We have prepared a large volume of pure, concentrated and homogenous zinc standard solution. This new standard solution is intended to be used as a primary reference standard for the zinc isotope community, and to serve as a replacement for the nearly exhausted current reference standard, the so-called JMC-Lyon Zn. The isotopic composition of this new zinc standard (AA-ETH Zn) has been determined through an inter-laboratory calibration exercise, calibrated against the existing JMC-Lyon standard, as well as the certified Zn reference standard IRMM-3702. The data show that the new standard is isotopically indistinguishable from the IRMM-3702 zinc standard, with a weighted δ^66/64Zn value of 0.28 ± 0.02‰ relative to JMC-Lyon. We suggest that this new standard be assigned a δ^66/64Zn value of +0.28‰ for reporting of future Zn isotope data, with the rationale that all existing published Zn isotope data are presented relative to the JMC-Lyon standard. Therefore our proposed presentation allows for a direct comparison with all previously published data, and that are directly traceable to a certified reference standard, IRMM-3702 Zn. This standard will be made freely available to all interested labs through contact with the corresponding author.

This work critically reviews stable isotope fractionation of essential (B, Mg, K, Ca, Fe, Ni, Cu, Zn, Mo), beneficial (Si), and non-essential (Cd, Tl) metals and metalloids in plants. The review (i) provides basic principles and methodologies for non-traditional isotope analyses, (ii) compiles isotope fractionation for uptake and translocation for each element and connects them to physiological processes, and (iii) interlinks knowledge from different elements to identify common and contrasting drivers of isotope fractionation. Different biological and physico-chemical processes drive isotope fractionation in plants. During uptake, Ca and Mg fractionate through root apoplast adsorption, Si through diffusion during membrane passage, Fe and Cu through reduction prior to membrane transport in strategy I plants, and Zn, Cu, and Cd through membrane transport. During translocation and utilization, isotopes fractionate through precipitation into insoluble forms, such as phytoliths (Si) or oxalate (Ca), structural binding to cell walls (Ca), and membrane transport and binding to soluble organic ligands (Zn, Cd). These processes can lead to similar (Cu, Fe) and opposing (Ca vs. Mg, Zn vs. Cd) isotope fractionation patterns of chemically similar elements in plants. Isotope fractionation in plants is influenced by biotic factors, such as phenological stages and plant genetics, as well as abiotic factors. Different nutrient supply induced shifts in isotope fractionation patterns for Mg, Cu, and Zn, suggesting that isotope process tracing can be used as a tool to detect and quantify different uptake pathways in response to abiotic stresses. However, the interpretation of isotope fractionation in plants is challenging because many isotope fractionation factors associated with specific processes are unknown and experiments are often exploratory. To overcome these limitations, fundamental geochemical research should expand the database of isotope fractionation factors and disentangle kinetic and equilibrium fractionation. In addition, plant growth studies should further shift toward hypothesis-driven experiments, for example, by integrating contrasting nutrient supplies, using established model plants, genetic approaches, and by combining isotope analyses with complementary speciation techniques. To fully exploit the potential of isotope process tracing in plants, the interdisciplinary expertise of plant and isotope geochemical scientists is required.

In response to new European Union regulations, studies are underway to mitigate accumulation of toxic cadmium (Cd) in cacao (Theobroma cacao, Tc). This study advances such research with Cd isotope analyses of 19 genetically diverse cacao clones and yeast transformed to express cacao natural resistance-associated macrophage protein (NRAMP5) and heavy metal ATPases (HMAs). The plants were enriched in light Cd isotopes relative to the hydroponic solution with Δ^114/110Cd_tot-sol = −0.22 ± 0.08‰. Leaves show a systematic enrichment of isotopically heavy Cd relative to total plants, in accord with closed-system isotope fractionation of Δ^114/110Cd_seq-mob = −0.13‰, by sequestering isotopically light Cd in roots/stems and mobilisation of remaining Cd to leaves. The findings demonstrate that (i) transfer of Cd between roots and leaves is primarily unidirectional; (ii) different clones utilise similar pathways for Cd sequestration, which differ from those of other studied plants; (iii) clones differ in their efficiency of Cd sequestration. Transgenic yeast that expresses TcNRAMP5 (T. cacao natural resistance-associated macrophage gene) had isotopically lighter Cd than did cacao. This suggests that NRAMP5 transporters constitute an important pathway for uptake of Cd by cacao. Cd isotope signatures of transgenic yeast expressing HMA-family proteins suggest that they may contribute to Cd sequestration. The data are the first to record isotope fractionation induced by transporter proteins in vivo.

