQMLKRYRNRQ]HUYLUDQMH

Water quality - Biochemical and physiological measurements on fish - Part 1: Sampling

of fish, handling and preservation of samples

Qualité de l'eau - Mesurages biochimiques et physiologiques sur poisson - Partie 1:

Échantillonnage des poissons, manipulation et conservation des échantillons
Ta slovenski standard je istoveten z: ISO 23893-1:2007
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 23893-1:2010 en,fr

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST ISO 23893-1:2010
---------------------- Page: 2 ----------------------
SIST ISO 23893-1:2010
INTERNATIONAL ISO
STANDARD 23893-1
First edition
2007-11-15
Water quality — Biochemical and
physiological measurements on fish —
Part 1:
Sampling of fish, handling and
preservation of samples
Qualité de l'eau — Mesurages biochimiques et physiologiques sur
poisson —
Partie 1: Échantillonnage des poissons, manipulation et conservation
des échantillons
Reference number
ISO 23893-1:2007(E)
ISO 2007
---------------------- Page: 3 ----------------------
SIST ISO 23893-1:2010
ISO 23893-1:2007(E)
PDF disclaimer

This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but

shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In

downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat

accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.

Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation

parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In

the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

COPYRIGHT PROTECTED DOCUMENT
© ISO 2007

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,

electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or

ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
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SIST ISO 23893-1:2010
ISO 23893-1:2007(E)
Contents Page

Foreword............................................................................................................................................................ iv

Introduction ........................................................................................................................................................ v

1 Scope ..................................................................................................................................................... 1

2 Principle................................................................................................................................................. 1

3 Equipment ............................................................................................................................................. 2

4 Fish sampling........................................................................................................................................ 3

4.1 Statistical aspects................................................................................................................................. 3

4.2 Frequency and season for sampling .................................................................................................. 3

4.3 Selection of sampling sites ................................................................................................................. 3

4.4 Sampling procedures ........................................................................................................................... 4

4.5 Handling of samples and analytical procedures ............................................................................... 6

4.6 Background information ...................................................................................................................... 7

5 Quality assurance................................................................................................................................. 7

5.1 General................................................................................................................................................... 7

5.2 Fish sampling........................................................................................................................................ 7

5.3 Tissue sampling.................................................................................................................................... 7

5.4 Biochemical/chemical analysis ........................................................................................................... 7

5.5 Evaluation.............................................................................................................................................. 7

6 Report .................................................................................................................................................... 8

6.1 General................................................................................................................................................... 8

6.2 Data logging, data hosting................................................................................................................... 8

6.3 Evaluation.............................................................................................................................................. 8

Annex A (informative) Summary of variables used as biomarkers in fish ................................................... 9

Annex B (informative) Guide to interpretation of biomarker responses with references......................... 12

Annex C (informative) Suggested report for fish sampling ......................................................................... 15

Annex D (informative) Suggested report for tissue sampling ..................................................................... 16

Bibliography ..................................................................................................................................................... 18

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SIST ISO 23893-1:2010
ISO 23893-1:2007(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies

(ISO member bodies). The work of preparing International Standards is normally carried out through ISO

technical committees. Each member body interested in a subject for which a technical committee has been

established has the right to be represented on that committee. International organizations, governmental and

non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the

International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 23893-1 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,

Biological methods.

ISO 23893 consists of the following parts, under the general title Water quality — Biochemical and

physiological measurements on fish:
⎯ Part 1: Sampling of fish, handling and preservation of samples

⎯ Part 2: Determination of ethoxyresorufin-O-deethylase (EROD) [Technical Specification]

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SIST ISO 23893-1:2010
ISO 23893-1:2007(E)
Introduction

Determination of biomarker responses can be used to detect toxicity of known as well as unknown pollutants,

when they occur singly or in combination. Therefore, measurement of biomarkers is a cost-effective way to

assess ecosystem health. In combination with determinations of occurring and suspected pollutants,

determinations of biomarkers can facilitate the interpretation of cause-effect relationships in the environment,

as well as in laboratory toxicity tests. Information on commonly used biomarkers and the interpretation of

biomarker responses is given in Annexes A and B, respectively.

Biomarkers like ethoxyresorufin-O-deethylase (EROD), metallothionein and vitellogenin are used to detect

and quantify sublethal effects of pollutants, especially in fish. However, many of the biochemical and

physiological variables that are used as biomarkers are sensitive not only to disturbances by the pollutants of

concern, but also by the normal biochemical and physiological adjustments made by the fish in response to

seasonal variation, its normal development and sexual maturation. Some variables can also be affected by

general stress to disturbances caused by the handling during fish and fish tissue sampling. Therefore,

standardisation of procedures used for sampling and handling of samples prior to determination of the

biochemical and physiological variables is important.

Sublethal responses at the individual level usually occur before effects are seen at the population and

community level. In the aquatic environment, fish are suitable for detection of physiological effects of

pollutants, because they are exposed both through the water and through their food organisms. Also, the

physiology and biochemistry of fishes is rather similar to that of humans and other vertebrates, making

comparisons with studies on mammals easier than for those with crustaceans and other invertebrates.

This part of ISO 23893 serves as guidance for sampling and a platform for determination of biomarkers in fish,

making it possible to use the measurements to:

describe the state of the environment regarding effects of anthropogenic compounds on the health of fish;

perform time-trend surveillance (monitoring);

provide reference data and material for assessment of effects from point sources;

evaluate and assess environmental threats;
provide background information for environmental measures;
follow up and assess effects of environmental corrective measures;

integrate the biomarker responses with other measurements (e.g. fish abundance, recruitment and

pollutant residues) in order to facilitate the interpretation of environmental status or impact.

