ISO 7346-2:1984
(Main)Water quality — Determination of the acute lethal toxicity of substances to a freshwater fish (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)) — Part 2: Semi-static method
Water quality — Determination of the acute lethal toxicity of substances to a freshwater fish (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)) — Part 2: Semi-static method
Qualité de l'eau — Détermination de la toxicité aiguë létale de substances vis-à-vis d'un poisson d'eau douce (Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)) — Partie 2: Méthode semi-statique
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International Standard @ 734612
INTERNATIONAI ORGANIZATION FOR STANDARDIZATIONOMEXAYHAPO~HAA OPrAHMBAUMR no CTAHflAPTM3AUMMOORGANlSATlON INTERNATIONALE DE NORMALISATION
0 Water quality - Determination of the acute lethal toxicity
of substances to a freshwater fish [Brachydanio rerio
Hamilton-Buchanan (Teleostei, Cyprinidae)] -
Part 2: Semi-static method
Qualité de I’eau - Détermination de la toxicité aiguë létale de substances vis-à-vis d’un poisson d’eau douce /Brachydanio rerjo
Hamilton- Buchanan I Teleostei, Cyprinidaell - Partie 2: Méthode semi-statique
First edition - 1984-12-01
-
- UDC 574.63/.64 Ref. No. IS0 7346/2-1984 (E)
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Descriptors : water, quality, tests, determination, toxicity, toxic substances, fresh water, fishes.
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5 Price based on 9 pages
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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, govern-
e
mental and non-governmental, in liaison with ISO, also take part in the work.
Draft International Standards adopted by the technical committees are circulated to
the member bodies for approval before their acceptance as International Standards by
the IS0 Council. They are approved in accordance with IS0 procedures requiring at
least 75 % approval by the member bodies voting.
International Standard IS0 7346/2 was prepared by Technical Committee
ISO/TC 147, Water quality.
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@ International Organization for Standardization, 1984 0
Printed in Switzerland
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INTERNATIONAL STANDARD IS0 7346/2-1984 (E)
Water quality - Determination of the acute lethal toxicity
of substances to a freshwater fish [Brachydanio rerio
Ha mi Iton- B uc ha na n (Teleostei, Cyprinidae)] -
Part 2: Semi-static method
O Introduction those substances whose concentrations can be maintained
satisfactorily throughout the test by renewal of the solutions
every 24 h.
The three parts of IS0 7346 describe methods of determining
the acute lethal toxicity of substances to the zebra fish
To assist in the preparation and maintenance of concentrations
(Brachydanio rerio Hamilton-Buchanan), but it must be
of substances which may be lethal at concentrations close to
emphasized that the recommended use of the zebra fish does
that of their aqueous solubility, a small volume of solvent may
not preclude the use of other species. The methodologies
be used, as specified in the methods.
presented here may also be used for other species of
freshwater, marine of brackish water fish, with appropriate
modifications of, for example, dilution water quality and the
1 Scope and field of application
temperature conditions of the test.
This part of IS0 7346 specifies a semi-static method for the
determination of the acute lethal toxicity of substances soluble
Within the three parts of IS0 7346, a choice can be made
in water under specified conditions to a species of freshwater
between static, semi-static and flow-through methods. The
fish [Brachydanio rerio Hamilton-Buchanan (Teleostei,
static test, described in IS0 7346/1, in which the solution is not
Cyprinidae) - common name, zebra fish1 in water of a
renewed, has the advantage of requiring simple apparatus
specified quality.
although the substance in the test vessel may become depleted
during the course of the test and the general quality of the
The method is applicable for assigning, for each test
water may deteriorate. The flow-through method, described in
substance, broad categories of acute lethal toxicity to
IS0 7346/3, in which the test solution is replaced almost con-
Brachydanio rerio under the test conditions.
tinuously, overcomes such problems but requires the use of
more complex apparatus. In the semi-static procedure, describ-
The results are insufficient by themselves to define water qual-
IS0 7346/2, the test solutions are renewed daily, this
ed in
ity standards for environmental protection.
method being a compromise between the other two.
