Novel Multi-isotope Tracer Approach to Test ZnO Nanoparticle and Soluble Zn Bioavailability in Joint Soil Exposures

Here we use two enriched stable isotopes, ⁶⁸Zn_en and ⁶⁴Zn_en (>99%), to prepare ⁶⁸ZnO nanoparticles (NPs) and soluble ⁶⁴ZnCl₂. The standard LUFA 2.2 test soil was dosed with ⁶⁸ZnO NPs and soluble ⁶⁴ZnCl₂ to 5 mg kg^-1 each, plus between 0 and 95 mg kg^-1 of soluble ZnCl₂ with a natural isotope composition. After 0, 1, 3, 6, and 12 months of soil incubation, earthworms (Eisenia andrei) were introduced for 72 h exposures. Analyses of soils, pore waters, and earthworm tissues using multiple collector inductively coupled plasma mass spectrometry allowed the simultaneous measurement of the diagnostic ⁶⁸Zn/⁶⁶Zn, ⁶⁴Zn/⁶⁶Zn, and ⁶⁸Zn/⁶⁴Zn ratios, from which the three different isotopic forms of Zn were quantified. Eisenia andrei was able to regulate Zn body concentrations with no difference observed between the different total dosing concentrations. The accumulation of labeled Zn by the earthworms showed a direct relationship with the proportion of labeled to total Zn in the pore water, which increased with longer soil incubation times and decreasing soil pH. The ⁶⁸Zn_en/⁶⁴Zn_en ratios determined for earthworms (1.09 ± 0.04), soils (1.09 ± 0.02), and pore waters (1.08 ± 0.02) indicate indistinguishable environmental distribution and uptake of the Zn forms, most likely due to rapid dissolution of the ZnO NPs.