Beltcheva et al. 2025 - clinoptilolite and cadmium toxicity in mice
Beltcheva and colleagues tested whether Na-exchanged, tribo-activated natural clinoptilolite from the Beli Plast deposit in Bulgaria could reduce cadmium toxicity in an ICR mouse model. This is primary toxicology and mitigation evidence for Cd bioaccumulation, excretion, hematology, oxidative stress, and micronucleus formation. It is not a food, ingredient, supplement, or consumer-product occurrence study.
Key numbers
Clinoptilolite material
The Beli Plast clinoptilolite-rich tuff was selected because it met the cited EU suitability criteria for sedimentary clinoptilolite used as a feed additive: greater than 80% clinoptilolite, clay minerals below 20%, and no quartz detected. The authors state that Pb and Cd were below the maximum permissible values of 60 ppm and 5 ppm, respectively, and As was below the detection limit. Exact trace-element values were placed in Supplementary Table S1 rather than the main article.
PXRD estimated the Beli Plast material as about 86 wt.% clinoptilolite, 7 wt.% opal-CT, 2 wt.% mica, 2 wt.% montmorillonite, and 2 wt.% plagioclase. Major oxide composition changed after Na exchange:
| Material | SiO2 | Al2O3 | Fe2O3(t) | MgO | CaO | Na2O | K2O | LOI | Total |
|---|---|---|---|---|---|---|---|---|---|
| Natural clinoptilolite tuff | 71.08 | 10.88 | 0.96 | 1.03 | 2.78 | 0.31 | 2.76 | 9.3 | 101.77 |
| Na-exchanged sample | 69.42 | 10.63 | 0.82 | 0.58 | 0.90 | 4.26 | 2.46 | 9.13 | 101.98 |
Electron-probe microanalysis of separate clinoptilolite grains gave Si/Al ratios from 4.22 to 4.71. The Na-exchanged and tribo-activated powder had multimodal particle sizes, with dominant laser-diffraction grains from 5 to 45 micrometers and DLS peaks near 260 nm and 1600 nm for finer suspended particles. BET surface area was 24.2 m2/g and total pore volume was reported as 0.051 cm2/g.
Exposure design
The study used four groups: control (C), clinoptilolite-only feed (Z), Cd(NO3)2 drinking water (CDw), and Cd(NO3)2 drinking water plus clinoptilolite-supplemented feed (CDwZ). The clinoptilolite diet contained 12.5 wt.% powdered modified natural clinoptilolite. The Cd exposure was 0.00125 M Cd(NO3)2 in drinking water and was described by the authors as an environmentally realistic intermediate dose for industrially polluted-area exposure simulation.
Sampling occurred at days 0, 15, 30, and 45. Each group was designed for 32 mice, yielding 128 planned animals. Two mice were removed because of severe distress or physiological imbalance, leaving 126 included animals.
Cadmium bioaccumulation and excretion
Cd concentrations in the livers and kidneys of control and clinoptilolite-only mice were below the method detection limit. Table 3 reports Cd in organs and feces as mg/kg dry weight:
| Day | CDw liver | CDw kidney | CDw feces | CDwZ liver | CDwZ kidney | CDwZ feces |
|---|---|---|---|---|---|---|
| 0 | 0.3 +/- 0.1 | 0.2 +/- 0.1 | 0 | 0.3 +/- 0.1 | 0.2 +/- 0.1 | 0 |
| 15 | 56.2 +/- 1.1 | 53.1 +/- 1.8 | 113.0 +/- 2.8 | 44.2 +/- 1.6 | 39.1 +/- 0.9 | 156.0 +/- 3.2 |
| 30 | 96.0 +/- 3.6 | 104.0 +/- 12.6 | 153.0 +/- 13.4 | 78.0 +/- 12.1 | 74.6 +/- 11.8 | 200.0 +/- 22.1 |
| 45 | 173.0 +/- 11.9 | 260.0 +/- 23.1 | 177.0 +/- 13.1 | 90.0 +/- 12.3 | 100.0 +/- 12.4 | 276.0 +/- 22.6 |
By day 45, the clinoptilolite-supplemented Cd group had about 48% lower liver Cd and 61% lower kidney Cd than the Cd-only group. Fecal Cd was higher in the CDwZ group; the authors summarize clinoptilolite-aided fecal excretion as rising from 28% to 36% across the experiment.
