Summary• The extent of isotopic discrimination of transition metals in biological processes is poorly understood but potentially has important applications in plant and biogeochemical studies.• Using multicollector inductively coupled plasma (ICP) mass spectrometry, we measured isotopic fractionation of zinc (Zn) during uptake from nutrient solutions by rice ( Oryza sativa ), lettuce ( Lactuca sativa ) and tomato ( Lycopersicon esculentum ) plants.• For all three species, the roots showed a similar extent of heavy Zn enrichment relative to the nutrient solution, probably reflecting preferential adsorption on external root surfaces. By contrast, a plant-species specific enrichment of the light Zn isotope occurred in the shoots, indicative of a biological, membrane-transport controlled uptake into plant cells. The extent of the fractionation in the shoots further depended on the Zn speciation in the nutrient solution.• The observed isotopic depletion in heavy Zn from root to shoot ( − 0.13 to − 0.26‰ per atomic mass unit) is equivalent to roughly a quarter of the total reported terrestrial variability of Zn isotopic compositions ( c. 0.84‰ per atomic mass unit). Plant uptake therefore represents an important source of isotopic variation in biogeochemical cycling of Zn.

Recent reports suggest that significant fractionation of stable metal isotopes occurs during biogeochemical cycling and that the uptake into higher plants is an important process. To test isotopic fractionation of copper (Cu) and zinc (Zn) during plant uptake and constrain its controls, we grew lettuce, tomato, rice and durum wheat under controlled conditions in nutrient solutions with variable metal speciation and iron (Fe) supply. The results show that the fractionation patterns of these two micronutrients are decoupled during the transport from nutrient solution to root. In roots, we found an enrichment of the heavier isotopes for Zn, in agreement with previous studies, but an enrichment of isotopically light Cu, suggesting a reduction of Cu(II) possibly at the surfaces of the root cell plasma membranes. This observation holds for both graminaceous and nongraminaceaous species and confirms that reduction is a predominant and ubiquitous mechanism for the acquisition of Cu into plants similar to the mechanism for the acquisition of iron (Fe) by the strategy I plant species. We propose two preliminary models of isotope fractionation processes of Cu and Zn in plants with different uptake strategies.

Two approaches to correct for mass discrimination effects associated with Cu and Zn isotopic measurements on two different MC-ICP-MS instruments (a Micromass IsoProbe and a VG Axiom) have been compared and assessed in detail: (1) sample-standard bracketing (SSB), and (2) the 'empirical external normalisation' (EEN) whereby a second element is used to simultaneously correct for mass discrimination. This has provided new insights into the mass discrimination behaviours of Cu and Zn under varying instrumental set-ups, and has allowed improvements to be made to the existing correction procedures. With the SSB approach, mass bias stability is a prerequisite, and matrix components must be removed from the analyte to avoid matrix-related mass discrimination effects. By comparison, the EEN approach requires a degree of mass bias instability, and automatically corrects for matrix-related mass discrimination effects. The EEN correction may therefore appear more robust. However, while the EEN correction yields high-precision 65 Cu/ 63 Cu and 66 Zn/ 64 Zn data, an as yet unidentified source of systematic drift in the 67 Zn and 68 Zn signals through time hinders analyses of ratios incorporating these isotopes. Using the EEN correction where analyte and spike ratios were measured sequentially within a peak-switching protocol led to a three-fold deterioration in precision relative to static measurements. This is consistent with mass bias drift on the scale of a single five-second-measurement integration. For relative 65 Cu/ 63 Cu and 66 Zn/ 64 Zn ratio measurements, the SSB and EEN corrections give long-term reproducibilities of less then ¡0.07% (2SD) for pure Cu and Zn reagents. This is sufficient for resolving mass-dependent isotopic variability in natural and anthropogenic materials.

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This paper presents an adapted anion exchange column chemistry protocol which allowed separation of high‐purity fractions of Cu and Zn from geological materials. Isobaric and non‐spectral interferences were virtually eliminated for consequent multiple‐collector ICP‐MS analysis of the isotopic composition of these metals. The procedure achieved ∼ 100% recoveries, thus ensuring the absence of column‐induced isotopic fractionation. By employing these techniques, we report isotopic analyses for Cu and Zn from five geological reference materials: BCR‐027 blende ore (BCR), δ65Cu = 0.52 ± 0.15‰ (n = 10) and δ66Zn = 0.33 ± 0.07‰ (n = 8); BCR‐030 calcined calamine ore (BCR), δ66Zn = ‐0.06 ± 0.09‰ (n = 8); BCR‐1 basalt (USGS), δ66Zn = 0.29 ± 0.12‰ (n = 8); NOD‐P‐1 manganese nodule (USGS), δ65Cu = 0.46 ± 0.08‰ (n = 10) and δ66Zn = 0.78 ± 0.09‰ (n = 9); SU‐1 Cu‐Co ore (CCRMP), δ65Cu = ‐0.018 ± 0.08‰ (n = 10) and δ66Zn = 0.13 ± 0.17‰ (n = 6). All uncertainties are ± 2s; copper isotope ratios are reported relative to NIST SRM‐976, and zinc isotope ratios relative to the Lyon‐group Johnson Matthey metal (batch 3‐0749 L) solution, JMC Zn. These values agree well with the limited data previously published, and with results reported for similar natural sample types. Samples were measured using a GVi IsoProbe MC‐ICP‐MS, based at the Natural History Museum, London. Long‐term measurement reproducibility has been assessed by repeat analyses of both single element and complex matrix samples, and was commonly better than ± 0.07‰ for both δ66Zn and δ65Cu.

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In an earlier study, we found that rice (Oryza sativa) grown in nutrient solution well-supplied with Zn preferentially took up light 64 Zn over 66 Zn, probably as a result of kinetic fractionation in membrane transport processes. Here, we measure isotope fractionation by rice in a submerged Zn-deficient soil with and without Zn fertilizer. We grew the same genotype as in the nutrient solution study plus low-Zn tolerant and intolerant lines from a recombinant inbred population. In contrast to the nutrient solution, in soil with Zn fertilizer we found little or heavy isotopic enrichment in the plants relative to plant-available Zn in the soil, and in soil without Zn fertilizer we found consistently heavy enrichment, particularly in the low-Zn tolerant line. These observations are only explicable by complexation of Zn by a complexing agent released from the roots and uptake of the complexed Zn by specific root transporters. We show with a mathematical model that, for realistic rates of secretion of the phytosiderophore deoxymugineic acid (DMA) by rice, and realistic parameters for the Zn-solubilizing effect of DMA in soil, solubilization and uptake by this mechanism is necessary and sufficient to account for the measured Zn uptake and the differences between genotypes.

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