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SIST ISO 23893-1:2010
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SIST ISO 23893-1:2010
INTERNATIONAL STANDARD ISO 23893-1:2007(E)
Water quality — Biochemical and physiological measurements
on fish —
Part 1:
Sampling of fish, handling and preservation of samples
1 Scope

This part of ISO 23893 provides guidance on how to sample fish for determination of biochemical and

physiological characteristics, such as the composition and enzyme activities of blood, liver, muscle and other

tissues in order to asses the health of fish in the field as well as in the laboratory. The biochemical and

physiological variables used for this purpose are often called biomarkers. This part of ISO 23893 includes

recommendations and methods for:
obtaining a site-specific sample of a representative number of fish;
sampling fish tissues in the field and in the laboratory; and

handling and preservation of samples prior to analysis of biochemical and physiological variables.

2 Principle

Fish of a suitable species, age (size), and sex are sampled at selected sites at a suitable time of the year in

order to reduce variability due to biological, geographical, and seasonal influences. Standardised sampling

and measurement procedures, and qualified staff are used for collection of samples, transport, storage, and

analysis. By these means, the results from time series of comparable data can be used to detect changes in

the environment that are caused by anthropogenic compounds.

Necessary permits for fish and fish tissue sampling shall be obtained to comply with national legislation. This

may include permits from the (land) owner of the fishing rights, regional environmental and fishery authorities,

and ethical (animal rights) authorities.

The health of fish can be assessed by determination of biochemical, physiological, histological, and

pathological methods. The subcellular and cellular variables are often called biomarkers. The primary toxic

effect of pollutants, which occurs at the subcellular level, results in a biochemical or physiological change. This

reaction is usually fast, and it can progress further and cause disturbances at higher levels of biological

organisation within the organism, resulting in changes at the cellular and tissue (organ) level (histological

changes). These can lead to disturbances of reproduction and growth, and can eventually cause death of the

organism. Monitoring of fish health can, therefore, serve as an early warning system for anthropogenic

disturbance. Through a combination with other measurements (integrated monitoring), it may be possible to

correlate biomarker responses with for instance pollutant residues, distance from point sources, and

ecological variables such as reproductive recruitment, which are known to be sensitive to pollutants.

In principle, this method can be applied to all species of fish from all types of environments (fresh, salt,

brackish, cold, and warm water) and in shallow as well as reasonably deep water habitats. However, it is

usually advantageous to restrict these methods to certain species of fish, which can be used as indicator

species for fish health. These species shall be stationary, readily available (catchable in most locations) and

reasonably resistant to handling stress. Their biology and physiology should be well known in order to make

the interpretation of data easier. Examples of such species are the perch (Perca fluviatilis) and the eelpout

(viviparous blenny, Zoarces viviparus), which are used for monitoring along the Swedish coast.

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Preferably the fish species used in the field should be suitable for keeping in the laboratory for toxicological

studies to investigate and confirm cause-effect relationships detected or suspected to take place in the field.

Procedures for organ and tissue sampling are essentially identical in both field and laboratory studies.

Procedures for collection of fish for field studies and for collection of organs are, therefore, described in

separate sections.
3 Equipment
3.1 Fish sampling equipment
3.1.1 Fishing boat, suitable for the area.
3.1.2 Clothing for outdoor work.
3.1.3 Lifejacket, of suitable size and buoyancy for each crew member.

3.1.4 Gill nets, made from textile or nylon fibres and of specified and suitable size for catching the desired

species and size and their gentle release into the fish chest used for storage.

3.1.5 Other equipment for fish capture, e.g. electroshocker and fyke nets, shall be described in enough

detail to allow interpretation and repeated sampling.

3.1.6 Global positioning system (GPS) instrument, for exact location of sampling sites.

3.1.7 Nautical map, for marking of sampling sites.
3.1.8 Knife and pair of scissors, for gentle removal of fish from gill nets.

3.1.9 Fish chest, made from wood or other inert material for storage of fish before tissue sampling.

3.1.10 Instruments for measurement of physical and chemical characteristics of water, e.g. thermometer,

pH meter, conductivity meter.

3.1.11 Equipment for determination of water depth, an echo-sounder or a calibrated line can be used to

determine the depth.
3.2 Tissue sampling equipment

3.2.1 Jetty, with easy access to fish chest and within 100 m of the field laboratory.

3.2.2 Landing net, suitable for the fish species and size.

3.2.3 Field laboratory, boathouse, garage or mobile laboratory supplied with electricity.

3.2.4 Stick (baton), for stunning the fish prior to sampling of blood.

3.2.5 Anaesthetic, to anaesthetise the fish (details on usage are given in 4.4.3).

3.2.6 Dissection equipment: forceps, scissors, scalpel, syringes, needles.
3.2.7 Ruler, for determination of body length.

3.2.8 Balance, for determination of body mass and tissue (liver, gonad, spleen) mass.

3.2.9 Centrifuge and tubes, for blood plasma.
3.2.10 Microscope slides, for preparation of blood smears.
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3.2.11 Sample containers, of suitable sizes for tissue samples (e.g. of plastic with snap locks).