Bioaccumulation coefficients, calculated as Cd45/Cd15, were reported as 3.07 liver, 4.90 kidney, and 1.54 feces for CDw mice, compared with 2.04 liver, 2.55 kidney, and 1.77 feces for CDwZ mice.
Growth, hematology, oxidative stress, and micronuclei
By day 45, body-mass gain was 39.15% in the clinoptilolite-only group, 42.52% in the Cd+clinoptilolite group, 37.34% in controls, and 22.0% in the Cd-only group. The average day-45 weight difference between CDw and CDwZ mice was about 5 g.
Selected hematology findings from Table 4:
| Endpoint | Cd-only pattern | Cd+clinoptilolite pattern |
|---|---|---|
| WBC | Elevated at days 15 and 30: 9.6 +/- 1.9 and 11.7 +/- 2.5 g/L | Lower than CDw at days 15 and 30: 4.6 +/- 2.0 and 6.0 +/- 1.3 g/L |
| Monocytes | Elevated in CDw at days 15, 30, and 45: 12.9 +/- 0.22, 10.4 +/- 0.62, and 12.4 +/- 0.18 x10^9/L | Lower in CDwZ: 2.1 +/- 0.06, 1.7 +/- 0.03, and 2.2 +/- 0.06 x10^9/L |
| Hemoglobin | Lower in CDw at days 15, 30, and 45: 36.3 +/- 24.0, 28.3 +/- 16.6, and 22.7 +/- 11.7 g/L | Rebounded in CDwZ: 45.0 +/- 5.9, 56.0 +/- 25.2, and 69.9 +/- 14.4 g/L |
| RBC | CDw declined to 1.8 +/- 1.2 x10^12/L by day 45 | CDwZ was 5.3 +/- 3.5 x10^12/L by day 45 |
For oxidative stress, Cd(NO3)2 increased MDA in liver and kidney by 41.38% and 40.79%, respectively, on day 15 compared with controls, while GSH decreased in liver and kidney. The authors report an inverse MDA-GSH relationship (p < 0.001, r = -0.947). Clinoptilolite supplementation increased GSH and decreased MDA relative to Cd-only mice, although levels did not fully return to control values.
The micronucleus assay showed a significant time-related increase in micronucleated erythrocytes in Cd-only mice. No elevated micronucleus levels were observed in the Cd+clinoptilolite group, where values stayed near control levels.
Methods (brief)
Clinoptilolite-rich tuffs from four Bulgarian deposits were screened, and Beli Plast material was selected for the animal experiment. The fraction below 0.15 mm was treated with repeated 1 M sodium nitrate exchange at 80 degrees C, washed, dried, and ball-milled. Mineral and chemical characterization used PXRD, ICP-OES, LA-ICP-MS, SEM/EPMA, laser diffraction, DLS, and nitrogen adsorption/desorption.
Male ICR (CD-1) mice were randomized into four groups with block randomization. Cd was administered as 0.00125 M Cd(NO3)2 in drinking water. Clinoptilolite was administered as 12.5 wt.% in standard rodent feed. Cd in organs and feces was measured by ICP-MS, with calibration over 0.01-500 micrograms/L and reference-material checks. Hematology used automated veterinary analysis. Oxidative stress was assessed by MDA/TBARS and total GSH in liver and kidney homogenates. Genotoxicity used an acridine-orange erythrocyte micronucleus assay, with 2000 erythrocytes counted per animal.
Implications
Certification: Do not use this source in product or ingredient occurrence pools. The source does not report market-sampled food, supplement, or consumer-product concentrations; it reports a controlled animal Cd exposure experiment and a material-suitability screen for the clinoptilolite sorbent.
App: Supports toxicology and mitigation context for Cd absorption, tissue distribution, fecal excretion, oxidative stress, hematology, and gut-binding sorbents. It may help explain why sorbent co-formulation is a mitigation mechanism distinct from reducing metal concentration in the underlying food matrix.
Courses: Useful for teaching the separation between exposure concentration, internal dose, excretion, biomarker response, and product occurrence.
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Verification notes
This page was built from the full PDF, including the abstract, clinoptilolite selection and preparation methods, animal-exposure design, analytical methods, Tables 1-4, Figures 1-7, discussion, conclusions, supplementary-materials note, and data-availability statement. Main-text trace-element values for the clinoptilolite material are limited to below-limit/below-detection statements; exact multi-element concentrations are in Supplementary Table S1. Products and ingredients are intentionally empty because no existing clinoptilolite ingredient page exists and the source is not a market occurrence study.
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 |