BRITISH STANDARD BS EN ISO

7346-2:1998
BS 6068-5.3:
1998

Water quality —
Determination of the
acute lethal toxicity of
substances to a fresh
water fish
[Brachydanio rerio
Hamilton-Buchanan
(Teleostei,
Cyprinidae)] —
Semi-static method

The European Standard EN ISO 7346-2:1997 has the status of a

British Standard

ICS 13.060.01
BS EN ISO 7346-2:1998

This British Standard, having

been prepared under the Amendments issued since publication
direction of the Health and
Environment Sector Board, was
published under the authority of
Amd. No. Date Comments
the Standards Board and comes
into effect on
15 January 1998

© BSI 03-1999

ISBN 0 580 28489 1

 BS EN ISO 7346-2:1998

Contents

Page
National foreword ii
Foreword 2
Foreword ii
Text of EN ISO 7346-2 1

© BSI 03-1999 i
BS EN ISO 7346-2:1998

National foreword

This British Standard is the English language version of EN ISO 7346-2:1997. It

is identical with ISO 7346-2:1996. It supersedes BS 6068-5.3:1985 which is
withdrawn.
The UK participation in its preparation was entrusted by Technical Committee
EH/3, Water quality, to Subcommittee EH/3/5, Biological methods, which has the
responsibility to:
— aid enquirers to understand the text;
— present to the responsible international/European committee any enquiries
on the interpretation, or proposals for change, and keep the UK interests
informed;
— monitor related international and European developments and promulgate
them in the UK.
A list of organizations represented on this subcommittee can be obtained on
request to its secretary.
BS EN ISO 7346-2 is one of a series of standards on water quality, others of which
have been, or will be, published as Sections of BS 6068. This standard has
therefore been given the secondary identifier BS 6068-5.3. The various Sections
of BS 6068 are comprised within Parts 1 to 7, which, together with Part 0, are
listed below.
— Part 0: Introduction;
— Part 1: Glossary;
— Part 2: Physical, chemical and biochemical methods;
— Part 3: Radiological methods;
— Part 4: Microbiological methods;
— Part 5: Biological methods;
— Part 6: Sampling;
— Part 7: Precision and accuracy.
Cross-references
Attention is drawn to the fact that CEN and CENELEC Standards normally
include an annex which lists normative references to international publications
with their corresponding European publications. The British Standards which
implement these international or European publications may be found in the BSI
Standards Catalogue under the section entitled “International Standards
Correspondence Index”, or by using the “Find” facility of the BSI Standards
Electronic Catalogue.
A British Standard does not purport to include all the necessary provisions of a
contract. Users of British Standards are responsible for their correct application.
Compliance with a British Standard does not of itself confer immunity
from legal obligations.

Summary of pages
This document comprises a front cover, an inside front cover, pages i and ii,
the EN title page, page 2, the ISO title page, page ii, pages 1 to 8,
and a back cover.
This standard has been updated (see copyright date) and may have had
amendments incorporated. This will be indicated in the amendment table on
the inside front cover.

ii © BSI 03-1999
EUROPEAN STANDARD EN ISO 7346-2
NORME EUROPÉENNE
November 1997
EUROPÄISCHE NORM

ICS 13.060.01

Descriptors: See ISO document

English version

Water quality — Determination of the acute lethal toxicity

of substances to a freshwater fish [Brachydanio rerio
Hamilton (Teleostei, Cyprinidae)] — Part 2: Semi-static
method
(ISO 7346-2:1996)

Qualité de l’eau — Détermination de la toxicité Wasserbeschaffenheit — Bestimmung der

aiguë létale de substances vis-à-vis d’un poisson akuten letalen Toxizität von Substanzen
d’eau douce [Brachydanio rerio gegenüber einem Süßwasserfisch
Hamilton-Buchanan (Téléostei, Cyprinidae)] — [Brachydanio rerio Hamilton-Buchanan
Partie 2: Méthode semi-statique (Teleostei, Cyprinidae)] —
(ISO 7346-2:1996) Teil 2: Semistatisches Verfahren
(ISO 7346-2:1996)

This European Standard was approved by CEN on 30 October 1997.