The method is also applicable when using certain other species
The flow-through method can be used for most types of of freshwater fish as the test organism.’)
substances, including those unstable in water, but the concen-
trations of the test substance are determined wherever poss-
The method may be adapted for use with other freshwater fish
ible. The static method is limited to the study of substances
and marine and brackish water fish with appropriate modifi-
whose tested concentrations remain relatively constant during
cation of the test conditions, particularly with respect to the
the test period. The semi-static method can be used for testing
quantity and quality of the dilution water and temperature.
The following species of freshwater fish can be used, in addition to Brachydanio rerio, without modification to this part of IS0 7346:
1)
- Cichlasoma nigrofasciatum (Teleostei, Cichlidae)
- Lepomis macrochirus (Teleostei, Centrarchidae)
- Oryzias latipes (Teleostei, Poeciliidae)
- Pimephales prornelas (Teleostei, Cyprinidae)
- Poecilia reticdata (Teleostei, Poeciliidae)
The results obtained from a test with one species cannot, however, be extrapolated to other species.
1
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IS0 7346/2-1984 (E)
2 Principle 3.2 Standard dilution water
Determination, under specified conditions, of the concentra-
The freshly prepared standard dilution water shall have a pH of
tions at which a substance is lethal to 50 % of a test population
7,8 f 0,2, and calcium hardness of approximately 250 mg/l,
of Brachydanio rerio after exposure periods of 24; 48; 72; and
expressed as calcium carbonate, and shall be prepared as
96 h to that substance in the ambient water. These median
follows.
lethal concentrations are designated the24 h LC50; 48 h LC50;
72 h LC50; and 96 h LC50.
Prepare the following solutions using distilled or deionized
water:
The test is carried out in two stages:
a) Calcium chloride solution
a) a preliminary test which gives an approximate indication
of the acute median lethal concentrations and serves to
determine the range of concentrations for the final test;
Dissolve 11,76 g of calcium chloride dihydrate (CaCI2.2H2O)
in water and dilute to 1 litre.
b) a final test, the results of which alone are recorded.
b) Magnesium sulfate solution
Where evidence is available to show that test concentrations
remain relatively constant (i.e. within about 20 % of the
Dissolve 4,93 g of magnesium sulfate heptahydrate
nominal values) throughout the test then either measured or
(MgS04.7H20) in water and dilute to 1 litre.
nominal concentrations may be used in the estimation of the
LC50. Where such analyses show that the concentrations pre-
c) Sodium hydrogen carbonate solution
sent remain relatively constant but are less than about 80 % of
the nominal values, then the analytical values shall be used in
estimating the LC50. Where evidence is not available to show
Dissolve 2,59 g of sodium hydrogen carbonate (NaHC0-J in
that the test concentrations remained at an acceptable level water and dilute to 1 litre.
throughout the test period or where it is known (or suspected)
that the concentrations of the test chemical have declined
d) Potassium chloride solution
significantly at any stage during the test then irrespective of
whether or not chemical analytical data are available the LC50
Dissolve 0,23 g of potassium chloride (KCI) in water and
cannot be defined, using this test method. In these cases the
dilute to 1 litre.
test is not necessarily invalidated but it can only be stated that
the LC50 of the substance is 6 x mg/l, the value, x, given
being estimated from the nominal concentrations used.
Mix 25 ml of each of these four solutions and dilute to 1 litre
with water.
3 Test organism and reagents
Aerate the dilution water until the concentration of dissolved
oxygen reaches its air saturation value (ASV) and the pH value
The reagents shall be of recognized analytical grade. The water is constant at 7,8 f 0,Z. If necessary, adjust the pH of the
used for the preparation of solutions shall be glass-distilled
solution by adding sodium hydroxide solution or hydrochloric
water or deionized water of at least equivalent purity.
acid. The dilution water thus prepared shall receive no further
forced aeration before use in the tests.
3.1 Test organism
3.3 Stock solutions of test substances
The test species shall be Brachydanio rerio Hamilton-Buchanan
(Teleostei, Cyprinidae), commonly known as the zebra fish.
