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<br />4 days (2 days for copepods) and fish for at <br />least 14 days before they are used in tests. They <br />should be held under stable conditions at a <br />constant temperature in uncontaminated, con- <br />stant-quality water in an aerated flow-through <br />system with a flow rate of at least two volume <br />additions per day. Higher flow rates are often <br />desirable (19) but static or renewal systems can <br />sometimes be used with small organisms. Wells <br />and springs are the preferred sources of fresh <br />water. Only as a last resort should a dechlorin- <br />ated water be used. When possible, the orga- <br />nisms should be held in dilution water and at <br />the temperature at which they are to be tested. <br />The temperatures listed in Table 6 are generally <br />good temperatures at which to hold the respec- <br />tive organisms. During long holding periods, <br />however, it is generally easier and safer to hold <br />fish at temperatures lower than those specified <br />in Table 6 because metabolic rate and the <br />number and severity of disease outbreaks are <br />reduced. <br />10.7 Acclimation-Organisms must be at <br />least partially acclimated to the test tempera- <br />ture and dilution water before beginning a test <br />with them. <br />10.7.1 Freshwater amphipods, caddisflies, <br />daphnids, burrowing mayflies, and midge lar- <br />vae should be acclimated to water quality and <br />temperature by rearing them in dilution water <br />at the test temperature. <br />10.7.2 Other organisms can be acclimated <br />(in a flow-through system with a flow rate of at <br />least two volume additions per day for flow- <br />through tests) simultaneously to the dilution <br />water and test temperature after transferring an <br />appropriate number of similar-length individ- <br />uals from a holding tank to an acclimation <br />tank. They should be acclimated to the dilution <br />water by gradually changing the water in the <br />acclimation tank from 100 % holding water to <br />100 % dilution water over a period of2 or more <br />days. All organisms must remain in 100 % di- <br />lution water for at least 2 days before use in <br />tests. All organisms should be acclimated to the <br />test temperature by changing the water tem- <br />perature at a rate not to exceed 30C within 24 <br />h for invertebrates and 30C within 72 h for <br />other organisms. They must be maintained at <br />the test temperature for at least 2 days before <br />they are used in tests. Complete acclimation <br />may take considerably longer; therefore, accli- <br /> <br />~~l~ <br /> <br />E 729 <br /> <br />mation times longer than the specified mini- <br />mum should be used when possible. <br />10.7.3 Young amphibian larvae and fish that <br />have been actively feeding for less than about <br />20 days, amphipods, daphnids, and midge lar- <br />vae must be fed, and all other insects may be <br />fed, up to the beginning of the test. All other <br />amphibian larvae and fish over 0.5 g each must <br />not be fed for 48 h, and all other invertebrates <br />over 0.5 g each must not be fed for 24 h, before <br />the beginning of the test. If adult amphipods or <br />daphnids are isolated before the beginning of <br />a test for the collection of young, they must be <br />fed. <br />10.8 Quality-All organisms in a test must <br />be of acceptable quality. <br />10.8.1 Organisms should be carefully ob- <br />served daily during holding and acclimation <br />for signs of disease, stress, physical damage, <br />and mortality. Injured, dead, and abnormal <br />individuals must be discarded. Visual exami- <br />nation of the behavior and external appearance <br />of the organisms should cover the following: <br />Are they eating? Is flipping, flashing, or erratic <br />swimming evident? Are they gasping at the <br />surface? Is their color normal? Is hyperventi- <br />lation, hemorrhaging, or excessive mucus pro- <br />duction evident? Are fin rot, fungus, or external <br />parasites present? Are burrowing organisms <br />burrowing? Are test organisms emaciated? If <br />the organisms show any signs of disease or <br />stress, remedial action should be taken. <br />10.8.2 Organisms may be used without anal- <br />yses (a) if they are reared using water and food <br />that support survival, growth, and reproduction <br />of aquatic organisms and meet the specifica- <br />tions of 8.2.2 and 10.4.2, respectively, or (b) if <br />they are obtained from a body of water that <br />does not receive a discharge or chemical treat- <br />ment and are maintained using water and foods <br />as above. Other organisms should not be used <br />unless it is shown that (a) they do not contain <br />elevated concentrations of chemicals to which <br />they were probably exposed and (b) either the <br />concentration of organic chlorine does not ex- <br />ceed 0.15 I-tg/g (wet weight) or the total concen- <br />tration of organochlorine pesticides plus PCBs <br />does not exceed 0.3 Mg/g (wet weight). Analysis <br />of a few representatives from a batch is suffi- <br />cient. Analysis of each shipment or before each <br />test is not necessary. <br />10.8.3 Organisms must not be used for a test <br /> <br />10 <br />