CEN members are bound to comply with the CEN/CENELEC Internal
Regulations which stipulate the conditions for giving this European Standard
the status of a national standard without any alteration. Up-to-date lists and
bibliographical references concerning such national standards may be obtained
on application to the Central Secretariat or to any CEN member.
This European Standard exists in three official versions (English, French,
German). A version in any other language made by translation under the
responsibility of a CEN member into its own language and notified to the
Central Secretariat has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium,
Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland,
Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden,
Switzerland and United Kingdom.

CEN
European Committee for Standardization
Comité Européen de Normalisation
Europäisches Komitee für Normung
Central Secretariat: rue de Stassart 36, B-1050 Brussels

© 1997 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national
Members. Ref. No. EN ISO 7346-2:1997 E
EN ISO 7346-2:1997

Foreword Contents
The text of the International Standard from Page
Technical Committee ISO/TC 147 “Water quality” of Foreword 2
the International Organization for Standardization
Foreword ii
(ISO) has been taken over as an European Standard
by Technical Committee CEN/TC 230 “Water Introduction 1
analysis”, the secretariat of which is held by DIN. 1 Scope 1
This European Standard shall be given the status of 2 Principle 1
a national standard, either by publication of an 3 Test organism and reagents 2
identical text or by endorsement, at the latest by
month of May 1998, and conflicting national 3.1 Test organism 2
standards shall be withdrawn at the latest by 3.2 Standard dilution water 2
May 1998. 3.3 Stock solutions of test substances 2
According to the CEN/CENELEC Internal 3.4 Test solutions 2
Regulations, the national standards organizations 4 Apparatus 3
of the following countries are bound to implement
this European Standard: Austria, Belgium, 5 Test environment 3
Czech Republic, Denmark, Finland, France, 6 Procedure 3
Germany, Greece, Iceland, Ireland, Italy, 6.1 Condition of the fish 3
Luxembourg, Netherlands, Norway, Portugal, 6.2 Limit test 3
Spain, Sweden, Switzerland and the United
Kingdom. 6.3 Preliminary test 3
6.4 Final test 4
Endorsement notice
7 Expression of results 4
The text of the International Standard 7.1 Validity 4
ISO 7346-2:1996 has been approved by CEN as a
European Standard without any modification. 7.2 Estimation of LC50 4
8 Test report 5
Annex A (informative) Environment
parameters for maintenance and breeding
of zebra fish (Brachydanio rerio
Hamilton-Buchanan) 6
Annex B (informative) Suggested form for
recording date 7
Annex C (informative) Bibliography 8
Figure 1 — Graphical interpolation of LC50
(liner scales) 5
Figure 2 — Graphical interpolation of LC 50
(logarithmic and probability scales) 5

2 © BSI 03-1999
EN ISO 7346-2:1997

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.
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.
International Standard ISO 7346-2 was prepared by Technical Committee
ISO/TC 147, Water quality, Subcommittee SC 5, Biological methods.
This second edition cancels and replaces the first edition (ISO 7346-2:1984), which
has been technically revised.
ISO 7346 consists of the following parts, under the general title Water quality —
Determination of the acute lethal toxicity of substances to a freshwater fish
[Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)]:
— Part 1: Static method;
— Part 2: Semi-static method;
— Part 3: Flow-through method.
Annex A, Annex B and Annex C of this part of ISO 7346 are for information only.

