Test feed development and methodological approaches allowing highly controlled dietary exposures to nano- and microparticulate contaminants in fish

There is increasing concern that particulate contaminants including manufactured nanomaterials and nano- and microplastics taken up and or accumulating in lower-trophic level aquatic organisms results in dietary exposure of fish feeding on these organisms. Controlled feeding studies can help advance our understanding of dietary uptake, bioaccumulation, and associated effects of (nano)particulate contaminants in fish, and also provide information about their likelihood to be transferred along the trophic chain and or to act as vector for other, surface-adsorbed pollutants. However, traditional approaches to prepare test feed for dietary exposure studies where commercial fish feed such as flakes, granules or pellets are soaked or spray-spiked with dissolved chemicals are not well suitable for (nano-)particulate contaminants. Microplastics, which often have sizes in the µm to mm range, and manufactured nanomaterials, in particular those which are soluble, such as metal/metal oxide nanoparticles, have to be incorporated • Here we describe a methodological approach to produce worm-shaped food packages, that is a practical diet, of controlled diameter and length (in the millimeter range), which allows to prepare food rations with a weight in the order of a few milligrams and to adjust the food rations to the individual body wet weight of small experimental fish with high accuracy (±0.5 mg) without the need for weighing/proportioning the feed using a scale. • The method can be used to prepare test feed with internally incorporated particulate contaminants, such as manufactured nanomaterials and nano- and microplastics, to assess the latter's dietary uptake, bioaccumulation and associated toxicity in fish. We described two protocol variations: One using dry starting material, such as feed flakes, and one using liquid starting material, such as worm homogenate. • The method has been developed for academic research environments with no access to specialized equipment for test feed preparation, and uses utensils and inexpensive plastic ware belonging to the standard inventory of ecotoxicological research laboratories.