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

be randomized; if replicate test chambers are
used for each treatment, random assignment of
one test chamber for each treatment in a row,
followed by random assignment of a second
test chamber for each treatment in another or
an extension of the same row, is more desirable
than total randomization. A representative
sample of the test organisms should be impar-
tially distributed to the test chambers by adding
to each test chamber no more than 20 % of the
number of test organisms to be placed in each
test chamber and repeating the process until
each test chamber contains the desired number
of test organisms. Alternatively, the organisms
can be assigned either by random assignment
of one organism to each test chamber, random
assignment of a second organism to each test
chamber, etc., or by total randomization. It is
often convenient to assign organisms to other
containers, and then add them to the test cham-
bers all at once.
11.1.4 It is desirable to repeat the test at a
later time to obtain information on the repro-
ducibility of the result.
11.2 Dissolved Oxygen:
11.2.1 For static tests the dissolved oxygen
concentration in each test chamber should be
between 60 % and 100 % saturation (12) during
the first 48 h of the test and should be between
40 % and 100 % saturation after 48 h. For flow-
through tests, the dissolved oxygen concentra-
tion in each test chamber should be between
60 % and 100 % saturation at all times during
the test.
11.2.2 Test solutions may be gently aerated
during static tests if the concentration of toxi-
cant in the aerated test chamber at the end of
the test is not more than 20 % lower than that
in a comparable unaerated test chamber. Test
solutions may be gently aerated during flow-
through tests if toxicant concentrations are
measured according to 11.9.2.2. Aeration that
is too vigorous can stress organisms. However,
since aeration readily occurs at the surface,
efficient aeration can be achieved with mini-
mum turbulence by using an air lift to transfer
solution from the bottom to the surface. The
aeration must be the same in all test chambers,
including the controls, at all times during the
test. When aeration is used, a simultaneous test
without aeration should be conducted to deter-
mine if aeration affects the results of the test.

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11.3 Temperature-The test temperature
should be selected from the series 7, 12, 17, 22,
27, and 320C. This series was selected because
it provides temperatures that better suit more
organisms and are more convenient for use by
investigators than any other series. The actual
test temperature must not deviate from the
selected test temperature by more than loe at
any time during the test. The temperatures
listed in Table 6 should be used as the selected
test temperatures for the species listed therein
whenever possible. Other temperatures from
the series may be used for studying the effect
of temperature on the results of toxicity tests.
Some life stages of some aquatic invertebrates
have rather narrow temperature requirements
and so the test temperature should be within
soe of the temperature ofthe water from which
they were obtained. They should be exposed at
the temperature listed in Table 6 if it is within
this range; otherwise, they should be exposed
at the temperature from the series 7, 12, 17,22,
27, and 320e that is closest to the listed tem-
perature and is within soe of the temperature
of the water from which they were obtained.
11.4 Loading:
11.4.1 The grams of organism (wet weight,
blotted dry) per litre of solution in the test
chambers must not be so high that it affects the
results of the test. Therefore, the loading must
be limited to ensure that (a) the concentrations
of dissolved oxygen and toxicant are not de-
creased below acceptable levels, (b) the concen-
tration of metabolic products does not increase
above acceptable levels, and (c) the organisms
are not stressed because of crowding. In the
shell deposition test, for which the flow-through
technique must be used, the maximum loading
depends mostly on the amount of food avail-
able in the dilution water and the flow rate.
11.4.2 For static tests with a species listed in
Table 6, the loading must not exceed 0.8 g/L
at or below the lower of (1) 170e and (2) the
temperature(s) listed for the species in Table 6
and must not exceed 0.5 g/L at higher temper-
atures. A lower loading or aeration (11.2.2)
must be used if the concentration of dissolved
oxygen falls below 60 % saturation during the
first 48 h of the test or below 40 % saturation
thereafter in any chamber containing live test
organisms.
11.4.3 For flow-through tests with a species

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