Descriptors: Water, quality, water pollution, tests, water tests, biological tests, determination,
toxicity.

ii
 EN ISO 7346-2:1997

Introduction 1 Scope
The three parts of ISO 7346 describe methods of This part of ISO 7346 specifies a semi-static method
determining the acute lethal toxicity of substances for the determination of the acute lethal toxicity of
to the zebra fish (Brachydanio rerio stable, non-volatile, single substances, soluble in
Hamilton-Buchanan) but it must be emphasized water under specified conditions, to a freshwater
that the recommended use of the zebra fish does not fish [Brachydanio rerio Hamilton-Buchanan
preclude the use of other species. The methodologies (Teleostei, Cyprinidae) — common name, zebra fish]
presented here may also be used for other species of in water of a specified quality.
freshwater, marine or brackish water fish, with The method is applicable for assigning, for each test
appropriate modifications of, for example, dilution substance, broad categories of acute lethal toxicity
water quality and the temperature conditions of the to Brachydanio rerio under the test conditions.
test.
The results are insufficient by themselves to define
Within the three parts of ISO 7346, a choice can be water quality standards for environmental
made between static, semi-static and flow-through protection.
methods. The static test, described in ISO 7346-1, in
The method is also applicable when using certain
which the solution is not renewed, has the
other species of freshwater fish as the test
advantage of requiring simple apparatus, although
organism1).
the substances in the test vessel may become
depleted during the course of the test and the The method may be adapted for use with other
general quality of the water may deteriorate. The freshwater fish and marine and brackish water fish
flow-through method, described in ISO 7346-3, in with appropriate modification of the test conditions,
which the test solution is replenished continuously, particularly with respect to the quantity and quality
overcomes such problems but requires the use of of the dilution water and the temperature.
more complex apparatus. In the semi-static
procedure, described in this part of ISO 7346, the 2 Principle
test solutions are renewed every 24 h or 48 h, this Determination, under specified conditions, of the
method being a compromise between the other two. concentrations at which a substance is lethal
The flow-through method can be used for most types to 50 % of a test population of Brachydanio rerio
of substances, including those unstable in water, after exposure periods of 24 h, 48 h, 72 h and 96 h
but the concentrations of the test substance are to that substance in the ambient water. These
determined wherever possible. The static method is median lethal concentrations are designated
limited to the study of substances whose tested the 24 h – LC50, 48 h – LC50, 72 h – LC50
concentrations remain relatively constant during and 96 h – LC50.
the test period. The semi-static method can be used The test is carried out in two stages:
for testing those substances whose concentrations a) a preliminary test which gives an approximate
can be maintained satisfactorily throughout the test indication of the acute median lethal
by renewal of the solutions every 24 h or 48 h. concentrations and serves to determine the range
Special arrangements may be necessary for of concentrations for the final test;
substances which are highly volatile.
b) a final test, the results of which alone are
To assist in the preparation and maintenance of reported.
concentrations of substances which may be lethal at
concentrations close to that of their aqueous
solubility, a small volume of solvent may be used, as
specified in the methods.

1)
The following species of freshwater fish can be used, in addition to Brachydanio rerio, without modification to this part of
ISO 7346.
— Lepomis macrochirus (Teleostei, Centrarchidae)
— Oryzias latipes (Teleostei, Poeciliidae)
— Pimephales promelas (Teleostei, Cyprinidae)
— Poecilia reticulata (Teleostei, Poeciliidae)

