9637

• UCREFRP Catalog Number: 9637
• Author: American Society For Testing And Materials.
• Title: Standard Practice For Conducting Acute Toxicity Tests With Fishes, Microinvertebrates, And Amphibians.
• USFW Year: 1980.
• USFW - Doc Type: Philadelphia, PA.
• Copyright Material: YES

if they appear to be diseased or otherwise
stressed or if more than 3 % die during the 48
h immediately preceding the test. If the orga-
nisms do not meet these criteria, they must be
either discarded or treated, held an additional
10 days, and reacclimated if necessary.
10.8.4 Organisms used in a test, including
those used in a control treatment, must not be
used in a later test.
10.8.5 Reference toxicants may be useful for
assessing the quality of test organisms. Many
compounds have been used or evaluated as
reference toxicants (20), but none has been
proven to be a reliable indicator of the overall
quality of any species or test results. Antimycin
has been found useful in detecting stressed
freshwater fish in some situations (21). A ref-
erence toxicant is more likely to be useful when
used in conjunction with a series of tests on
toxicants that all probably have the same mode
of action as the reference toxicant.

11. Procedure

11.1 Experimental Design:
11.1.1 Acute toxicity tests usually consist of
one or more control treatments (11.1.2) and a
geometric series of at least five toxicant concen-
trations. Five toxicant concentrations will often
provide LC50 or EC50 values for several du-
rations and is a reasonable compromise be-
tween the cost of the test and the risk of all the
concentrations being either too high or too low
(12.2). The concentration of toxicant in each
treatment, except for the highest concentration
and the control(s), should be at least 60 % of
the next higher one, so that the test will usually
provide reasonably useful information about
the LC50 or EC50. If the toxicity ofthe toxicant
to the test organisms is fairly well-known, use
of fewer than six treatments or a dilution factor
higher than 60 %, or both, may be desirable.
However, as the total range of the toxicant
concentrations is decreased, the risk of all the
concentrations being too high or too low is
increased with little reduction in cost.
11.1.1.1 If it is only necessary to determine
whether an LC50 or EC50 is above or below a
specified concentration, only a control and one
concentration are necessary, although at least
30 organisms should be used per treatment.
Two additional concentrations at about one
half and two times the specified concentration

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with fewer test organisms may be desired to
increase confidence in the results.
11.1.1.2 If an LC or EC value near the ex-
tremes of toxicity, such as an LC5 or an LC95,
is to be calculated, at least one treatment must
have killed or affected a percentage of test
organisms, other than 0 % and 100 %, near the
percentage for which the LC or EC is to be
calculated. This requirement might be met in
a test to determine an LC50 or EC50, but
special tests with appropriate toxicant concen-
trations and more test organisms per treatment
will often be necessary. Other ways of provid-
ing information concerning extremes oftoxicity
are to report the highest concentration of toxi-
cant that actually killed or affected no greater
a percentage of the test organisms than did the
control treatment or to report the lowest con-
centration of toxicant that actually killed or
affected all test organisms exposed to it. These
alternatives are normally more reliable than
reporting a calculated result such as an LC5 or
an LC95 unless several percent kills or effects
are obtained close to 5 % or 95 %.
11.1.2 Every test requires a control treat-
ment consisting of the same dilution water,
conditions, procedures, and organisms as are
used in the other treatments, except that none
of the toxicant being tested is added to the
dilution water. If any solvent other than water
(9.1) is present in any of the test chambers, a
solvent control is also required. The solvent
control must be identical to the regular control,
except that the highest amount of solvent pres-
ent in any other treatment is added to this
treatment. If the toxicant is a mixture, formu-
lation, or commerical product, none of the
ingredients is considered a solvent unless an
extra amount is used to prepare the stock so-
lution.
11.1.3 In static tests at least 10 organisms,
and in flow-through tests at least 20 organisms,
must be exposed to each treatment, but they
may be divided between two or more test cham-
bers. If each toxicant concentration is more
than 60 % of the next higher one, proportion-
ately fewer organisms per toxicant concentra-
tion, but not the control treatment, may be
used. The use of more organisms and replicate
test chambers for each treatment is desirable.
Replicate test chambers must not have water
connections between them. Treatments should.

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