3.2.12 Marking pen, waterproof and freezeproof.

3.2.13 Vacuum flask with liquid nitrogen, for rapid freezing and temporary storage of samples.

3.2.14 Container with solid carbon dioxide, for transport of deep-frozen tissue samples between field

laboratory and analytical laboratory.
3.3 Biomarker determination equipment for the field laboratory
3.3.1 Haematocrit tubes and centrifuge, if required.
3.3.2 Blood glucose meter, if required.
3.3.3 Haemoglobin meter, if required.
4 Fish sampling
4.1 Statistical aspects

Feral fish, like other wild animals, are affected by a number of natural factors besides those caused by

anthropogenic load. Important natural factors for fish are climate, hydrology, oxygen and salinity (abiotic

factors), as well as age, size, sex, maturation, nutritional status, parasites and diseases (biotic factors). All

these factors can contribute to the overall variability of the measured response variables. In order to detect

temporal changes in trend monitoring and geographical variation in mapping of potential disturbance, all the

abiotic and biotic factors mentioned above shall be reduced in importance as much as possible.

4.2 Frequency and season for sampling

Fish should be sampled once a year during the autumn period in order to avoid the effects of rapid changes in

physiological conditions due to the reproduction season. During the autumn, most species of fish are not

reproducing, and the conditions to get enough fish by stationary gear like gill nets (3.1.4) and fyke nets (3.1.5)

are still good because the fish are still active. More frequent sampling at other times of the year does generally

not provide any new information in trend monitoring.

In Sweden, perch for fish-health monitoring is sampled by gill nets in September, and eelpout by fyke nets in

November. The most suitable period differs between countries and regions due to differences in climate. Often

only sexually mature fish of one sex (e.g. females for perch and eelpout, and males for chub and zebrafish)

within a certain size interval are used for each species in order to minimise the influence of sex and size.

4.3 Selection of sampling sites

In fish-health monitoring, it is of utmost importance to have as much detailed information as possible about the

anthropogenic load on sites to be used as reference locations. These sites should be monitored regularly,

preferably each year, in order to detect any large-scale impact from diffuse sources of pollution.

Fish-health monitoring can also be applied on a local scale. The locations of the sampling sites should then be

determined by the objectives, which are usually related to the location of point sources of pollution. A suitable

number of sites should be placed in a gradient from the local discharge point, or at sites which should be

protected from disturbances. A reference site with a biotope, which is as similar as possible to the recipient,

should also be selected.

Another aspect to be considered in the selection of sampling sites is availability of fish and reasonably easy

access to the sampling site, or at least to the site where the fish is to be killed for taking the samples [the fish

chest site (3.1.9)].
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4.4 Sampling procedures
4.4.1 General

The number of fish should be sufficient in order to detect a predetermined change in the response variable

within a certain number of years. An experienced statistician can give advice on this. It should also be

considered that an additional number of fish to be sampled does not necessarily add much to the total cost of

the monitoring programme. For example for perch and eelpout, 25 females each, with a total length of 20 cm

to 30 cm, are sampled at each station in the Swedish monitoring programme. This number fulfils the statistical

need for determination of differences between stations for all the monitoring response variables used in that

programme. These are presented in Annexes A and B. If more stations are used, as in mapping of

disturbance from a point source, a lower number (10 to 20) should be used at each station. By these means,

more sites can be included at the same overall cost. The sex of the fish shall be determined and recorded,

and a sufficient number of the sex to be used shall be sampled. For most variables, females are the preferred

sex, but in some studies males should be used (e.g. for determination of vitellogenin in blood plasma).

4.4.2 Fish sampling

Fish can be caught by several methods (reviewed in Reference [3]) like angling and electric fishing gear

(Reference [1]), if they are killed immediately on site, sampled directly and samples are handled appropriately.

However, in most long-term monitoring programmes, adult fish are captured by gill nets (3.1.4), traps or fyke

nets (3.1.5) in order to get a sufficient sample of fish of suitable size and sex.

In order to avoid unnecessary stress on the fish when they are caught and killed for tissue sampling, they

should first be brought to a fish chest (3.1.9) and kept there for 2 days to 4 days before they are killed. This

stabilises stress-sensitive response variables like blood glucose, blood lactate and haematocrit.

In the field, fishes should preferably be caught by gill nets or fyke nets and kept alive through frequent

sampling carried out with the fishing equipment. However, other fishing techniques may also be used to

collect fish. Reference to the method used or a detailed description shall be given in a report in such cases.

The intention is that the fish are sampled from predetermined sampling sites by suitable fishing gear, e.g. gill

nets for perch and fyke nets for eelpout. Gill nets shall be made from suitable material that facilitates the

removal of fish with a minimum of damage. The mesh shall be adjusted to the species and size of the fish to

be used in the study. For perch of 20 cm to 30 cm body length, a mesh size of 30 mm to 33 mm is suitable.

The gill nets used for sampling of fish for population studies, as described in Reference [2], are multi-mesh

gill nets, and these are not the same as the nets used in this part of ISO 23893. Ordinary fyke nets can be

used to catch eelpout.

The nets should be set 3 days to 5 days before the fish tissues are to be sampled, in order to keep the fish in

the fish chest for 2 days to 4 days prior to sampling the tissues. The gill nets shall be sampled frequently, and

at least every 12 h, in order to collect as many live fishes as possible. They should be set at sunset and

collected during sunrise. This also means that the laboratory staff sampling the tissues shall maintain contact

with the local fishermen involved in the fishery to check that enough fish of suitable size is available before

they arrive.
An example of a sampling form is given in Annex C.
4.4.3 Fish tissue sampling

The fish shall be collected from the fish chest (3.1.9) one by one by a landing net (3.2.2), taking care to disturb

the remaining fish as little as possible. Tissue sampling shall be performed in a locality that is less than 100 m

from the fish chest. The sampling locality (3.2.3) shall have electricity and adequate lighting, and be

reasonably comfortable, so that the staff can operate safely and under suitable working conditions. A

boathouse, a garage or a caravan is a suitable locality.
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After its capture, the fish is either stunned by a blow on the back of the head with a wooden rod or a rubber

baton (3.2.4) or anaesthetised with a suitable anaesthetic (3.2.5) such as MS-222 (tricaine methanesulfonate

or ethyl 3-aminobenzoate methanesulfonate) . Then the samples should be taken in the following order:

blood, bile, liver, spleen, muscle, gonads, and other tissues (see Figure 1).
Key
1 brain
2 gills
3 muscle
4 backbone
5 blood
6 spleen
7 liver
8 kidney

Figure 1 — Sampling of blood from caudal blood vessels using a heparinised syringe

If some tissues are not needed then just proceed to the next item.