© BSI 03-1999 1
EN ISO 7346-2:1997

Where evidence is available to show that test 3.2 Standard dilution water
concentrations remain relatively constant The freshly prepared standard dilution water shall
(i.e. within about 20 % of the nominal values) have a pH of 7,8 ± 0,2, and a calcium hardness of
throughout the test, then either measured or approximately 250 mg/l, expressed as calcium
nominal concentrations are used in the estimation carbonate, and shall contain the following
of the LC50. Where such analyses show that the concentrations of salts dissolved in distilled or
concentrations present remain relatively constant deionized water:
but are less than about 80 %, or greater than 120 %,
of the nominal values, then the analytical values are 294,0 mg/l CaCl2.2H2O
used in estimating the LC50. Where evidence is not 123,3 mg/l MgSO4.7H2O
available to show that the test concentrations
63,0 mg/l NaHCO3
remained at an acceptable level throughout the test
period, or where it is known (or suspected) that the 5,5 mg/l KCl
concentrations of the test chemical have declined Aerate the dilution water until the concentration of
significantly at any stage during the test, then, dissolved oxygen reaches at least 90 % of its air
irrespective of whether or not chemical analytical saturation value (ASV) and the pH is constant
data are available, the LC50 cannot be defined at 7,8 ± 0,2. If necessary, adjust the pH of the
using this test method. In these cases, the test is not solution by adding sodium hydroxide solution or
necessarily invalidated but it can only be stated that hydrochloric acid. The dilution water thus prepared
the LC50 of the substance is k x mg/l, the value, x, shall receive no further forced aeration before use in
being estimated from the nominal concentrations the tests.
used.
3.3 Stock solutions of test substances
3 Test organism and reagents A stock solution of the test substance should be
prepared by dissolving a known amount of test
The reagents shall be of recognized analytical grade. substance in a defined volume of dilution water,
The water used for the preparation of solutions shall deionized water or glass-distilled water. The stock
be glass-distilled water or deionized water of at least solution should be prepared at a frequency
equivalent purity. appropriate to the stability of the test substance. To
3.1 Test organism enable stock solutions to be prepared and to assist in
The test species shall be Brachydanio rerio their transfer to the test vessels, substances of low
Hamilton-Buchanan (Teleostei, Cyprinidae), aqueous solubility may be dissolved or dispersed by
commonly known as the zebra fish. Each test fish suitable means, including ultrasonic devices and
shall have a total length of 30 mm ± 5 mm, which, organic solvents of low toxicity to fish. If any such
in principle, corresponds to a mass of 0,3 g ± 0,1 g. organic solvent is used, its concentration in the test
They shall be selected from a population of a single solution shall not exceed 0,1 ml/l, or the volume
stock. This stock should have been acclimatized and, containing 0,1 g/l, whichever is the greater. Where a
in any case, maintained for at least 7 d prior to the solvent is used, two sets of controls, one containing
test in dilution water, continuously aerated using solvent at the maximum concentration used in any
bubbled air (see 3.2), under conditions of water test vessel and one without solvent or test
quality and illumination similar to those used in the substance, shall be included.
test. They shall be fed as normal up to the 24 h 3.4 Test solutions
period immediately preceding the test. Test solutions are prepared by adding appropriate
Test fish shall be free of overt disease or visible amounts of the stock solution of the test substance
malformation. They shall not receive treatment for to the dilution water to give the required
disease during the test or in the 2 weeks preceding concentrations. It is recommended that, when a
the, test. Subsequent to the test, fish remaining stock solution is prepared in distilled or deionized
alive should be suitably disposed of. water, no more than 100 ml of stock solution should
Environmental conditions for the maintenance and be added per 10 litres of dilution water.
breeding of zebra fish are given in Annex A.

