Cadmium, zinc, and copper leaching rates determined in large monolith lysimeters.

Soil mass balances are used to assess the risk of trace metals that are inadvertently applied with fertilizers into agroecosystems. The accuracy of such balances is limited by leaching rates, as they are difficult to measure. Here, we used monolith lysimeters to precisely determine Cd, Cu, and Zn leaching rates in 2021 and 2022. The large lysimeters (n = 12, 1 m diameter, 1.35 m depth) included one soil type (cambisol, weakly acidic) and distinct cropping systems with three experimental replicates. Stable isotope tracers were applied to determine the direct transfer of these trace metals from the soil surface into the seepage water. The annual leaching rates ranged from 0.04 to 0.30 for Cd, 2.65 to 11.7 for Cu, and 7.27 to 39.0 g (ha a)-1 for Zn. These leaching rates were up to four times higher in the year with several heavy rain periods compared to the dry year. Monthly resolved data revealed that distinct climatic conditions in combination with crop development have a strong impact on trace metal leaching rates. In contrast, fertilization strategy (e.g., conventional vs. organic) had a minor effect on leaching rates. Trace metal leaching rates were up to 10 times smaller than fertilizer inputs and had therefore a minor impact on soil mass balances. This was further confirmed with isotope source tracing that showed that only small fractions of Cd, Cu, and Zn were directly transferred from the soil surface to the leached seepage water within two years (< 0.07 %). A comparison with models that predict Cd leaching rates in the EU suggests that the models overestimate the Cd soil output with seepage water. Hence, monolith lysimeters can help to refine leaching models and thereby also soil mass balances that are used to assess the risk of trace metals inputs with fertilizers.

67Zn and 111Cd labelled green manure to determine the fate and dynamics of zinc and cadmium in soil-fertilizer-crop systems.

Isotope source tracing enables to accurately determine the fate of nutrients that are applied with fertilizers to soils. While this approach is well established for major nutrients such as nitrogen, it is not yet established for trace metals. Here, we aimed to determine the fate of the micronutrient zinc (Zn) and the contaminant cadmium (Cd) that were applied with an organic fertilizer to a soil-wheat system. A pot study was conducted in which wheat was grown on an alkaline soil. The soils received green manure and/or soluble Zn fertilizer and were compared with non-fertilized control treatments (n = 4 experimental replicates). The green manure was labelled with the stable isotopes 67Zn and 111Cd. For an efficient sample throughput, a method was provided and validated to determine enriched stable isotope ratios (67Zn:66Zn and 111Cd:110Cd) and the Zn and Cd concentrations in one analytical run. To this end, single collector ICP-MS analyses and stable isotope mass balances calculations were combined. Applying this method revealed that the addition of green manure increased neither Zn nor Cd concentrations in wheat grains due to biomass dilution effects. Isotope source tracing showed that the largest fraction of these metals in the wheat shoots derived from the soil in all treatments (Zn 87-99 %, Cd 94-98 %). Moreover, the addition of green manure increased the transfer of Zn and Cd from soil to wheat by a factor 1.9 for both elements. This increased transfer was likely related to a nitrogen fertilization effect that increased root and shoot biomass and thereby the soil exploration of the wheat. This study demonstrated how the fate and dynamics of multiple trace metals can be efficiently determined in soil-fertilizer-crop systems using isotope source tracing.

In Vitro Fossilization for High Spatial Resolution Quantification of Elements in Plant-Tissue Using LA-ICP-TOFMS.

Laser ablation in combination with an inductively coupled plasma time-of-flight mass spectrometer (LA-ICP-TOFMS) is an upcoming method for rapid quantitative element mapping of various samples. While widespread in geological applications, quantification of elements in biotissues remains challenging. In this study, a proof-of-concept sample preparation method is presented in which plant-tissues are fossilized in order to solidify the complex biotissue matrix into a mineral-like matrix. This process enables quantification of elements by using silicone as an internal standard for normalization while also providing consistent ablation processes similar to minerals to reduce image blurring. Furthermore, it allows us to generate a quantitative image of the element composition at high spatial resolution. The feasibility of the approach is demonstrated on leaves of sunflowers (Helianthus annuus), soy beans (Glycine max), and corn (Zea mays) as representatives for common crops, which were grown on both nonspiked and cadmium-spiked agricultural soil. The quantitative results achieved during imaging were validated with digestion of whole leaves followed by ICP-OES analysis. LA-ICP-TOFMS element mapping of conventionally dried samples can provide misleading trends due to the irregular ablation behavior of biotissue because high signals caused by high ablation rates are falsely interpreted as enrichment of elements. Fossilization provides the opportunity to correct such phenomena by standardization with Si as an internal standard. The method demonstrated here allows for quantitative image acquisition without time-consuming sample preparation steps by using comparatively safe chemicals. The diversity of tested samples suggests that this sample preparation method is well-suited to achieve reproducible and quantitative element maps of various plant samples.