The often high Cd concentrations of cacao beans are a serious concern for producers in Latin America due to the implementation of stricter Cd limits for cocoa products by the European Union in 2019. This is the first investigation to employ coupled Cd isotope and concentration measurements to study soil – cacao systems. Analyses were carried out for 29 samples of soils, soil amendments and cacao tree organs from organic farms in Ecuador that harvest three distinct cacao cultivars. The majority of soils from 0–80 cm depth have very similar δ^114/110Cd of about −0.1‰ to 0‰. Two 0–5 cm topsoils, however, have high Cd concentrations coupled with heavy Cd isotope compositions of δ^114/110Cd ≈ 0.2%, possibly indicating Cd additions from the tree litter used as organic fertilizer. Whilst cacao leaves, pods and beans are ubiquitously enriched in Cd relative to soils there are distinct Cd isotope signatures. The leaves and pods are isotopically heavier than the soils, with similar Δ^114/110Cd_leaf–soil values of 0.22 ± 0.07‰ to 0.41 ± 0.09‰. In contrast, the data reveal differences in Δ^114/110Cd_bean–leaf that may be linked to distinct cacao cultivars. In detail, Δ^114/110Cd_bean–leaf values of −0.34‰ to −0.40‰ were obtained for Nacional cacao from two farms, whilst CCN-51 hybrid cacao from a third farm showed no fractionation within error (−0.08 ± 0.13‰). As such, further work to investigate whether Cd isotopes are indeed useful for tracing sources of Cd enrichments in soils and to inform genetic efforts to reduce the Cd burden of cocoa is indicated.

Biological reference materials with well-characterised stable isotope compositions are lacking in the field of ‘isotope biochemistry’, which seeks to understand bodily processes that rely on essential metals by determining metal stable isotope ratios. Here, we present Zn stable isotope data for six biological reference materials with certified trace metal concentrations: fish muscle, bovine muscle, pig kidney, human hair, human blood serum and human urine. Replicate analyses of multiple aliquots of each material achieved reproducibilities (2sd) of 0.04–0.13‰ for δ^66/64Zn (which denotes the deviation of the ⁶⁶Zn/⁶⁴Zn ratio of a sample from a pure Zn reference material in parts per 1000). This implies only very minor isotopic heterogeneities within the samples, rendering them suitable as quality control materials for Zn isotope analyses. This endorsement is reinforced by (i) the close agreement of our Zn isotope data for two of the samples (bovine muscle and human blood serum) to previously published results for different batches of the same material and (ii) the similarity of the isotopic data for the samples (δ^66/64Zn ≈ –0.8 to 0.0‰) to previously published Zn isotope results for similar biological materials. Further tests revealed that the applied Zn separation procedure is sufficiently effective to enable accurate data acquisition even at low mass resolving power (M/ΔM ≈ 400), as measurements and analyses conducted at much higher mass resolution (M/ΔM ≈ 8500) delivered essentially identical results.

Breast, prostate, and pancreatic cancers alter the zinc (Zn) metabolism. Combined analyses of urinary Zn concentrations [Zn] and Zn stable isotope compositions (δ⁶⁶Zn) may provide a non-invasive approach for tracing malignancy-induced Zn dyshomeostasis. In this study, we measured [Zn] and δ⁶⁶Zn in urine from prostate (n = 22), breast (n = 16), and from women with benign breast disease (n = 14) and compared those with age-matched healthy controls (22–49 years or 50+ years) and published data for pancreatic cancer (n = 17). Our results show that cancer-induced changes are reflected in higher urinary [Zn] and lower urinary δ⁶⁶Zn for pancreatic and prostate cancer and benign breast disease when compared with healthy controls. For prostate cancer, the progression of low [Zn] and high δ⁶⁶Zn for patients of low-risk disease toward high [Zn] and low δ⁶⁶Zn for the higher risk patients demonstrates that [Zn] and δ⁶⁶Zn in urine could serve as a reliable prognostic tool. Urinary excretion of isotopically light Zn by patients with prostatic and pancreatic cancer is probably the result of increased reactive oxygen species in cancerous cells, which limits the scavenging of hydroxyl radicals and thus facilitates the oxidation of metalloproteins with sulfur-rich ligands. Urine from breast cancer patients shows undistinguishable δ⁶⁶Zn to healthy controls, implying that the expression of metalloproteins with sulfur-rich ligands is stronger in breast cancer tissues. In conclusion, urinary δ⁶⁶Zn may provide a non-invasive diagnostic tool for pancreatic cancer and support disease prognosis for prostate cancer. These findings should translate to comprehensive transverse and longitudinal cohort studies in future.