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sensitive than older individuals. Among fresh- water organisms, daphnids should be in the first instar; amphipods, stoneflies, and mayflies in an early instar; and midges in the second or third ins tar. Decapod crustaceans bearing eggs and polychaetes with visible developing eggs in the coleom should not be used. 10.3.3 Amphibians-Young larvae should be used whenever possible. 10.4 Care and Handling-Organisms must be cared for and handled properly (19) so they are not unnecessarily stressed. 10.4.1 To avoid unnecessary stress, orga- nisms should not be subjected to rapid changes in temperature or water quality. In general, aquatic organisms should not be subjected to more than a 30C change in water temperature in any 12-h period. Between use with different groups of test organisms, holding and accli- mation tanks should be sterilized with an io- dophor or with 200 mg of hypochlorite per litre for I h, brushed well once during the hour, and then rinsed well. Although iodophors are not very acutely toxic to most aquatic animals, hypochlorite is. Hypochlorite can be removed by flushing or by reaction with sodium thiosul- fate or sodium sulfite. When fish are first brought into a facility, they should be quaran- tined for at least 7 days and until they appear disease-free. To maintain organisms in good condition during holding and acclimation, crowding should be avoided, and the dissolved oxygen concentration should be maintained be- tween 60 % and 100 % saturation (12); gentle aeration may be used if necessary. Water that may be contaminated with undesirable micro- organisms should be passed through a properly maintained ultraviolet sterilizer equipped with an intensity meter (13), and the un-ionized ammonia concentration in the holding and ac- climation tanks should be less than 20 JLg/L. 10.4.2 At least once daily (however, see 10.7.3 for exceptions) organisms should be fed. A batch of food may be used without analysis if it has been shown that the batch will support survival, growth, and reproduction of at least one species of aquatic animal. Other batches should not be used unless it has been shown that the concentration of organic chlorine does not exceed 0.15 JLg/g (wet weight) or the total concentration of organochlorine pesticides plus PCBs does not exceed 0.3 JLg/g (wet weight). ~~l~ E 729 Analysis of all batches of food for the toxicant to be tested is desirable. 10.4.3 Tanks should be scraped or brushed as needed. 10.4.4 Organisms should be handled as little as possible. When handling is necessary, it should be done as gently, carefully, and quickly as possible, so that organisms are not unneces- sarily stressed. Organisms that touch dry sur- faces or are dropped or injured during handling must be discarded. Dip nets are best for han- dling organisms over 0.5 g each. Such nets are commercially available, or can be made from small-mesh nylon netting, nylon or silk bolting cloth, plankton netting, or similar knotless ma- terial. Nets coated with urethane resin are best for handling catfish. Fire-polished smooth glass tubes with rubber bulbs should be used for transferring smaller organisms such as daph- nids and midge larvae. Equipment used to handle aquatic organisms should be sterilized between uses by autoclaving or by treatment with an iodophor, 200 mg of hypochlorite per litre, or 30 % formalin plus I % benzalkonium chloride for at least 1 h. Hands should be washed or sterilized before handling or feeding test organisms. 10.5 Disease Treatment-Freshwater fish may be chemically treated to cure or prevent diseases by using the treatments listed in Table 7, but if they are severely diseased, the entire lot should be destroyed immediately. Until ac- ceptable treatments have been proven effective, all other diseased animals should be discarded immediately. Generally organisms should not be treated during the first 16 h after they arrive at a facility because they are probably stressed due to collection or transportation, and because they may have been treated with drugs during transit. However, immediate prophylactic treat- ment is necessary in some situations, such as for treatment of bluegills for columnaris (see Table 7, External bacteria) during hot weather. Organisms must not be used in tests for at least 10 days after treatment. Tanks and test cham- bers that may be contaminated with undesira- ble microorganisms should be sterilized by treatment with an iodophor or with 200 mg of hypochlorite per litre for at least I h. 10.6 Holding-After collection or transpor- tation, invertebrates and amphibians must have been in holding or acclimation tanks for at least 9