From soil to cacao bean: Unravelling the pathways of cadmium translocation in a high Cd accumulating cultivar of Theobroma cacao L.

The research on strategies to reduce cadmium (Cd) accumulation in cacao beans is currently limited by a lack of understanding of the Cd transfer pathways within the cacao tree. Here, we elucidated the transfer of Cd from soil to the nib (seed) in a high Cd accumulating cacao cultivar. Here, we elucidated the transfer of Cd from soil to the nib (seed) in a high Cd accumulating cacao cultivar through Cd stable isotope fractionation, speciation (X-Ray Absorption Spectroscopy), and localization (Laser Ablation Inductively Coupled Plasma Mass Spectrometry). The plant Cd concentrations were 10-28 higher than the topsoil Cd concentrations and increased as placenta< nib< testa< pod husk< root< leaf< branch. The retention of Cd in the roots was low. Light Cd isotopes were retained in the roots whilst heavier Cd isotopes were transported to the shoots (Δ 114/110 Cd shoot-root = 0.27 ± 0.02 ‰ (weighted average ± standard deviation)). Leaf Cd isotopes were heavier than Cd in the branches (Δ 114/110 Cd IF3 leaves-branch = 0.18 ± 0.01 ‰), confirming typical trends observed in annual crops. Nibs and branches were statistically not distinguishable (Δ 114/110 Cd nib-branch = -0.08‰ ± 0.06 ‰), contrary to the leaves and nibs (Δ 114/110 Cd nib-IF3 leaves = -0.25‰ ± 0.05 ‰). These isotope fractionation patterns alluded to a more direct transfer from branches to nibs rather than from leaves to nibs. The largest fraction (57%) of total plant Cd was present in the branches where it was primarily bound to carboxyl-ligands (60-100%) and mainly localized in the phloem rays and phelloderm of the bark. Cadmium in the nibs was mainly bound to oxygen ligands (60-90%), with phytate as the most plausible ligand. The weight of evidence suggested that Cd was transferred like other nutrients from root to shoot and accumulated in the phloem rays and phelloderm of the branches to reduce the transfer to foliage. Finally, the data indicated that the main contribution of nib Cd was from the phloem tissues of the branch rather than from leaf remobilization. This study extended the limited knowledge on Cd accumulation in perennial, woody crops and revealed that the Cd pathways in cacao are markedly different than in annual crops.

Tracing the fate of phosphorus fertilizer derived cadmium in soil-fertilizer-wheat systems using enriched stable isotope labeling.

Applying mineral phosphorus (P) fertilizers introduces a considerable input of the toxic heavy metal cadmium (Cd) into arable soils. This study investigates the fate of P fertilizer derived Cd (Cddff) in soil-wheat systems using a novel combination of enriched stable Cd isotope mass balances, sequential extractions, and Bayesian isotope mixing models. We applied an enriched 111Cd labeled mineral P fertilizer to arable soils from two long-term field trials with distinct soil properties (a strongly acidic pH and a neutral pH) and distinct past mineral P fertilizer application rates. We then cultivated wheat in a pot trial on these two soils. In the neutral soil, Cd concentrations in the soil and the wheat increased with increasing past mineral P fertilizer application rates. This was not the case in the strongly acidic soil. Less than 2.3% of freshly applied Cddff was taken up by the whole wheat plant. Most of the Cddff remained in the soil and was predominantly (>95% of freshly applied Cddff) partitioned into the easily mobilizable acetic acid soluble fraction (F1) and the potentially mobile reducible fraction (F2). Soil pH was the determining factor for the partitioning of Cddff into F1, as revealed through a recovery of about 40% of freshly applied Cddff in F1 in the neutral pH soil compared with about 60% in the strongly acidic soil. Isotope mixing models showed that F1 was the predominant source of Cd for wheat on both soils and that it contributed to over 80% of the Cd that was taken up by wheat. By tracing the fate of Cddff in entire soil-plant systems using different isotope source tracing approaches, we show that the majority of Cddff remains mobilizable and is potentially plant available in the subsequent crop cycle.