2 © BSI 03-1999
 EN ISO 7346-2:1997

4 Apparatus Maintain the temperature of the water in the stock

tanks at 23 °C ± 1 °C (4.2).
All materials which may come into contact with any
liquid into which the fish are to be placed, or with 6.2 Limit test
which they may come into contact, shall be inert and Using the procedures described in this part of
should not absorb the test substance significantly. ISO 7346, a limit test may be performed at the limit
Usual laboratory equipment and the following. of aqueous solubility under the conditions of the test
4.1 Test vessels, of sufficient capacity (which may or at 100 mg/l, whichever is the lower, in order to
need to be greater than 10 litres), with a large area demonstrate that the 96 h – LC50 is greater than
of interface between the air and the test medium (of this concentration. If no fish die in the limit test, no
about 800 cm2 for 10 litres of medium) and equipped further testing is required.
with a securely fixed and close-fitting cover. The Perform the limit test using 10 fish, with the same
volume of the test vessels should be sufficient that a number in the control(s).
loading rate of 1 g of fish per litre of water should NOTE 1 Binominal theory dictates that, when 10 fish are used,
not be exceeded at any time during the test. with zero mortality there is a 99,9 % confidence that
the 96 h – LC50 is greater than the limit-test concentration. If
Before use, the test vessels shall be cleaned mortalities occur, a complete study (see 6.3 and 6.4) may need to
thoroughly, for example with a non-ionic detergent be considered. If sub-lethal effects are observed, these should be
(followed by acid and solvent washes for substances recorded.
expected to adsorb strongly to the vessel). 6.3 Preliminary test
4.2 Temperature control equipment, to regulate the Add at least 2,5 litres, preferably 5 litres, of
temperature of the test solutions and the water in standard dilution water (3.2) to each of six test
the stock tanks to 23°C ± 1 °C by a suitable method. vessels (4.1) and aerate if necessary to restore the
4.3 Dip-net, made of nylon or of another chemically concentration of dissolved oxygen to at least 90 % of
inert material, for the control vessels and another its air saturation value.
for all the test vessels (4.1). Prepare test solutions by adding appropriate
amounts of stock solution of the test substance (3.3)
5 Test environment to five of the vessels in order to obtain an adequate
geometric range of concentrations, for
The preparation and storage of solutions, the
example 1 000 mg/l; 100 mg/l; 10 mg/l; 1 mg/l
holding of fish, and all the manipulations and tests
shall be carried out in premises with an atmosphere and 0,1 mg/l. Nothing is added to the sixth vessel,
free from harmful concentrations of airborne which serves as a control. The solutions shall be
adjusted to and maintained at 23 °C ± 1 °C (4.2) and
contaminants.
shall not be forcibly aerated during the test.
Take care to avoid any unwanted disturbance that
Place three fish in each vessel.
may change the behaviour of the fish. Carry out all
tests under normal laboratory illumination with a After 24 h or 48 h, prepare new test solutions in new
daily photoperiod of 12 h to 16 h. test vessels and transfer the live fish to them
without delay. Repeat every 24 h or 48 h throughout
6 Procedure the preliminary test.
6.1 Condition of the fish At least twice a day, note the number of dead fish
and the dissolved oxygen concentration in each
Whenever there is a change of stock population, vessel. Remove the dead fish.
carry out a toxicity test using the method specified
If there are insufficient data for establishing the
in this part of ISO 7346 using a suitable reference
chemical [e.g. potassium dichromate (K2Cr2O7)]. range of concentrations required for the final test,
The results of such tests shall be in reasonable repeat this preliminary test with alternative ranges
of concentrations.
agreement with results obtained previously in the
same laboratory.
Test fish shall not have been used for any previous
testing procedure.