1) The body mass is determined to the nearest gram and the total body length is determined to the nearest

millimeter.

2) Blood is taken by a heparinised syringe from the caudal vessels (see Figure 1).

3) The fish is decapitated.
4) The body cavity is cut open, taking care not to damage the gall bladder.

1) MS-222 is an example of a suitable product available commercially. This information is given for the convenience of

users of this part of ISO 23893, and does not constitute an endorsement of this product by ISO.

Tricaine methanesulfonate is probably the most widely used fish anaesthetic, even if it is rather expensive. A dilution of

1:1 000 is lethal in 5 min to 10 min. Other commonly used anaesthetics for fish are quinaldine (2-methylquinoline), for

which a dilution of 1:20 000 is lethal in 5 min to 10 min, and benzocaine (ethyl 4-aminobenzoate) which can be used in the

field by dissolving 0,2 g in 5 ml acetone (to facilitate solubility) and adding it to 8 l of water (Reference [11]). It should be

noted that the use of anaesthetics may affect certain biomarkers.
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5) Bile is sampled using a syringe (no heparin needed).
6) The liver is removed and weighed to the nearest 0,1 g.
7) The gonads are removed and weighed to the nearest 0,1 g.
8) The spleen is removed and weighed to the nearest 0,1 g.
9) The intestinal tract is removed and weighed to determine somatic mass.
10) A piece of muscle tissue is removed from a predetermined place.

11) Otoliths or other tissues (opercular bone, scales) suitable for determination of age are taken.

12) Samples of tissues for histological examination are put into vessels with preservative.

13) The remaining part is sometimes stored frozen for later chemical analysis or for additional biochemical or

physiological analysis.

All tissue samples taken for biochemical measurements (liver and muscle) shall be processed further

immediately by taking subsamples for different analyses. The subsamples shall have been taken from the

same part of the organ for each variable to be analysed. This is crucial because enzyme activities may differ

within an organ (like the liver). The anatomy of the liver may differ between species of fish. This may require

standardisation within national and regional monitoring programmes in order to reduce variations due to

micro-anatomical variation. It is especially important for the liver, which is often used for determination of

several biochemical variables and also for histological examination.

Blood samples shall be processed directly to determine haematocrit by centrifugation, haemoglobin by

spectrophotometry, and to produce blood plasma by centrifugation. Blood smears for differential counts of

blood cells shall be prepared directly. Determination of blood glucose and lactate may be determined on site,

or samples of blood for glucose and lactate may be subsampled and processed further in an analytical

laboratory.

Samples of blood plasma, bile and other tissues shall be placed in labelled containers (3.2.11) and frozen

directly in liquid nitrogen or in contact with solid carbon dioxide. Labelling of containers shall be tested to be

sure that they withstand the freezing and processing of samples. Samples for histological examinati

...

NORME ISO
INTERNATIONALE 23893-1
Première édition
2007-11-15
Qualité de l'eau — Mesurages
biochimiques et physiologiques sur
poisson —
Partie 1:
Échantillonnage des poissons,
manipulation et conservation des
échantillons
Water quality — Biochemical and physiological measurements on
fish —
Part 1: Sampling of fish, handling and preservation of samples
Numéro de référence
ISO 23893-1:2007(F)
ISO 2007
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ISO 23893-1:2007(F)
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ISO 23893-1:2007(F)
Sommaire Page

Avant-propos..................................................................................................................................................... iv

Introduction ........................................................................................................................................................ v

1 Domaine d'application.......................................................................................................................... 1

2 Principe.................................................................................................................................................. 1

3 Équipement ........................................................................................................................................... 2

4 Échantillonnage des poissons ............................................................................................................ 3

4.1 Aspects statistiques............................................................................................................................. 3

4.2 Fréquence et saison de l'échantillonnage ......................................................................................... 4

4.3 Sélection des sites d'échantillonnage................................................................................................ 4

4.4 Modes opératoires d'échantillonnage ................................................................................................ 4

4.5 Manipulation des échantillons et modes opératoires analytiques.................................................. 8

4.6 Informations de base............................................................................................................................ 8

5 Assurance qualité................................................................................................................................. 8

5.1 Généralités ............................................................................................................................................ 8

5.2 Échantillonnage des poissons ............................................................................................................ 8

5.3 Échantillonnage des tissus ................................................................................................................. 9

5.4 Analyse biochimique/chimique ........................................................................................................... 9

5.5 Évaluation.............................................................................................................................................. 9

6 Rapport .................................................................................................................................................. 9

6.1 Généralités ............................................................................................................................................ 9

6.2 Enregistrement chronologique des données, hébergement des données .................................... 9

6.3 Évaluation.............................................................................................................................................. 9

Annexe A (informative) Récapitulatif des variables utilisées comme marqueurs biologiques chez

les poissons ........................................................................................................................................ 10

Annexe B (informative) Guide sur l'interprétation des réponses des marqueurs biologiques avec

références............................................................................................................................................ 13

Annexe C (informative) Suggestion pour le rapport d'échantillonnage des poissons ............................. 16

Annexe D (informative) Suggestion pour le rapport d'échantillonnage des tissus .................................. 17

Bibliographie .................................................................................................................................................... 19

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ISO 23893-1:2007(F)
Avant-propos

L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux de

normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général confiée

aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du

comité technique créé à cet effet. Les organisations internationales, gouvernementales et non

gouvernementales, en liaison avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec

la Commission électrotechnique internationale (CEI) en ce qui concerne la normalisation électrotechnique.

Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI,

Partie 2.

La tâche principale des comités techniques est d'élaborer les Normes internationales. Les projets de Normes

internationales adoptés par les comités techniques sont soumis aux comités membres pour vote. Leur

publication comme Normes internationales requiert l'approbation de 75 % au moins des comités membres

votants.

L'attention est appelée sur le fait que certains des éléments du présent document peuvent faire l'objet de

droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable de ne

pas avoir identifié de tels droits de propriété et averti de leur existence.

L'ISO 23893-1 a été élaborée par le comité technique ISO/TC 147, Qualité de l'eau, sous-comité SC 5,

Méthodes biologiques.

L'ISO 23893 comprend les parties suivantes, présentées sous le titre général Qualité de l'eau — Mesurages

biochimiques et physiologiques sur poisson:

⎯ Partie 1: Échantillonnage des poissons, manipulation et conservation des échantillons

⎯ Partie 2: Dosage de l'éthoxyrésorufine-O-dééthylase (EROD) [Spécification technique]

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ISO 23893-1:2007(F)
Introduction

L'interprétation des réponses des marqueurs biologiques peut être utilisée pour détecter la toxicité de

polluants connus ou inconnus, isolément ou en combinaison. Par conséquent, la mesure des indicateurs

biologiques constitue une manière rentable pour évaluer l'état de santé d'un écosystème. Associées à des

dosages de polluants présents et soupçonnés, les dosages de marqueurs biologiques peuvent faciliter

l'interprétation des relations de cause à effet dans l'environnement, ainsi que dans les essais de toxicité

réalisés en laboratoire. Les Annexes A et B fournissent respectivement des informations sur les marqueurs

biologiques couramment utilisés et sur l'interprétation des réponses des marqueurs biologiques.

Les marqueurs biologiques comme l'éthoxyrésorufine-O-dééthylase (EROD), la métallothionéine et la

vitellogénine sont employés pour détecter et quantifier les effets sublétaux des polluants, notamment chez les

poissons. Toutefois, un grand nombre des variables biochimiques et physiologiques utilisées en tant que

marqueurs biologiques sont sensibles non seulement aux perturbations causées par les polluants concernés,

mais également aux réactions biochimiques et physiologiques normales des poissons qui varient avec les

variations saisonnières, leur développement normal et leur maturation sexuelle. Certaines variables peuvent

également être affectées par un stress d'ordre général lié aux perturbations causées par la manipulation

durant l'échantillonnage des poissons et de leurs tissus. Par conséquent, la normalisation des modes

opératoires employés pour l'échantillonnage et la manipulation des échantillons avant le dosage des variables

biochimiques et physiologiques est importante.

Les réponses sublétales observées au niveau individuel se produisent habituellement avant que des effets

soient constatés au niveau de la population et des peuplements. En milieu aquatique, les poissons constituent

les meilleurs candidats pour la détection des effets physiologiques des polluants car ils y sont exposés à la

fois via la colonne d'eau et leurs proies. En outre, la physiologie et la biochimie des poissons sont

relativement similaires à celles des humains et autres vertébrés. Par conséquent, il est plus facile de réaliser

des comparaisons avec des études portant sur les mammifères qu'avec celles concernant les crustacés et

autres invertébrés.

La présente partie de l'ISO 23893 sert de guide pour l'échantillonnage et de plate-forme pour le dosage des

marqueurs biologiques chez les poissons, rendant ainsi possible l'utilisation des mesures pour

décrire l'état du milieu par rapport aux effets des composés anthropiques sur l'état de santé des

poissons;
réaliser un contrôle en fonction du temps (surveillance);

fournir des données et matériaux de référence pour l'évaluation des effets depuis des sources de

pollution;
évaluer et quantifier les menaces environnementales;
fournir des informations de base pour les mesures environnementales;
suivre et évaluer les effets des mesures environnementales correctives;

intégrer les réponses des marqueurs biologiques à d'autres mesures (par exemple la quantité de

poissons, le recrutement et les résidus des polluants) afin de faciliter l'interprétation de l'état ou de

l'impact environnemental.
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NORME INTERNATIONALE ISO 23893-1:2007(F)
Qualité de l'eau — Mesurages biochimiques et physiologiques
sur poisson —
Partie 1:
Échantillonnage des poissons, manipulation et conservation
des échantillons
1 Domaine d'application

La présente partie de l'ISO 23893 fournit des indications sur la manière d'échantillonner des poissons pour

déterminer leurs caractéristiques biochimiques et physiologiques, telles que la composition et les activités

enzymatiques du sang, du foie, des muscles et d'autres tissus afin d'évaluer l'état de santé des poissons

aussi bien sur le terrain qu'en laboratoire. Les variables biochimiques et physiologiques utilisées dans ce but

sont souvent appelées marqueurs biologiques. La présente partie de l'ISO 23893 inclut des recommandations

et des méthodes concernant:

la manière d'obtenir un échantillon spécifique du site pour un nombre représentatif de poissons,

l'échantillonnage des tissus de poissons sur le terrain et en laboratoire, et

la manipulation et la conservation des échantillons avant analyse des variables biochimiques et

physiologiques.
2 Principe

Les poissons appartenant à une espèce appropriée et ayant un âge (taille) et un sexe adaptés sont prélevés à

des sites choisis à un moment adapté de l'année afin de réduire la variabilité due à des influences biologiques,

géographiques et saisonnières. Des modes opératoires d'échantillonnage et de mesure normalisés, et un

personnel qualifié, sont utilisés pour le prélèvement, le transport, le stockage et l'analyse des échantillons.

