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30 JOSEPH B. HUNN AND ROSALIE A. SCHNICK Sample Collection for Suspected Toxic Substances "It is an old axiom that the result of any test can be no better than the sample on which it is per- formed" (APHA et al. 1985). When a toxic substance is suspected as the possible cause of a fish kill, it is critical that the investigators collect samples prop- erly, use appropriate containers, follow preservation and storage methods that are consistent with ac- cepted methodology, and ship samples properly and promptly. The following sections discuss proper pro- cedures for the collection, handling, storage, and shipment of samples for fish, water, sediments, in- vertebrates, and plants. An essential element is a field log in which there is an entry for each sample collected for analysis, its identification number, the site where collected, the date, and the name or initials of the collector. These entries provide backup identification if sam- ple labels are damaged, are lost, or if confusion develops over when and where certain samples were taken. Fish Samples A representative size series of moribund or recent- ly dead fish of each species affected should be col- lected. If possible, healthy fish of the same species and sizes from the unaffected area should also be collected to provide background data. Methods that are used to preserve the various samples should always be noted on the label. For samples to be analyzed for pesticides or other toxic organic sub- stances, the whole fish should be rinsed with clean water, wrapped in aluminum foil (with the dull side toward the specimen), and frozen as quickly as possi- ble. Samples to be analyzed for metals or other elements should be collected separately, placed in polyethylene bags, and frozen. Subsamples of tissues such as brain, gills, or blood that are needed for special analyses should be taken immediately after sampling and frozen in separate clean glass con- tainers. Special analyses may include measurements of enzymatic activity (e.g., acetylcholinesterase in brain or Na, K-ATPase in gill tissue) or metals in gill tissue (e.g., Cd, Hg, Zn, or Cu). Tissues for histological examination should be taken from moribund fish-never from dead fish (in which postmortem changes are likely to have oc- curred). Fish that have been dead longer than 10-15 minutes are not suitable specimens. Tissue samples for histological examination should not be frozen. It is imperative that tissue specimens be placed into a suitable fixative as soon as possible, preferably at a ratio of 1 part tissue to 10 parts fixative. A 10070 solution of buffered neutral formalin is readily avail- able and is an acceptable fixative. Check with the histopathologist who will do the tissue analyses for his or her choice of fixative and for other instruc- tions on fixation techniques. Fish tissues that were preserved in a fixative for histological examination should be transferred to 7007o ethanol for storage. They can then be held for a year or more if the solu- tion is renewed periodically. For further informa- tion, see Morrison and Smith (1981) or Yasutake (1987). For analytical purposes, it is better to collect several small fish than one large fish from each species that is affected. The numbers collected, amount of tissue needed, and preservation tech- niques depend on the types of analyses to be per- formed. The following general guidelines apply: Inorganic analyses Per sample: at least three fish or as many as needed to provide 100 g of whole body tissue as the minimum total sample; collect three samples for each species from each site Organic analyses Per sample: at least three fish or as many as needed to provide 250 g of whole body tissue as the minimum total sample; collect three samples for each species from each site If it is suspected that the causative agent is a volatile substance, about 100 g of tissue should be placed in containers that can be sealed airtight and frozen. The composite samples of three or more fish should be separately wrapped in foil and placed in a single bag, properly labeled, and frozen. Samples of all types should be frozen as quickly as possible and kept frozen at - 20 ° C or lower until analyzed. For a large kill with many species, the investigator must select the species to be collected. Samples should include representatives from all trophic levels that are affected-for example, herbivores, omni- vores, forage fish, and predators. It is critical that the same species of fish (and preferably of the same sizes) be sampled from the control or reference area as from the kill area. The numbers and types of samples collected will depend on the extent of the L