Each test fish shall have a total length of 30 + 5 mm and a
A stock solution of the test substance should be prepared by
mass of 0,3 f 0,l g. They shall be selected from a population
dissolving a known amount of test substance in a defined
of a single stock. This stock should have been acclimated and,
volume of dilution water, deionized water or glass-distilled
in any case, maintained for at least 2 weeks prior to the test in
water. The stock solution should be prepared daily except
dilution water, continuously aerated (using bubbled air) (see
where it is known that the material is stable in solution, in
3.21, under conditions of water quality and illumination similar
which case sufficient solution for use over 2 days may be
to those used in the test. They shall be fed as normal up to the
prepared. To enable stock solutions to be prepared and to
24 h period immediately preceding the test.
assist in their transfer to the test vessels, substances of low
aqueous solubility may be dissolved or dispersed by suitable
Test fish shall be free of overt disease or visible malformation.
means, including ultrasonic devices and using organic solvents
They shall not receive treatment for disease during the test or in
of low toxicity to fish. If any such organic solvent is used, its
the 2 weeks preceding the test.
concentration in the test solution shall not exceed 0,l mV1, and
two sets of controls, one containing solvent at the maximum
Environmental conditions for the maintenance and breeding of
concentration used in any test vessel and one without solvent
zebra fish are given in annex A.
or test substance, shall be included.
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IS0 7346/2-1984 (E)
3.4 Test solutions 6.2 Preliminary test
Add at least 2,5 I, preferably 5 I, of standard dilution water (3.2)
Test solutions are prepared by adding appropriate amounts of
to each of six vessels and aerate if necessary to restore the con-
the stock solution of the test substance to the dilution water to
centration of dissolved oxygen to its air saturation value.
give the required concentrations. It is recommended that when
Prepare test solutions by adding appropriate amounts of stock
a stock solution is prepared in distilled or deionized water, no
solution of the test substance (3.3) to five of the vessels in
more than 100 ml of stock solution should be added per 10 I of
order to obtain an adequate range of concentrations, for
dilution water.
example 1 000: 100; 10; 1; and 0,l mg/l. Nothing is added to
the sixth vessel, which serves as a control. The solutions
should be adjusted to and maintained at 23 f 1 OC.
4 Apparatus
All materials which may come into contact with any liquid into Place five fish in each vessel.
which the fish are to be placed, or with which they may come
At least twice a day for a suitable period, note the numbers of
into contact, shall be inert and should not absorb the test
substance significantly. dead fish and the dissolved oxygen concentration in each
vessel. Remove dead fish.
of nylon
Usual laboratory equipment [including a dip-net, made
If there are insufficient data for establishing the range of con-
or of another chemically inert material, for the control vessels
centrations required for the final test, repeat this preliminary
and another for all the test vessels (4.111, and
test with alternative ranges of concentrations.
4.1 Test vessels
6.3 Final test
Test vessels shall have sufficient capacity (which may need to be
Select at least five concentrations, forming an approximately
greater than 10 I) with a large area of interface between the air
geometric series, for example 8; 4; 2; 1 ; and 0,5 mg/l, be-
and the test medium (of about 800 cm2for 10 I of medium) and
tween, but including, the lowest concentration killing ail the
shall be equipped with a securely fixed and close-fitting cover.
fish in the preliminary test, and the highest non-lethal concen-
tration in 48 h. This selected series of concentration should
Before use, new test vessels shall be carefully washed and then
provide the possibility of obtaining mortalities of between 20
rinsed successively with water and the dilution water. At the
80 % in at least three consecutive concentrations of the
and
end of the test, the vessels shall be emptied, cleaned by
geometric series used, for estimation of the LC 50.
appropriate means, rinsed with water to remove all traces of
the test substance and cleaning aid, and dried.
In some instances, a narrower range of concentrations may be
required to provide the necessary data and for others a wider
Test vessels shall be rinsed with dilution water just before use.
range may be needed.
4.2 Temperature control equipment
Take at least six test vessel
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