Grâce à ces moyens, les résultats issus de séries temporelles de données comparables peuvent être utilisés

pour détecter des modifications environnementales causées par des composés anthropiques.

Les autorisations nécessaires au prélèvement des poissons et de leurs tissus doivent être obtenues

conformément à la législation nationale. Ceux-ci peuvent inclure les permis à obtenir auprès du propriétaire

(foncier) des droits de pêche, des autorités régionales environnementales et de pêche et des autorités

éthiques (droits des animaux).

L'état de santé des poissons peut être évalué par la détermination des méthodes biochimiques,

physiologiques, histologiques et pathologiques. Les variables subcellulaires et cellulaires constituent souvent

des marqueurs biologiques. L'effet toxique primaire associé aux polluants se produisant au niveau sub-

cellulaire entraîne une modification biochimique ou physiologique. Cette réaction est habituellement rapide et

peut progresser par la suite pour provoquer des perturbations à des niveaux supérieurs de l'organisation

biologique à l'intérieur de l'organisme, ce qui entraîne des modifications aux niveaux cellulaires et tissulaires

(organes) (modifications histologiques). Ces modifications peuvent entraîner des troubles au niveau de la

reproduction et de la croissance, et peuvent éventuellement provoquer la destruction de l'organisme. La

surveillance de l'état de santé des poissons peut, par conséquent, servir de système d'alerte avancée par

rapport aux perturbations anthropiques. Associé à d'autres mesures (surveillance intégrée), il peut s'avérer

possible d'établir une corrélation entre les réponses des marqueurs biologiques et, par exemple, les résidus

© ISO 2007 – Tous droits réservés 1
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ISO 23893-1:2007(F)

de polluants, la distance par rapport aux sources et les variables écologiques, tel que le recrutement, qui sont

reconnues comme étant sensibles aux polluants.

En principe, cette méthode peut être appliquée à toutes les espèces de poissons provenant de tous types

d'environnements (eau douce, eau salée, eau saumâtre, eau froide ou chaude), ainsi que dans des habitats à

faible profondeur ou profondeur raisonnablement importante. Toutefois, il est habituellement avantageux de

limiter ces méthodes à certaines espèces de poissons pouvant être utilisées comme espèces indicatrices de

l'état de santé des poissons. Ces espèces doivent être sédentaires, facilement disponibles (capturables dans

une majorité de lieux) et raisonnablement résistantes au stress lié aux manipulations. Il convient que leur

biologie et physiologie soient bien connues afin de rendre l'interprétation des données plus facile. Des

exemples de telles espèces sont la perche (Perca fluviatilis) et la blennie vivipare (Zoarces viviparous), qui

sont utilisées pour la surveillance le long des côtes suédoises.

Il convient, de préférence, qu'il soit possible de maintenir les espèces de poissons, utilisées sur le terrain, en

laboratoire pour des études toxicologiques afin d'analyser et de confirmer les relations de cause à effet qui

sont détectées, ou dont on soupçonne l'existence, sur le terrain. Les modes opératoires d'échantillonnage des

organes et des tissus sont essentiellement identiques dans les études de terrain et en laboratoire. Les modes

opératoires de prélèvement des poissons pour les études de terrain et pour le prélèvement des organes sont,

par conséquent, décrits séparément.
3 Équipement
3.1 Équipement pour l'échantillonnage des poissons
3.1.1 Bateau de pêche, adapté au milieu.
3.1.2 Vêtements, adaptés au travail en extérieur.

3.1.3 Gilet de sauvetage, de taille et de flottabilité appropriées pour chaque membre d'équipage.

3.1.4 Filets maillants, fabriqués en fibres textiles ou nylon, ayant les dimensions spécifiées et appropriées

pour pêcher les espèces souhaitées de la taille choisie, et pour relâcher les poissons en douceur dans le

coffre à poissons employé pour leur conservation.

3.1.5 Autres équipements de pêche, par exemple les dispositifs à électrochocs et les verveux à ailes, qui

doivent être décrits de manière suffisamment détaillée pour permettre l'interprétation et des échantillonnages

répétés.

3.1.6 Système de positionnement par satellite ou GPS, pour localiser les sites d'échantillonnage avec

précision.
3.1.7 Carte marine, pour marquer les sites d'échantillonnage.

3.1.8 Couteau et paire de ciseaux, pour prélever en douceur les poissons dans les filets maillants.

3.1.9 Coffre à poissons, fabriqué en bois ou en d'autres matériaux inertes, destiné à la conservation des

poissons avant prélèvement de leurs tissus.

3.1.10 Instruments de mesure des propriétés physiques et chimiques de l'eau, par exemple thermomètre,

pH-mètre, conductimètre.