Changes of Cadmium Storage Forms and Isotope Ratios in Rice During Grain Filling.

Rice poses a major source of the toxic contaminant cadmium (Cd) for humans. Here, we elucidated the role of Cd storage forms (i.e., the chemical Cd speciation) on the dynamics of Cd within rice. In a pot trial, we grew rice on a Cd-contaminated soil in upland conditions and sampled roots and shoots parts at flowering and maturity. Cd concentrations, isotope ratios, Cd speciation (X-ray absorption spectroscopy), and micronutrient concentrations were analyzed. During grain filling, Cd and preferentially light Cd isotopes were strongly retained in roots where the Cd storage form did not change (Cd bound to thiols, Cd-S = 100%). In the same period, no net change of Cd mass occurred in roots and shoots, and the shoots became enriched in heavy isotopes (Δ114/110Cd maturity-flowering = 0.14 ± 0.04‰). These results are consistent with a sequestration of Cd in root vacuoles that includes strong binding of Cd to thiol containing ligands that favor light isotopes, with a small fraction of Cd strongly enriched in heavy isotopes being transferred to shoots during grain filling. The Cd speciation in the shoots changed from predominantly Cd-S (72%) to Cd bound to O ligands (Cd-O, 80%) during grain filling. Cd-O may represent Cd binding to organic acids in vacuoles and/or binding to cell walls in the apoplast. Despite this change of ligands, which was attributed to plant senescence, Cd was largely immobile in the shoots since only 0.77% of Cd in the shoots were transferred into the grains. Thus, both storage forms (Cd-S and Cd-O) contributed to the retention of Cd in the straw. Cd was mainly bound to S in nodes I and grains (Cd-S > 84%), and these organs were strongly enriched in heavy isotopes compared to straw (Δ114/110Cd grains/nodes- straw = 0.66-0.72‰) and flag leaves (Δ114/110Cd grains/nodes-flag leaves = 0.49-0.52‰). Hence, xylem to phloem transfer in the node favors heavy isotopes, and the Cd-S form may persist during the transfer of Cd from node to grain. This study highlights the importance of Cd storage forms during its journey to grain and potentially into the food chain.

Cadmium transfer in contaminated soil-rice systems: Insights from solid-state speciation analysis and stable isotope fractionation.

Initial Cadmium (Cd) isotope fractionation studies in cereals ascribed the retention of Cd and its light isotopes to the binding of Cd to sulfur (S). To better understand the relation of Cd binding to S and Cd isotope fractionation in soils and plants, we combined isotope and XAS speciation analyses in soil-rice systems that were rich in Cd and S. The systems included distinct water management (flooded vs. non-flooded) and rice accessions with (excluder) and without (non-excluder) functional membrane transporter OsHMA3 that transports Cd into root vacuoles. Initially, 13% of Cd in the soil was bound to S. Through soil flooding, the proportion of Cd bound to S increased to 100%. Soil flooding enriched the rice plants towards heavy isotopes (δ114/110Cd = -0.37 to -0.39%) compared to the plants that grew on non-flooded soils (δ114/110Cd = -0.45 to -0.56%) suggesting that preferentially light Cd isotopes precipitated into Cd sulfides. Isotope compositions in CaCl2 root extracts indicated that the root surface contributed to the isotope shift between soil and plant during soil flooding. In rice roots, Cd was fully bound to S in all treatments. The roots in the excluder rice strongly retained Cd and its lights isotopes while heavy isotopes were transported to the shoots (Δ114/110Cdshoot-root 0.16-0.19‰). The non-excluder rice accumulated Cd in shoots and the apparent difference in isotope composition between roots and shoots was smaller than that of the excluder rice (Δ114/110Cdshoot-root -0.02 to 0.08‰). We ascribe the retention of light Cd isotopes in the roots of the excluder rice to the membrane transport of Cd by OsHMA3 and/or chelating Cd-S complexes in the vacuole. Cd-S was the major binding form in flooded soils and rice roots and partly contributed to the immobilization of Cd and its light isotopes in soil-rice systems.