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EN ISO 7346-2:1997

6.4 Final test If the substance is shown to be stable over the period
Select at least five concentrations, forming an of exposure, (i.e. losses of less than 20 % of the initial
approximately geometric series, for measured concentration are expected or have been
example 8 mg/l; 4 mg/l; 2 mg/l; 1 mg/l and 0,5 mg/l, demonstrated) then, if possible, measure the
between, but including, the lowest concentration concentrations of the test substance in the test
killing all the fish in the preliminary test, and the vessels at least at the beginning and end of the first
highest non-lethal concentration in 96 h. This and final renewal periods. If the test substance is
selected series of concentrations shall provide the shown to be unstable over the period of exposure,
possibility of obtaining mortalities of between 0 % then if possible measure the concentration of the
and 100 % in at least two consecutive test substance in the test vessels at the beginning
concentrations of the geometric series used, which is and end of each renewal period throughout the
necessary for an estimation of the LC50 using the duration of the test.
probit method. Measure the dissolved oxygen concentration, the pH
In some instances, a narrower range of and temperature in each vessel at least at the
concentrations may be required to provide the beginning of the test and immediately before and
necessary data and in others a wider range may be after the renewal of the test solution.
needed. A suggested form which is suitable for recording the
Take at least six test vessels (4.1) and into each data is given in Annex B.
pour, for example, 10 litres of standard dilution
water (3.2). Nothing is added to one of these (the 7 Expression of results
control) but, to the remainder, add the different 7.1 Validity
amounts of stock solution (3.3) required to give the
The results shall be considered valid if the following
particular range of concentrations of test substance
requirements are fulfilled:
which has been selected for testing. If an organic
solvent has been used to dissolve a substance, a) the dissolved oxygen concentration in the test
prepare a second control with the standard dilution solutions during the test was at least 60 % ASV;
water containing sufficient of the organic solvent to b) the concentrations of the test substance were
give the maximum concentration at which this not known (or suspected) to have declined
solvent is present in any of the test solutions. When significantly throughout the test (but
the test solution (3.4) has been adjusted see clause 2);
to 23 °C ± 1 °C (4.2), place at least seven fish in each c) the mortality of the control fish did not
of the vessels, as follows. exceed 10 % or one per tank;
Select the fish at random from the stock and place d) d) the proportion of control fish showing
them at random in the test vessels, without delay, abnormal behaviour did not exceed 10 % or one
using a small mesh dip-net of soft inert per tank;
material (4.3). Discard any fish dropped or
e) the 24 h – LC50 of the reference chemical
otherwise mishandled during the transfer. In a
[e.g. potassium dichromate (K2Cr2O7)] for the
given test, add all the fish within a period of 30 min.
stock of fish was in reasonable agreement with
After 24 h or 48 h, prepare new test solutions in new results obtained previously in the same
test vessels and transfer the live fish to them laboratory.
without delay. The renewal of test solutions and
7.2 Estimation of LC50
transfer of fish shall be repeated every 24 h or 48 h
during the test. In order to avoid significant transfer Where a simple graphical estimation of the LC50 is
of test substances between test vessels via the considered adequate, this can be obtained by
dip-net (4.3), the transfer of fish should begin with plotting mortality (expressed as a percentage of test
the lowest concentration and proceed towards the fish in each test vessel) against concentration of test
highest concentration. substance. Using axes with linear scales, this will
The solutions shall not be forcibly aerated. Record produce a sigmoid relationship from which the LC50
the number of dead fish in each vessel at least daily can be derived by interpolating the concentration
over the period of the test. Remove each dead fish expected to cause 50 % mortality (see Figure 1).
from the vessel as soon as possible. Observations It is more appropriate to plot the data on graph
can be made more frequently, for example to enable paper having axes with logarithmic and probability
median periods of survival to be calculated for each scales. Data plotted in this way should produce a
concentration. linear relationship from which the LC50 can be
Note any abnormal behaviour of the fish. interpolated as above (see Figure 2).

4 © BSI 03-1999
 EN ISO 7346-2:1997

Where estimation of slope and 95 % confidence f) a tabulated list showing the nominal
limits of this and the LC50 are required, and it is concentrations tested (with chemical analytical
recommended that these statistics are frequently values, where available), and the total percentage
valuable in expressing results, the data can be mortalities in each 24 h, 48 h, 72 h and 96 h after
analysed graphically ([2] in Annex C). the start of the test;
Where computing facilities are available, probit g) the LC50 values and confidence limits, if
analysis ,can be applied ([1] in Annex C). available, at 24 h, 48 h, 72 h and 96 h, of the
If insufficient data are available to estimate the substance tested; reference should be made to the
LC50 at 24 h and 48 h and, if available, at 72 h method of calculation, and the method of
and 96 h, record the minimum concentration in chemical analysis, where applicable;
which 100 % mortality occurred and the maximum h) the slope of the concentration-response curve
concentration giving 0 % mortality (and its 95 % confidence limit if available);
at 24 h; 48 h; 72 h and 96 h. These concentrations i) a graphical illustration of the
will indicate the limits within which the LC50 concentration-response relationship;
probably lies.
j) any unusual reactions by the fish under the test
conditions and any visible external effects
produced by the test substance;
k) any deviation from the procedure specified in
this part of ISO 7346, and the reason for it.