3.1.11 Équipement permettant de déterminer la profondeur d'eau; il est possible d'utiliser un

échosondeur ou une ligne calibrée pour déterminer la profondeur.
2 © ISO 2007 – Tous droits réservés
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ISO 23893-1:2007(F)
3.2 Équipement pour l'échantillonnage des tissus

3.2.1 Appontement, permettant un accès facile au coffre à poissons, situé à moins de 100 m du

laboratoire de terrain.
3.2.2 Épuisette, adaptée à l'espèce et à la taille de poisson.

3.2.3 Laboratoire de terrain, par exemple un hangar à bateaux, un garage ou un laboratoire mobile,

pourvu d'une alimentation électrique.
3.2.4 Bâton, pour assommer les poissons avant de leur prélever du sang.

3.2.5 Anesthésique, pour anesthésier les poissons (les détails d'utilisation sont donnés en 4.4.3).

3.2.6 Équipement de dissection: paire de pinces, ciseaux, scalpel, seringues et aiguilles.

3.2.7 Règle, pour déterminer la taille des poissons.

3.2.8 Balance, pour déterminer la masse du corps des poisson et celle de leurs organes (foie, gonades,

rate, etc.) et tissus.
3.2.9 Centrifugeuse, pour le plasma sanguin, et tubes.
3.2.10 Lames de verre, pour la préparation de frottis sanguins.

3.2.11 Flaconnages pour échantillons, de tailles adaptées aux échantillons de tissus (par exemple en

plastique avec des verrous d'accrochage).
3.2.12 Feutre marqueur, résistant à l'eau et à la congélation.

3.2.13 Bouteille isotherme contenant de l'azote liquide, pour la congélation rapide et la conservation

temporaire des échantillons.

3.2.14 Récipient contenant de la neige carbonique (dioxyde de carbone solide), pour le transport des

échantillons de tissus surgelés entre le laboratoire de terrain et le laboratoire analytique.

3.3 Équipement servant au dosage des marqueurs biologiques dans le laboratoire de

terrain
3.3.1 Tubes et centrifugeuse à hématocrite, si nécessaire.
3.3.2 Glucosimètre, si nécessaire.
3.3.3 Appareil de mesure de l'hémoglobine, si nécessaire.
4 Échantillonnage des poissons
4.1 Aspects statistiques

Les poissons sauvages, à l'image des autres animaux, sont affectés par un certain nombre de facteurs

naturels en plus des facteurs dus à la charge anthropique. Les facteurs naturels importants pour les poissons

incluent le climat, l'hydrologie, l'oxygène et la salinité (facteurs abiotiques) ainsi que l'âge, la taille, le sexe, la

maturation, l'état nutritionnel, les parasites et les maladies (facteurs biotiques). Tous ces facteurs peuvent

contribuer à la variabilité globale des variables mesurées, caractérisant la réponse. Afin de détecter les

changements temporels affectant la surveillance des tendances et la variation géographique dans la

cartographie des perturbations potentielles, le nombre de tous les facteurs abiotiques et biotiques mentionnés

ci-dessus doit être réduit autant que possible.
© ISO 2007 – Tous droits réservés 3
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ISO 23893-1:2007(F)
4.2 Fréquence et saison de l'échantillonnage

Il convient d'échantillonner les poissons une fois par an en automne afin d'éviter les effets associés à des

modifications rapides des états physiologiques dues à la saison de reproduction. Durant l'automne, la plupart

des espèces de poissons ne sont pas en période de reproduction et les conditions permettant de capturer un

nombre suffisant de poissons en utilisant un équipement fixe tel que les filets maillants (3.1.4) et les verveux à

ailes (3.1.5) sont encore bonnes car les poissons sont encore actifs. Un échantillonnage plus fréquent à

d'autres périodes de l'année ne fournit généralement pas de nouvelles informations concernant la surveillance

des tendances.

En Suède, la perche servant à la surveillance de l'état de santé des poissons est prélevée à l'aide de filets

maillants en septembre, et la blennie vivipare à l'aide de verveux à ailes en novembre. La période la plus

appropriée varie selon les pays et les régions en raison des différences de climats. Le plus souvent, seuls les

poissons sexuellement matures correspondant à un seul sexe (par exemple les femelles pour la perche et la

blennie vivipare et les mâles pour le chevaine et le danio zébré) dans un certain intervalle de taille sont

utilisés pour chaque espèce afin de minimiser l'influence du sexe et de la taille.

4.3 Sélection des sites d'échantillonnage

En matière de surveillance de l'état de santé des poissons, il est d'une importance capitale de disposer

d'informations aussi détaillées que possible concernant la charge anthropique des sites devant être utilisés

comme lieux de référence. Il convient que ces sites soient surveillés régulièrement, et de préférence chaque

année, afin de détecter tout impact à grande échelle provenant de sources de pollution diffuse.

La surveillance de l'état de santé des poissons peut également être réalisée à une échelle locale. Il convient

alors que les emplacements des sites d'échantillonnage soient déterminés par les objectifs qui sont

habituellement liés à l'emplacement des sources de pollution. Il est recommandé qu'un nombre approprié de

sites soient disposés selon un gradient à partir du point de rejet local, ou à des sites qu'il convient de protéger

contre les perturbations. Il convient également de choisir un site de référence dont le biotope est aussi

similaire que possible à celui du site pollué.

Un autre aspect à prendre en compte dans la sélection des sites d'échantillonnage concerne la disponibilité

des poissons et des conditions d'accès relativement faciles au site de prélèvement, ou au moins au site où le

poisson doit être sacrifié pour le prélèvement des échantillons [le site du coffre à poissons (3.1.9)].

