SIST ISO 23893-1:2010
Water quality - Biochemical and physiological measurements on fish - Part 1: Sampling of fish, handling and preservation of samples
Water quality - Biochemical and physiological measurements on fish - Part 1: Sampling of fish, handling and preservation of samples
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.
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 mani�re d'�chantillonner des poissons pour d�terminer leurs caract�ristiques biochimiques et physiologiques, telles que la composition et les activit�s enzymatiques du sang, du foie, des muscles et d'autres tissus afin d'�valuer l'�tat de sant� des poissons aussi bien sur le terrain qu'en laboratoire. Les variables biochimiques et physiologiques utilis�es dans ce but sont souvent appel�es marqueurs biologiques.
L'ISO 23893-1:2007 inclut des recommandations et des m�thodes concernant la mani�re d'obtenir un �chantillon sp�cifique du site pour un nombre repr�sentatif de poissons, l'�chantillonnage des tissus de poissons sur le terrain et en laboratoire, et la manipulation et la conservation des �chantillons 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
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Standards Content (Sample)
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
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
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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
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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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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.
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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.
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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)].
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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
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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
1)
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
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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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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
ii © ISO 2007 – All rights reserved
---------------------- Page: 4 ----------------------
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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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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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
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
1)
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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ii © ISO 2007 – Tous droits réservés
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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
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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.
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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.
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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).
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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.
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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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