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<br />the EC50 is based on immobilization that is
<br />defined as lack of movement and lack of re-
<br />sponse to gentle prodding. Because some juve-
<br />nile and adult bivalve molluscs can close their
<br />valves for extended periods of time and thus
<br />prevent exposure to the toxicant, acute lethality
<br />tests should not be conducted with them. An
<br />EC50 based on shell deposition can be deter-
<br />mined with bivalve molluscs, especially oysters,
<br />under conditions that promote rapid growth
<br />(22). An EC50 based on abnormal development
<br />and an LC50 can be determined with bivalve
<br />mollusc larvae, but special procedures must be
<br />used (23). I[ loss of equilibrium, defmed as the
<br />inability to make coordinated movement and
<br />maintain a normal upright position, or immo-
<br />bilization occurs in a test, an EC50 based on
<br />the effect should be calculated if enough orga-
<br />nisms are affected. Other effects can be used to
<br />determine an EC50, but the effect and its defi-
<br />nition must always be reported. General obser-
<br />vations on such things as erratic swimming, loss
<br />of reflex, excitability, discoloration, changes in
<br />behavior, excessive mucus production, hyper-
<br />ventilation, opaque eyes, curved spine, hemor-
<br />rhaging, molting, cessation of burrowing by
<br />shrimp and crabs, and cannibalism should be
<br />reported.
<br />11.8.2 Procedures used to determine
<br />whether test organisms are dead, immobilized,
<br />or otherwise affected, and to remove dead or-
<br />ganisms must not stress live test organisms.
<br />Movement of test chambers and prodding must
<br />be done very gently. Fish exposed to some
<br />organophosphorus compounds seem to be very
<br />sensitive to lights being turned on.
<br />11.8.3 The number of dead or affected or-
<br />ganisms in each test chamber must be counted
<br />every 24 h after the beginning of the test;
<br />burrowing organisms, however, do not have to
<br />be counted until the end of the test. More
<br />frequent observations are desirable, especially
<br />near the beginning of the test. Especially for
<br />flow-through tests, counts should be conducted
<br />at enough appropriate times during the test to
<br />defme the shape of the toxicity curve (LC50 or
<br />EC50 versus time). A suggested schedule is to
<br />count the number of dead or affected organisms
<br />in each chamber 3, 6, 12, and 24 h after the
<br />beginning of the test and twice a day thereafter
<br />to the end of the test. Obtaining data that
<br />defme the shape of the toxicity curve is more
<br />important than obtaining data at prespecified
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<br />times other than every 24 h.
<br />11.8.4 Dead organisms must be removed as
<br />soon as they are observed, or once every 24 h,
<br />in static tests and at least once every 24 h in
<br />flow-through tests except when loading is less
<br />than one tenth the maximum specified in 11.4.
<br />11.8.5 The weights (wet weight, blotted dry)
<br />and lengths (standard lengths offish) of the test
<br />organisms should be determined by weighing,
<br />measuring, and discarding representative or-
<br />ganisms before the test or the control organisms
<br />after the test.
<br />11.9 Chemical and Physical Data:
<br />11.9.1 Static Tests-I[ a freshwater dilution
<br />water is used, hardness, alkalinity, pH, and
<br />conductivity must be measured, and measure-
<br />ment of calcium, magnesium, sodium, potas-
<br />sium, chloride and sulfate is desirable. I[ a
<br />saltwater dilution water is used, salinity and
<br />pH must be measured. In both waters, mea-
<br />surement of particulate matter and TOC (or
<br />COD in freshwater) is desirable. The dissolved
<br />oxygen concentration must be measured at the
<br />beginning and end of the test and every 48 h
<br />during the test in the control and the high,
<br />medium, and low toxicant concentrations as
<br />long as live organisms are present. The pH
<br />should be measured at the beginning and end
<br />of the test in the control and the high, medium,
<br />and low toxicant concentrations. I[ possible,
<br />the concentration of toxicant should be mea-
<br />sured at the beginning and end of the test in all
<br />test chambers. Measurement of degradation
<br />products of the toxicant is desirable.
<br />11.9.2 Flow-Through Tests:
<br />11.9.2.1 Analysis of Water Quality-Certain
<br />measurements must be performed at least once
<br />every 30 days or at the beginning of the test, if
<br />data are available to show that the quality of
<br />the dilution water is constant, and daily if such
<br />data are not available. For a freshwater dilution
<br />water, hardness, alkalinity, pH, conductivity,
<br />TOC or COD, and particulate matter must be
<br />measured, and measurement of calcium, mag-
<br />nesium, sodium, potassium, chloride, and sul-
<br />fate is desirable. For a saltwater dilution water,
<br />salinity, pH, TOC, and particulate matter must
<br />be measured. The dissolved oxygen concentra-
<br />tion must be measured at the beginning and
<br />end of the test and every 48 h in between in the
<br />control and the high, medium, and low toxicant
<br />concentrations as long as . live organisms are
<br />present. The pH should be measured at least
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