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

listed in Table 6, loading in the test chambers
must not exceed 1 g/L of solution passing
through the chamber in 24 h and must not
exceed 10 g/L of solution in the chamber at
any time at or below the lower of (1) l70C and
(2) the temperature(s) listed in Table 6 and
must be lower if aggression occurs; at higher
temperatures, loading must not exceed 0.5 g/L.
day or 5 g/L. A lower loading or faster flow
rate or both must be used if necessary to meet
the following three criteria at all times during
the test in each chamber containing live test
organisms: (a) the concentration of dissolved
oxygen must not fall below 60 % saturation (see
also 11.2.2); (b) the concentration of un-ionized
ammonia should not exceed 20 J.lg/L (15); and
(c) the concentration of toxicant must not be
lowered more than 20 % because of uptake by
the test organisms.
11.4.4 Comparable loadings should be used
for other species.
11.5 Beginning the Test:
11.5.1 Static tests are begun either by (a)
placing the toxicant in the test chambers 18 to
24 h after the test organisms are added or (b)
placing the test organisms in the chambers
within 30 min after the toxicant is added to the
dilution water. Alternative (a) allows the or-
ganisms to partially acclimate to the chambers
and precludes loss of toxicant due to hydrolysis,
sorption, or evaporation before addition of or-
ganisms. In alternative (a) dilution water con-
taining organisms may be gently aerated in the
chambers, but aeration must be stopped before
addition of toxicant, except as in 11.2.2. Alter-
native (b) conserves dissolved oxygen and pre-
vents exposure of the test organism to the tox-
icant before it is evenly dispersed; this alterna-
tive must be used when tests are conducted on
aged solutions of toxicant in dilution water and
when tests are conducted with daphnids or
midge larvae. A gentle swirl with a glass rod is
usually sufficient to disperse the toxicant.
11.5.2 Flow-through tests are begun either
by (a) placing test organisms in test chambers
after test solutions have been flowing through
the chambers long enough so that toxicant
concentrations have become constant or (b)
activating the toxicant-metering device in the
metering system several days after organisms
were placed in test chambers that had dilution
water flowing through them. Alternative (a)

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allows the investigator to study the properties
of the toxicant (11.9.2.2) and the operation of
the metering system immediately before the
beginning of the test, whereas alternative (b)
allows the organisms to partially acclimate to
the chambers before the beginning of the test.
The initial effects on the organisms using alter-
native (b) will depend on flow rate, mixing,
and the ability of the organism to adjust to the
changing conditions. Alternative (b) should not
be used for tests lasting less than 48 h, unless
the flow rate is above fifteen volume additions
per 24 h.
11.6 Feeding-Organisms should not be fed
while in test chambers. If cannibalistic orga-
nisms cannot be physically restrained or sepa-
rated, however, minimal feeding is necessary.
Organisms should not be fed during acute tox-
icity tests or for a time before them when
possible (10.7.3), because fecal matter and
uneaten food will decrease the dissolved oxygen
concentration and alter the biological activity
of some toxicants. These problems are most
severe with the static technique, but can also
be important with the flow-through technique.
11.7 Duration- A test begins when the test
organisms are first exposed to toxicant. Daph-
nids and midge larvae should be exposed to
toxicant for 48 h. All other organisms should
be exposed for 96 h in static tests and for at
least 96 h in flow-through tests. When flow-
through tests are conducted with organisms
over 0.5 g each, the shape of the toxicity curve,
that is, LC50 or EC50 versus time, should be
determined for at least 8 days to determine
whether additional organisms are affected or
killed after 96 h.
11.8 Biological Data:
11.8.1 Death is the adverse effect most often
used in acute toxicity tests with aquatic orga-
nisms. The criteria for death are usually lack of
movement, especially the absence of respiratory
movements in fish and shrimp, and lack of
reaction to gentle prodding. If polychaetes
break into two parts, the anterior portion
should be checked for death. Because death is
not easily determined for some invertebrates,
an EC50 is usually determined rather than an
LC50. For daphnids and midge larvae, the
EC50 is based on immobilization that is defmed
as the lack of movement except for minor
activity of appendages. For crabs and crayfish

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