Using isotopes to trace freshly applied cadmium through mineral phosphorus fertilization in soil-fertilizer-plant systems.

Applications of mineral phosphorus (P) fertilizer can lead to cadmium (Cd) accumulation in soils and can increase Cd concentrations in edible crop parts. To determine the fate of freshly applied Cd, a Cd source tracing experiment was conducted in three soil-fertilizer-wheat systems by using a mineral P fertilizer labeled with the radio isotope 109Cd and by exploiting natural differences in Cd stable isotope compositions (δ114/110Cd). Source tracing with stable isotopes overestimated the proportion of Cd in plants derived from the P fertilizer, because the isotope ratios of the sources were not sufficiently distinct from those of the soils. Despite indistinguishable extractable Cd pools between control and treatments, the addition of P fertilizer resulted in a more negative apparent isotope fractionation between soil and wheat. Overall, the radio isotope approach provided more robust results and revealed that 6.5 to 15% of the Cd in the shoot derived from the fertilizer. From the introduced Cd, a maximum of 2.2% reached the wheat shoots, whilst 97.8% remained in the roots and soils. The low recoveries of the fertilizer derived Cd suggest that continuous P fertilizer application in the past decades can lead to a build-up of a residual Cd pool in soils.

Zinc isotope fractionation during grain filling of wheat and a comparison of zinc and cadmium isotope ratios in identical soil-plant systems.

Remobilization of zinc (Zn) from shoot to grain contributes significantly to Zn grain concentrations and thereby to food quality. On the other hand, strong accumulation of cadmium (Cd) in grain is detrimental for food quality. Zinc concentrations and isotope ratios were measured in wheat shoots (Triticum aestivum) at different growth stages to elucidate Zn pathways and processes in the shoot during grain filling. Zinc mass significantly decreased while heavy Zn isotopes accumulated in straw during grain filling (Δ66 Znfull maturity-flowering  = 0.21-0.31‰). Three quarters of the Zn mass in the shoot moved to the grains, which were enriched in light Zn isotopes relative to the straw (Δ66 Zngrain-straw -0.21 to -0.31‰). Light Zn isotopes accumulated in phloem sinks while heavy isotopes were retained in phloem sources likely because of apoplastic retention and compartmentalization. Unlike for Zn, an accumulation of heavy Cd isotopes in grains has previously been shown. The opposing isotope fractionation of Zn and Cd might be caused by distinct affinities of Zn and Cd to oxygen, nitrogen, and sulfur ligands. Thus, combined Zn and Cd isotope analysis provides a novel tool to study biochemical processes that separate these elements in plants.

Cadmium Isotope Fractionation in Soil-Wheat Systems.

Analyses of stable metal isotope ratios constitute a novel tool in order to improve our understanding of biogeochemical processes in soil-plant systems. In this study, we used such measurements to assess Cd uptake and transport in wheat grown on three agricultural soils under controlled conditions. Isotope ratios of Cd were determined in the bulk C and A horizons, in the Ca(NO3)2-extractable Cd soil pool, and in roots, straw, and grains. The Ca(NO3)2-extractable Cd was isotopically heavier than the Cd in the bulk A horizon (Δ(114/110)Cdextract-Ahorizon = 0.16 to 0.45‰). The wheat plants were slightly enriched in light isotopes relative to the Ca(NO3)2-extractable Cd or showed no significant difference (Δ(114/110)Cdwheat-extract = -0.21 to 0.03‰). Among the plant parts, Cd isotopes were markedly fractionated: straw was isotopically heavier than roots (Δ(114/110)Cdstraw-root = 0.21 to 0.41‰), and grains were heavier than straw (Δ(114/110)Cdgrain-straw = 0.10 to 0.51‰). We suggest that the enrichment of heavy isotopes in the wheat grains was caused by mechanisms avoiding the accumulation of Cd in grains, such as the chelation of light Cd isotopes by thiol-containing peptides in roots and straw. These results demonstrate that Cd isotopes are significantly and systematically fractionated in soil-wheat systems, and the fractionation patterns provide information on the biogeochemical processes in these systems.