Figure 1 — Graphical interpolation of LC50

(linear scales)

8 Test report
The test report shall include the following
information:
a) a reference to this part of ISO 7346;
b) the chemical identity and any additional
available information about the test substance
(e.g. water solubility, volatility, octanol/water
partition coefficient, degradation rate);
c) the method of preparing the dilution water,
stock solutions and test solutions;
d) all chemical, biological and physical data
pertaining to the test and not otherwise specified
in this part of ISO 7346, including details of the Figure 2 — Graphical interpolation of LC50
(logarithmic and probability scales)
acclimatization conditions of the test fish, and the
mass of fish, in grams per litre;
e) the data taken into account when assessing the
validity of the test:
1) concentration of dissolved oxygen;
2) mortality observed among control fish;
3) proportion of control fish showing abnormal
behaviour;
4) LC50 of the reference substance;

© BSI 03-1999 5
EN ISO 7346-2:1997

Annex A (informative) A.4 Conditioning

Environmental parameters for This period lasts for approximately 2 weeks. Males
maintenance and breeding of zebra and females are separated and fed on live food. This
fish (Brachydanio rerio consists of white worms (enchytraeids), Daphnia
Hamilton-Buchanan) and brine shrimps (Artemia). The density of
stocking during conditioning is kept below 30 fish in
A.1 General tanks of capacity 70 litres.
The species originates from the Coromandel coast of At the end of 2 weeks, the males possess a deep
India where it inhabits fast flowing streams. It is a golden sheen and the females are greatly distended
common aquarium fish, so that information about with ova.
procedures for its care and culture can be found in
A.5 Breeding stage
standard reference books on tropical fish culture. Its
biology has recently been reviewed by Laale [5]. The spawning tank can be set up as follows.
The fish rarely exceeds 45 mm in length. The body Fill an empty tank with fresh tap water aged
is cylindrical with 7 to 9 dark blue horizontal stripes at 27 °C for 48 h and place a plastic cage inside the
on silver. These stripes run into the caudal and anal tank under the lip, allowing the fish a swimming
fins. The back is olive green. Males are slimmer space of volume about 1 litre. Place six females in
than females and possess a golden sheen. Females the basket in the morning and feed with freeze-dried
are more silvery and the abdomen is distended, brine shrimps.
particularly prior to spawning. Add nine males to the basket in the evening and
A.2 Environmental parameters feed the fish once more with freeze-dried brine
shrimps before the lights are switched off.
The fish are capable of withstanding wide ranges of
temperature, pH and water hardness. Axelrod [4] Spawning is induced by the morning light and is
states a temperature range of 15,5 °C to 43,3 °C and completed after the lights have been switched on for
a pH of 6,6 to 7,2. Fish may be bred, reared and approximately 4 h. The eggs, which are
maintained in tap water with a total hardness as non-adhesive, fall through the mesh, out of reach of
high as 300 mg/l (as calcium carbonate) and a pH the adults.
of 7,7 to 8,2. The temperature is maintained When the females are exhausted of eggs, remove the
at 26 °C ± 1 °C and raised to 27 °C ± 1 °C to induce adults and leave the eggs to hatch.
spawning. A.6 Development of fry
A.3 Materials and methods The eggs hatch in 4 d to 5 d, and the fry or alevins
The fish may readily be spawned in glass tanks of adhere to the side of the tank and remain motionless
capacity about 70 litres. The fry are later for 24 h to 48 h. When the fry become
transferred to a tank of capacity 200 litres. free-swimming, feed them on suitable proprietary
Since the adult fish are avid egg eaters, a method of fish food of small particle size. At 3 weeks, the fry
protecting newly laid eggs and young fish is can be fed newly hatched brine shrimps and growth
necessary. One method, used successfully, is to then becomes more rapid. After 1 month, they can
confine the adult fish in mesh cages in the water so be transferred to a 200 litre tank and fed on a
that, as the female lays her eggs, these fall through mixture of live and proprietary foods. The fish are
the mesh to the bottom of the tank out of reach of the sexually mature at 3 months and attain a length
adults. of 3,5 cm. It should be noted that spontaneous
abnormalities in the developing larvae have been
The mesh cages are made of plastics netting
observed in certain strains ([9] in Annex C).
with 3 mm mesh, of dimensions
approximately 250 mm × 250 mm × 80 mm. They Further studies ([7] in Annex C) indicate that a
are clipped to the lips of the tanks so that the whole dietary factor is responsible for the deformities and
of the upper edge of the cage is above water with the that the zebra fish is especially susceptible to this
mesh dropping 60 mm into the water. An factor (other species breed normally when fed the
undergravel filter system should not be used to same proprietary fish food).
cleanse the water because it is likely to damage the
eggs. The tanks should be illuminated for 8 h per
day.

