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Factors influencing the design and execution of a secondary hazard study are presented using the anticoagulant rodenticide, difethialone, as a model. Frequently, regulatory agencies require a determination of the potential hazard posed to non-target species by the application of pesticides. This hazard can occur through: 1) primary hazard, direct consumption of the bait or formulation containing the pesticide, or 2) secondary hazard, indirect consumption of the pesticide via ingestion of biological matrices such as animals or plants containing pesticide residues. In making this determination, the selection of appropriate test species as well as routes and level of exposure are critical. For the example study of the estimation of secondary hazards associated with difethialone, albino rats served as the representative species for the primary target animal. Ferrets and magpies served as representative mammalian and avian secondary scavenger species, respectively.