A laboratory preparation procedure for studying bioaccumulation of U and its subcellular form in earthworms (Diplocardia spp.)

Uranium (U) is a ubiquitous trace element in soils. With increasing in application of U in nuclear energy and nuclear weapon, a large amount of U was dissipated into the environment including soil and water. Earthworm may be an eco-indicator for U bioaccumulation, transformation and transport across the ecosystem. There have been a variety of methods preformed to assess the bioaccumulation of uranium in small organisms such as earthworms, including uranium speciation, subcellular separation, and total U accumulation. All methods require an initial grinding preparation process that allows for the further fractionation of metals and metalloids in earthworms. The slime like mucus that coats the body of a worm presents a challenge in the disintegration and dissolution of the worm body. In order to analyze U subcellular forms, we developed a reliable and effective procedure to grind the worm body into a uniform fine suspension. We conducted a comparative study of disintegration of worms with 3 grinding techniques (agate mortar, liquid nitrogen freezing then agate mortar, and direct sonication) that would assist U subcellular analyses and bioaccumulation. The essences of this new development was as follows: • A scheme for preparation of earthworm samples for investigation of subcellular U forms in earthworms from U.S. army weapon test range soil with various U forms. • The direct sonication of earthworms was found to be the most proficient process in achieving the best preparation for U subcellular analyses with the high precision.