Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)) - Part 3: Flow-through method (ISO 7346-3:1996)

Specifies a flow-through method for the determination of the acute lethal toxicity of stable, non-volatile, single substances, soluble in water under specified conditions, to a freshwater fish (Brachydanio rerio - zebra fish). Replaces the first edition.

Wasserbeschaffenheit - Bestimmung der akuten letalen Toxizität von Substanzen gegenüber einem Süßwasserfisch (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) - Teil 3: Durchflußverfahren (ISO 7346-3:1996)

Dieser Teil der ISO 7346 legt ein Durchflußverfahren  zur Bestimmung der akuten letalen Konzentration von stabilen, nicht-flüchtigen, unter gegebenen Bedingungen wasserlöslichen Einzelsubstanzen gegenüber einem Süßwasserfisch (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)) - Trivialnahme Zebrabärbling) in Wasser einer festgelegten Beschaffenheit fest.

Qualité de l'eau - Détermination de la toxicité aiguë létale de subtances vis-à-vis d'un poisson d'eau douce (Brachydanio rerio Hamilton-Buchanan (Téléostei, Cyprinidae)( - Partie 3: Méthode avec renouvellement continu (ISO 7346-3:1996)

La présente partie de l'ISO 7346 prescrit une méthode avec renouvellement continu pour la détermination de la toxicité aiguë létale de substances simples, stables, non volatiles, solubles dans l'eau dans les conditions prescrites, vis-à-vis d'un poisson d'eau douce [Brachydanio rerio Hamilton-Buchanan (Téléostei, Cyprinidae) -- nom commun, poisson-zèbre], dans une eau de qualité donnée. La méthode est applicable pour chaque substance à expérimenter, pour donner une idée globale de la toxicité aiguë létale vis-à-vis du Brachydanio rerio, dans les conditions de l'essai. Les résultats sont en eux-mêmes insuffisants pour définir des normes de qualité de l'eau en vue de la protection de l'environnement. La méthode est également applicable à certaines autres espèces de poissons d'eau douce comme organismes pour essai1). La méthode peut être adaptée pour d'autres poissons d'eau douce, marine ou saumâtre avec des modifications appropriées des conditions d'essai, concernant notamment la quantité et la qualité de l'eau de dilution et la température.

Kakovost vode - Ugotavljanje akutne smrtne strupenosti snovi s sladkovodnimi ribami (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)) - 3. del: Pretočna metoda (ISO 7346-3:1996)

General Information

Status
Published
Publication Date
18-Nov-1997
Technical Committee
Drafting Committee
Current Stage
9093 - Decision to confirm - Review Enquiry
Due Date
21-May-2008
Completion Date
21-May-2008

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SLOVENSKI STANDARD
SIST EN ISO 7346-3:1998
01-maj-1998
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SIST ISO 7346-3:1997
.DNRYRVWYRGH8JRWDYOMDQMHDNXWQHVPUWQHVWUXSHQRVWLVQRYLVVODGNRYRGQLPL
ULEDPL %UDFK\GDQLRUHULR+DPLOWRQ%XFKDQDQ 7HOHRVWHL&\SULQLGDH GHO
3UHWRþQDPHWRGD ,62

Water quality - Determination of the acute lethal toxicity of substances to a freshwater

fish (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)) - Part 3: Flow-

through method (ISO 7346-3:1996)
Wasserbeschaffenheit - Bestimmung der akuten letalen Toxizität von Substanzen
gegenüber einem Süßwasserfisch (Brachydanio rerio Hamilton-Buchanan (Teleostei,
Cyprinidae) - Teil 3: Durchflußverfahren (ISO 7346-3:1996)

Qualité de l'eau - Détermination de la toxicité aiguë létale de subtances vis-a-vis d'un

poisson d'eau douce (Brachydanio rerio Hamilton-Buchanan (Téléostei, Cyprinidae)( -

Partie 3: Méthode avec renouvellement continu (ISO 7346-3:1996)
Ta slovenski standard je istoveten z: EN ISO 7346-3:1997
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 7346-3:1998 en

