Makarova et al. 2021 - Trifolium phytoextraction amendments
Makarova and colleagues tested whether K2HEDP, plant growth regulators, and iron chelate could improve or redirect Cd, Ni, and Cu phytoextraction by white clover seedlings compared with Na2EDTA. This is primary laboratory phytoremediation evidence in plant shoots and roots, not occurrence evidence for a food, ingredient, supplement, or consumer product.
Key numbers
Experimental contamination and amendments
The simulated multi-metal substrate was universal soil plus fertilizer, spiked per vegetation pot with Ni(NO3)2.6H2O at 38.2 mg, CuSO4.5H2O at 23.18 mg, and Cd(NO3)2.4H2O at 15.65 mg. The authors state these additions corresponded to five times the Russian soil maximum permissible concentration for each metal.
Each pot received 20 Trifolium repens L. seeds. Amendments were applied in triplicate variants: no amendment, Na2EDTA, K2HEDP, K2HEDP + GA + IAA, and K2HEDP + GA + IAA + Na(FeEDDHA), plus clean-soil controls. Chelating-agent doses were split across five daily additions from day 20 to day 25 after planting. Plants were harvested on day 31 and separated into shoots and roots.
Shoot and root concentrations
Table 4 reports seedling organ mass and Ni, Cu, and Cd concentrations. Concentrations are in ug/g, and the relative standard deviation for all concentration elements did not exceed 0.3 ug/g.
| Variant | Shoot mass (g) | Shoot Ni | Shoot Cu | Shoot Cd | Root mass (g) | Root Ni | Root Cu | Root Cd |
|---|---|---|---|---|---|---|---|---|
| Clean soil | 0.278 | 6.4 | 2.1 | 0.19 | 0.024 | 25.3 | 5.6 | 1.4 |
| Ni/Cu/Cd, no amendment | 0.24 | 9.1 | 6.7 | 10.1 | 0.014 | 19.0 | 17.4 | 189 |
| Ni/Cu/Cd + Na2EDTA | 0.10 | 54.3 | 40.4 | 32.9 | 0.009 | 27.0 | 41.0 | 56.7 |
| Ni/Cu/Cd + K2HEDP | 0.14 | 13.5 | 7.6 | 3.7 | 0.007 | 49.9 | 27.5 | 123 |
| Ni/Cu/Cd + K2HEDP + GA + IAA | 0.18 | 7.8 | 5.2 | 10.4 | 0.012 | 28.0 | 13.3 | 70.9 |
| Ni/Cu/Cd + K2HEDP + GA + IAA + Na(FeEDDHA) | 0.23 | 9.7 | 4.2 | 8.5 | 0.010 | 26.0 | 12.6 | 144 |
The authors report that Na2EDTA produced the strongest increase in shoot uptake: shoot concentrations increased by 5.96x for Ni, 6.03x for Cu, and 3.26x for Cd compared with the no-amendment contaminated treatment. In roots, Na2EDTA increased Ni and Cu by 1.42x and 2.36x, respectively, but root Cd was 3.33x lower than the no-amendment contaminated treatment.
Accumulation and translocation
Na2EDTA gave the highest shoot-to-root translocation coefficient for Ni and also increased Cu distribution into shoots. The paper reports total Ni accumulation across three pots of 5.4 ug for Na2EDTA and 2.5 ug for K2HEDP + GA + IAA + Na(FeEDDHA). Total Cu accumulation was highest with Na2EDTA at 4.2 ug; the no-amendment treatment accumulated 1.9 ug Cu.
Cd accumulated most intensely in roots without amendments. The no-amendment contaminated plants accumulated 5.1 ug Cd across three pots, while Na2EDTA accumulated 3.6 ug and K2HEDP + PGRs + iron chelate accumulated 3.4 ug. K2HEDP reduced Cd in both shoots and roots relative to the no-amendment treatment and decreased the Cd translocation coefficient.
Across whole seedlings, total accumulation with Na2EDTA reached 13.88 ug, 48% higher than no amendment. K2HEDP alone had an inhibitory or stabilizing effect, while K2HEDP + GA + IAA and K2HEDP + GA + IAA + Na(FeEDDHA) produced total bioaccumulation values 11% and 44% higher than K2HEDP alone because biomass improved.
Plant condition
The visual plant-condition table reports high shoot turgor and green leaves for clean soil, no-amendment contaminated soil, K2HEDP, K2HEDP + GA + IAA, and K2HEDP + GA + IAA + Na(FeEDDHA). Na2EDTA-treated seedlings had low turgor, significant growth suppression, chlorosis, and pale-green leaf plates. The authors interpret this as a strong negative effect of Na2EDTA on plant growth and viability.
Methods (brief)
The pot experiment followed ISO 22030:2005 for chronic toxicity in higher plants. Universal soil was mixed with NPK fertilizer and spiked with Ni, Cu, and Cd salts. White clover seedlings were harvested 31 days after planting. Samples were washed, separated into shoots and roots, digested by autoclaved acid decomposition, and analyzed by ICP methods: AES-ICP for selected light elements and MS-ICP for Ni, Cu, Cd, and a broader elemental panel.
The study also compared Ni behavior under polymetallic contamination with Ni-only contamination. The absolute Ni content in white-clover organs was higher when the soil was not also contaminated with Cu and Cd. In both polymetallic and Ni-only settings, Na2EDTA showed a clearer positive effect on Ni uptake than K2HEDP.
Implications
Certification: Do not use this source in product occurrence pools. The matrix is white-clover tissue from a laboratory phytoremediation experiment, not an edible commodity or consumer product.
App: Context for remediation notes involving chelator-assisted phytoextraction, EDTA risk tradeoffs, K2HEDP, and growth-regulator/iron-chelate biomass support.
Courses: Useful for teaching how a treatment can increase metal translocation while reducing plant health, and why root/shoot partitioning must be preserved when routing remediation evidence.
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Verification notes
This page was built from the full PDF, including the abstract, problem formulation, materials and methods, Tables 1-4, Figures 1-9, discussion, conclusions, and conflict statement. Products and ingredients are intentionally empty because the source reports simulated-soil phytoextraction and white-clover tissue concentrations only. The reported metal values are total elemental concentrations in plant organs after acid digestion; no speciation split is provided for Cd, Ni, or Cu.
Page history
The five most recent substantive edits to this page. The full version history lives in git; when DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.
| Commit | Date | Description |
|---|---|---|
| c1aef38 | 2026-06-02 | audit-queue: hamid2021-bacterial-plant-biostimulants-review → audited-promote |