6 © BSI 03-1999
 EN ISO 7346-2:1997

Annex B (informative)
Suggested form for recording data

Laboratory Operator
Sample No. Date of start of test
Substance
Purity
Impurities
If a formulation is being tested, the identity of the components
Method of preparing the stock solution Stock solution concentration (mg/l)
Maximum concentration of solvent in test vessels (ml/l)
Method of chemical analysis
Control vessels
1 Dilution water only
Determinands Time from start of test (h)
0
Dissolved oxygen concentration (% ASV ) a

pH
Temperature (°C)
Number of dead fish

2 Dilution water and ml/l solvent

Determinands Time from start of test (h)

0
Dissolved oxygen concentration (% ASVa)
pH
Temperature (°C)
Number of dead fish
Test vessel No.
Initial (measured or calculated) concentration of mg/l
test substance

Determinands Time from start of test (h)

0
Test substance concentration
[mg/l (by analysis)]
Dissolved oxygen concentration (% ASVa)
pH
Temperature (°C)
Number of dead fish
a
Air saturation value.

© BSI 03-1999 7
EN ISO 7346-2:1997

Annex C (informative) [6] MERTENS, J. Year–round controlled mass

Bibliography reproduction of the zebra fish. Aquaculture 2 (1973),
pp. 245–249.
[1] FINNEY, D.J. Statistical Methods in Biological
Assay, Wycombe, United Kingdom, Griffin (1978). [7] NEWSOME, C.S. and PIRON, R.D. Aetiology of
skeletal deformities in the Zebra Danio fish
[2] LITCHFIELD, J.T. and WILCOXON, F. A simplified (Brachydanio rerio Hamilton-Buchanan). J. Fish
method for evaluating dose-effect experiment. Biol. 21 (1982), pp. 231–237.
J. Pharmacol. Exp. Ther. 96 (1949), pp. 99–113.
[8] NIIMI, A.J. and LAHAM, Q.N. Influence of
[3] STEPHAN, C.E. Methods for calculating an LC50. breeding time interval on egg number, mortality
Aquatic Toxicology and Hazard Evaluation. ASTM and hatching of the zebra fish (Brachydanio rerio).
(1977), ST, p. 634. Can. J. Zool. 52 (1974), pp. 515–517.
[4] AXELROD, H.P. Breeding Aquarium Fishes [9] PIRON, R.D. Spontaneous skeletal deformities in
Book 1. T.F.H. Publication, 1967. the zebra fish (Brachydanio rerio) bred for fish
[5] LAALE, H.W. The biology and use of zebra fish toxicity tests. J. Fish Biol. 13 (1978), pp. 79–84.
(Brachydanio rerio) in fisheries research. A
literature review. J. Fish Biol. 10 (2) (1977),
pp. 121–173.

8 © BSI 03-1999
blank
BS EN ISO
7346-2:1998
BS 6068-5.3: BSI — British Standards Institution
1998
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