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

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SIST EN ISO 7346-3:1998
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SIST EN ISO 7346-3:1998
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SIST EN ISO 7346-3:1998
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SIST EN ISO 7346-3:1998
INTERNATIONAL
Is0
STANDARD
7346-3
Second edition
1996-06-I 5
Water quality - Determination of the
acute lethal toxicity of substances to a
freshwater fish [ Brachydanio rerio
Hamilton-Buchanan (Teleostei,
Cyprinidae)] -
Part 3:
Flow-through method
Qua/it@ de I’eau - Dktermination de la toxicit aigue ktale de substances
vis-&vis d ’un Poisson d ’eau deuce [Brachydanio rerio Hamilton-Buchanan
(Tkkostei, Cyprinidae)] -
Partie 3: M&hode avec renouvellement continu
Reference number
IS0 7346-3:1996(E)
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SIST EN ISO 7346-3:1998
IS0 7346-3: 1996(E)
Foreword
IS0 (the International Organization for Standardization) is a worldwide
federation of national standards bodies (IS0 member bodies). The work
of preparing International Standards is normally carried out through IS0
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. IS0
collaborates closely with the International Electrotechnical Commission
(I EC) 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 IS0 7346-3 was prepared by Technical Committee ~
ISOnC 147, Water quality, Subcommittee SC 5, Biological methods.
This second edition cancels and replaces the first edition
(IS0 7346-3:1984), which has been technically revised.
IS0 7346 consists of the following parts, under the general title VVater
Determination of the acute lethal toxicity of substances to a
quality -
freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei,
Cyprinidae)]:
- Part I: Static method
- Part 2: Semi-static method
- Part 3: Flow-through method
Annexes A, B and C of this part of IS0 7346 are for information only.
0 IS0 1996

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 the publisher.
International Organization for Standardization
Case Postale 56 l CH-1211 Geneve 20 l Switzerland
Printed in Switzerland
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SIST EN ISO 7346-3:1998
0 IS0
IS0 7346-3: 1996(E)
Introduction
The three parts of IS0 7346 describe methods of determining the acute
lethal toxicity of substances to the zebra fish (Brachydanio rerio
Hamilton-Buchanan) but it must be emphasized that the recommended
use of the zebra fish does not preclude the use of other species. The
methodologies presented here may also be used for other species of
freshwater, marine or brackish water fish, with appropriate modifications
of, for example, dilution water quality and the temperature conditions of
the test.
Within the three parts of IS0 7346, a choice can be made between static,
semi-static and flow-through methods. The static test, described in
IS0 7346-1, in which the solution is not renewed, has the advantage of
requiring simple apparatus, although the substances in the test vessel may
become depleted during the course of the test and the general quality of
the water may deteriorate. The flow-through method, described in this
part of IS0 7346, in which the test solution is replenished continuously,
overcomes such problems but requires the use of more complex appar-
atus. In the semi-static procedure, described in IS0 7346-2, the test sol-
utions are renewed every 24 h or 48 h, this method being a compromise
between the other two.
The flow-through method can be used for most types of substances, in-
cluding those unstable in water, but the concentrations of the test sub-
stance are determined wherever possible. The static method is limited to
the study of substances whose tested concentrations remain relatively
constant during the test period. The semi-static method can be used for
testing those substances whose concentrations can be maintained satis-
factorily throughout the test by renewal of the solutions every 24 h or
48 h. Special arrangements may be necessary for substances which are
highly volatile.
To assist in the preparation and maintenance of concentrations of sub-
stances which may be lethal at concentrations close to that of their
aqueous solu.bility, a small volume of solvent may be used, as specified in
the methods.
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SIST EN ISO 7346-3:1998
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SIST EN ISO 7346-3:1998
INTERNATIONAL STANDARD 0 IS0 IS0 7346-3: 1996(E)
Water quality - Determination of the acute lethal
toxicity of substances to a freshwater fish [5~~hydanio
rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] -
Part 3:
Flow-through method
with appropriate modification of the test conditions,
1 Scope
particularly with respect to the quantity and quality of
the dilution water and the temperature.
This part of IS0 7346 specifies a flow-through method
for the determination of the acute lethal toxicity of
stable, non-volatile, single substances, soluble in
2 Principle
water under specified conditions, to a freshwater fish
[ Brachydanio rerio Hamilton-Buchanan (Teleostei,
Determination, under specified conditions, of the
Cyprinidae) - common name, zebra fish] in water of
concentrations at which a substance is lethal to
a specified quality.
50 % of a test population of Brachydanio rerio after
exposure periods of 24 h, 48 h, 72 h and 96 h to that
The method is applicable for assigning, for each test
substance in the ambient water. These median lethal
substance, broad categories of acute lethal toxicity to
concentrations are designated the 24 h - LC50,
Brachydanio rerio under the test conditions.
48 h - LC50, 72 h - LC50 and 96 h - LC50.
The results are insufficient by themselves to define
The test is carried out in two stages:
water quality standards for environmental protection.
a) a preliminary test which gives an approximate in-
The method is also applicable when using certain
dication of the acute median lethal concentrations
other species of freshwater fish as the test
and serves to determine the range of concen-
organism?
trations for the final test;
b) a final test, the results of which alone are re-
The method may be adapted for use with other
ported.
freshwater fish and marine and brackish water fish

