M1136

Snails were cultured from hatching to the adulthood in aquaria containing metal-free reconstituted water [20 (link)]. Control and experimental female snails were maintained in 6 L aquaria with reconstituted water [10 (link)] which was changed weekly. Each age group (4, 5, 6 and 7 months old) cultured in reconstituted water was divided before sacrifice in four experimental subgroups that were exposed for 4 weeks to 6 μg/L of TBT [NOECL reported in 26 (link)] as tributyltin chloride (Sigma-Aldrich, T50202) or for six weeks to each of the following compounds, 2 μg/L of Hg as HgCl₂ (Sigma-Aldrich, M1136), 10 μg/L of As as Na₃AsO₄ 7H₂0 (Sigma–Aldrich, S9663), or 30 μg/L of U as UO₂CH₂COOH (Ted Pella Inc.19481) (Figure 1, orange lines). These elements were selected because of their potential toxicity to humans and the environment and it were used at the maximum contaminant levels (MCLs) for human consumption allowed by EPA regulations. Exposure conditions were semi static and each compound was added after changing the aquarium water. No snails died during the exposure period. Shell length, PL, PSL, and DI were measured as described above.

Mice were perfused with PBS at 8 weeks of age. Brains were dissected and immersed in Golgi staining solution which contained 50% potassium dichromate (MP 021563389), 5% mercuric chloride (Sigma‐Aldrich M1136), and 5% potassium chromate (Sigma‐Aldrich 529508) and protected from light for 7 days. When performing Golgi staining, brain sections were washed with distilled water, dehydrated with ethanol, and then treated with ammonia (3:1). The sections were subsequently washed and incubated in 5% sodium thiosulfate for 10 min and then dehydrated with degraded ethanol and clarified with xylene. In the end, the sections were observed under bright field of Olympus FV1000. The images were taken by z‐stack scanning with the excitation wavelength of 405 nm, and the virtual color was converted into green color. For dendrites branching, Sholl analysis was conducted. For spine density, dendrites of pyramidal neurons in CA region were reconstructed and analyzed by using IMARIS.