4.4 Modes opératoires d'échantillonnage
4.4.1 Généralités

Il convient que le nombre de poissons soit suffisant pour permettre la détection d'un changement

prédéterminé au niveau de la variable suivie au cours d'un certain nombre d'années. Un statisticien

expérimenté peut fournir des conseils sur ce sujet. Il est également recommandé de considérer qu'un nombre

supplémentaire de poissons à prélever n'implique pas nécessairement une augmentation supplémentaire

importante du coût du programme de surveillance. Dans le cas de la perche et de la blennie vivipare, par

exemple, 25 femelles de chaque espèce, de longueur totale comprise entre 20 cm et 30 cm, sont prélevées à

chaque station dans le cadre du programme de surveillance suédois. Ce nombre satisfait aux exigences

statistiques concernant la détermination des différences existant entre les stations pour l'ensemble des

variables de réponse de surveillance employées dans ce programme. Celles-ci sont présentées dans les

Annexes A et B. Si davantage de stations sont employées, comme dans le cas de la cartographie des

perturbations à partir de la source de pollution, il convient d'utiliser un nombre plus faible (10 à 20) au niveau

de chaque station. De cette manière, un nombre plus important de sites peut être inclus pour le même coût

global. Le sexe des poissons doit être déterminé et enregistré et un nombre suffisant de poissons du sexe

devant être utilisé doit être échantillonné. Pour la plupart des variables, les femelles sont le sexe préféré, mais

dans certaines études il convient d'utiliser des mâles (par exemple pour la détermination du taux de

vitellogénine dans le plasma sanguin).
4 © ISO 2007 – Tous droits réservés
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ISO 23893-1:2007(F)
4.4.2 Échantillonnage des poissons

Les poissons peuvent être capturés en utilisant plusieurs méthodes (voir la Référence [3]), comme la pêche à

[1]

la ligne et l'équipement de pêche électrique s'ils sont tués immédiatement sur site, échantillonnés

directement et que les échantillons sont manipulés de manière appropriée. Toutefois, dans la plupart des

programmes de surveillance à long terme, les poissons adultes sont capturés à l'aide de filets maillants

(3.1.4), de pièges ou de verveux à ailes (3.1.5) afin d'obtenir un nombre suffisant d'échantillons de poissons

de taille et de sexe adaptés.

Afin d'éviter tout stress inutile aux poissons lors de la capture et de leur mise à mort pour l'échantillonnage

des tissus, il convient de les déposer tout d'abord dans un coffre à poissons (3.1.9) où ils sont maintenus

durant 2 j à 4 j avant d'être sacrifiés. Ceci permet de stabiliser les variables de réponse sensibles au stress,

tels que le taux de glucose et de lactate dans le sang et l'hématocrite.

Sur le terrain, il est préférable de capturer les poissons à l'aide de filets maillants ou de verveux à ailes et de

les conserver en vie lors des fréquents échantillonnages effectués avec l'équipement de pêche. Toutefois,

d'autres techniques de pêche peuvent être employées pour récolter les poissons. Dans de tels cas, la

méthode utilisée ou une description détaillée doit être référencée dans un rapport. Le but est de prélever les

poissons sur des sites d'échantillonnage prédéterminés à l'aide d'un équipement de pêche approprié, par

exemple les filets maillants pour la perche et les verveux à ailes pour la blennie vivipare. Les filets maillants

doivent être réalisés avec un matériau approprié facilitant le prélèvement du poisson avec un minimum de

dégâts. La maille doit être adaptée à l'espèce et à la taille du poisson devant être utilisé pour l'étude. Pour une

perche ayant une taille de 20 cm à 30 cm, une dimension de maille de 30 mm à 33 mm est convenable. Les

filets maillants employés pour l'échantillonnage des poissons pour les études de populations, tels que décrits

à la Référence [2], sont des filets maillants multimailles et ils sont différents des filets utilisés dans la présente

partie de l'ISO 23893. Les verveux à ailes ordinaires peuvent servir à pêcher la blennie vivipare.

Il convient de mettre les filets en place 3 j à 5 j avant l'échantillonnage des tissus de poissons afin que les

poissons soient conservés dans le coffre à poissons durant 2 j à 4 j avant le prélèvement des tissus. Les filets

maillants doivent faire l'objet d'échantillonnages fréquents, au moins toutes les 12 h, afin de permettre la

récolte du plus grand nombre possible de poissons vivants. Il convient de les mettre en place au coucher du

soleil et de les relever au lever du soleil. Ceci signifie également que le personnel du laboratoire procédant

aux prélèvements de tissus doit rester en contact avec les pêcheurs locaux en charge de la zone de pêche

pour vérifier qu'un nombre suffisant de poissons de la taille adaptée soit disponible avant leur arrivée.

Un exemple de formulaire d'échantillonnage des poissons est fourni en Annexe C.
4.4.3 Échantillonnage des tissus

Les poissons doivent être prélevés un à un dans le coffre à poissons (3.1.9) à l'aide d'une épuisette (3.2.2),

en prenant soin de stresser le moins possible les poissons restants. L'échantillonnage des tissus doit être

réalisé à moins de 100 m du coffre. Le lieu d'échantillonnage (3.2.3) doit être pourvu d'électricité et d'un

éclairage adéquat, et être raisonnablement confortable de sorte que le personnel puisse travailler en sécurité

dans des conditions de travail appropriées. Un hangar à bateaux, un garage ou une caravane constitue un

lieu approprié.
© ISO 2007 – Tous droits réservés 5
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ISO 23893-1:2007(F)

Suite à sa capture, le poisson est soit assommé par un coup porté à l'arrière de la tête avec un bâton (3

...

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