1) The following species of freshwater fish can be used, in addition to Brachydanio rerio, without modification to this part of

IS0 7346.
- Lepomis macrochirus (Teleostei, Centrarchidae)
- Oryzias latipes (Teleostei, Poeciliidae)
- Pimephales promelas (Teleostei, Cyprinidae)
- Poecilia reticulata (Teleostei, Poeciliidae)
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SIST EN ISO 7346-3:1998
IS0 7346=3:1996(E) 0 IS0
Where evidence is available to show that test con-
3.2 Standard dilution water
centrations remain relatively constant (i.e. within

about 20 % of the nominal values) throughout the The freshly prepared standard dilution water shall

test, then either measured or nominal concentrations
have a pH of 7,8 + 0,2, and a calcium hardness of
are used in the estimation of the LC50. Where such
approximately 250 mg/l, expressed as calcium
analyses show that the concentrations present remain
carbonate, and shall contain the following concen-
relatively constant but are less than about 80 %, or
trations of salts dissolved in distilled or deionized
greater than 120 %, of the nominal values, then the
water:
analytical values are used in estimating the LC50.
294,0 mg/ I CaCI,.2H,O
Where evidence is not available to show that the test
concentrations remained at an acceptable level
123,3 mg/ I MgSO,.7H,O
throughout the test period, or where it is known (or
suspected) that the concentrations of the test chemi-
NaHCO,
63,0 mg/l
cal have declined significantly at any stage during the
test, then, irrespective of whether or not chemical
5,5 mg/l KCI
analytical data are available, the LC50 cannot be de-
fined using this test method. In these cases, the test
Aerate the dilution water until the concentration of
is not necessarily invalidated but it can only be stated
dissolved oxygen reaches at least 90 % of its air
that the LC50 of the substance is < x mg/l, the value,
saturation value (ASV) and the pH is constant at
X, being estimated from the nominal concentrations
7,8 + 0,2. If necessary, adjust the pH of the solution
used.
by adding sodium hydroxide solution or hydrochloric
acid. The dilution water thus prepared shall receive
no further forced aeration before use in the tests.
3 Test organism and reagents
3.3 Stock solutions of test substances
The reagents shall be of recognized analytical grade.
The water used for the preparation of solutions shall
A stock solution of the test substance should be pre-
be glass-distilled water or deionized water of at least
pared by dissolving a known amount of test sub-
equivalent purity.
stance in a defined volume of dilution water,
deionized water or glass-distilled water. The stock
solution should be prepared at a frequency appropri-
ate to the stability of the test substance. To enable
3.1 Test organism
stock solutions to be prepared and to assist in their
transfer to the test vessels, substances of low
The test species shall be Brachydanio rerio Hamilton-
aqueous solubility may be dissolved or dispersed by
Buchanan (Teleostei, Cyprinidae), commonly known
suitable means, including ultrasonic devices and or-
as the zebra fish. Each test fish shall have a total
ganic solvents of low toxicity to fish. If any such or-
length of 30 mm + 5 mm, which, in principle, corre-
ganic solvent is used, its concentration in the test
sponds to a mass of 0,3 g + 0,l g. They shall be
solution shall not exceed 0,l ml/l, or the volume con-
selected from a population cf a single stock. This
taining 0,l g/l, whichever is the greater. Where a sol-
stock should have been acclimatized and, in any case,
vent is used, two sets of controls, one containing
maintained for at least 7 d prior to the test in dilution
solvent at the maximum concentration used in any
water, continuously aerated using bubbled air (see
test vessel and one without solvent or test substance,
3.2), under conditions of water quality and illumination
shall be included.
similar to those used in the test. They shall be fed as
normal up to the 24 h period immediately preceding
the test.
3.4 Test solutions
Test fish shall be free of overt disease or visible
Test solutions are prepared by adding appropriate
malformation. They shall not receive treatment for
amounts of the stock solution of the test substance
disease during the test or in the 2 weeks preceding
to the dilution water to give the required concen-
the test. Subsequent to the test, fish remaining alive
trations. It is recommended that, when a stock sol-
should be suitably disposed of.
ution is prepared in distilled or deionized water, no
maintena nce and more than JO0 ml of stock solution should be added
Enviro nmental conditions for the
per 10 litres of dilution water.
breedi ng of ze b ra fish are given in annex A.
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SIST EN ISO 7346-3:1998
0 IS0 IS0 734693:1996( E)
4 Apparatus 6 Procedure
All materials which may come into contact with any
6.1 Condition of the fish
liquid into which the fish are to be placed, or with
which they may come into contact, shall be inert and
Whenever there is a change of stock population, carry
should not absorb the test substance significantly.
out a toxicity test using the method specified in this
part of IS0 7346 using a suitable reference chemical
Usual laboratory equipment and the following.
[e.g. potassium dichromate (K,Cr,O,)]. The results of
such tests shall be in reasonable agreement with re-
sults obtained previously in the same laboratory.
4.1 Test vessels, which may be of different forms,
suitable for the properties of the test material and the
Test fish shall not have been used for any previous
need to achieve the validity criteria [especially
testing procedure.
7.1 b)]. For volatile materials, an enclosed system may
Maintain the temperature of the water in the stock
be required. When non-volatile chemicals are being
tanks at 23 “C + 1 “C (4.2).
tested, the vessels may be open topped. The volume
of the test vessels should be sufficient that a loading
rate of I,5 g of fish per litre of water should not be
6.2 Limit test
exceeded at any time during the test.
Using the procedures described in this part of
Before use, the test vessels shall be cleaned
IS0 7346, a limit test may be performed at the limit
thoroughly, for example with a non-ionic detergent
of aqueous solubility under the conditions of the test
(followed by acid and solvent washes for substances
or at 100 mg/l, whichever is the lower, in order to
expected to adsorb strongly to the vessel).
demonstrate that the 96 h - LC50 is greater than this
concentration. If no fish die in the limit test, no further
testing is required.
4.2 Temperature control equipment, to regulate

the temperature of the test solutions and the water Perform the limit test using 10 fish, with the same

in the stock tanks to 23 “C + 1 “C by a suitable number in the control(s).
method.
NOTE 1 Binominal theory dictates that, when 10 fish are
used, with zero mortality there is a 99,9 % confidence that
the 96 h - LC50 is greater than the limit-test concentration.
4.3 Solution replacement equipment, dosing and
If mortalities occur, a complete study (see 6.3 and 6.4) may
mixing apparatus capable of maintaining the required
need to be considered. If sub-lethal effects are observed,
concentrations of the test substance in the test flasks
these should be recorded.
to within 20 %, and set to renew the test solutions in
the flasks at a rate which is sufficient to prevent the
6.3 Preliminary test
concentration of oxygen in the vessels from falling
below 60 % ASV.
6.3.1 If possible, use the flow-through method for
the preliminary test, adopting the same range of con-
centrations, number of fish per test solution concen-
4.4 Dip-net, made of nylon or of another chemically
tration and method of observation of the fish as
inert material, for the control vessels and another for
described in 6.3.2 fo
...

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