Beltcheva et al. 2025 - Modified clinoptilolite as oral Cd sorbent in ICR mice

Beltcheva and colleagues ran a 45-day in vivo experiment testing whether Na-exchanged and tribo-activated natural clinoptilolite from the Beli Plast deposit in Bulgaria, administered as a 12.5 wt.% dietary supplement, mitigates cadmium toxicity in male ICR (CD-1) mice exposed to environmentally relevant Cd through drinking water. This is primary remediation-mechanism evidence in a mammalian model, not food or product occurrence data: the measured endpoints are Cd accumulation in liver, kidney, and feces (ICP-MS), hematological indices, oxidative stress markers (MDA and GSH in liver and kidney homogenates), body and organ mass, and genotoxicity (peripheral-blood micronucleus frequency).

Key numbers

Experimental design

Four groups of 32 mice each (total n = 128, reduced to 126 after two distress-related exclusions), four sampling time points (day 0, 15, 30, 45). Effect size 0.4, alpha 0.05, power 0.95; eight mice per time point per group. Cd(NO3)2 concentration in drinking water 0.00125 M. Clinoptilolite-supplemented food was 12.5 wt.% Na-exchanged tribo-activated Beli Plast clinoptilolite. Acclimatisation seven days; ambient 20-22 C, humidity 45-60%, 12:12 h light:dark; ad libitum standard pelleted rodent food and water.

Clinoptilolite material characterisation

Beli Plast tuff mineral composition (PXRD, wt.%): clinoptilolite ~86, opal-CT ~7, mica (10 A) ~2, montmorillonite ~2, plagioclase ~2.

Chemical composition of natural vs Na-exchanged sample (ICP-OES, wt.%):

Si/Al ratios from EPMA on seven grains ranged 4.22 to 4.71. BET specific surface area of Na-exchanged tribo-activated material 24.2 m2/g; total pore volume 0.051 cm3/g. Particle size distribution multimodal with a fine population near 260 nm and a coarse population averaging 1696.6 nm (range 1600-1800 nm). Beli Plast Pb and Cd levels were below the EU Regulation 744/2012 feed-additive permissible maxima of 60 ppm and 5 ppm respectively, and As was below the detection limit (Table S1).

Cadmium bioaccumulation (Table 3, mg/kg dry weight, mean +/- SD)

C and Z group livers and kidneys were below the ICP-MS detection limit at all time points. CDw and CDwZ groups:

By day 45, Na-modified clinoptilolite supplementation produced a roughly 48% reduction in liver Cd, 61% reduction in kidney Cd, and 36% increase in faecal Cd excretion in the CDwZ group compared to the CDw group (all p < 0.001 to p < 0.0001 per Table 3 footnote). Liver-Cd reduction by clinoptilolite ranged from 19% on day 15 to 48% on day 45; kidney-Cd reduction ranged from 26% to 61% over the same interval. Faecal Cd excretion attributable to clinoptilolite-mediated trapping rose from 28% to 36% across the experiment.

Bioaccumulation coefficients (Cd45/Cd15): CDw liver 3.07, kidney 4.90, feces 1.54; CDwZ liver 2.04, kidney 2.55, feces 1.77.

Growth (Figure 4)

Body-mass percentage gain over 45 days: Z group 41.68%, CDwZ 42.52%, C 37.34%, CDw 22.0%. CDw mice lost weight by day 15 then partially rebounded; final day-45 body mass in CDw was 12.89% lower than in C and Z controls (p < 0.001). CDwZ recovered to within ~5.0 g (~15%) of CDw by day 45 (p < 0.05).

Organ indices (Figure 5)

Liver index: CDw declined significantly by day 45 vs the day-0 control baseline (p < 0.0001); Z and CDwZ liver indices normalised to control values by days 30 and 45. Kidney index: CDw on day 45 was significantly lower than C on both day 0 and day 45 (p < 0.001); Z group did not differ from control.

Hematology (Table 4, mean +/- SD, n = 32 per group at study start)

Headline values at day 45:

CDw mice exhibited hypochromic microcytic anaemia: significantly decreased Hb, RBC, and Hct relative to day-0 controls (p < 0.05) and elevated WBC and MO leukocyte markers. CDwZ values returned toward normal erythrogram and leukogram ranges by day 15 and held those values through day 45.

Oxidative stress (Figure 6)

Liver MDA at day 15 rose by 41.38% in CDw vs control (p < 0.01); kidney MDA by 40.79% (p < 0.01). Per the paper text, liver and kidney GSH in CDw fell by 185.71% and 235.04% respectively at day 15 relative to control (the source uses the percentage-decrease wording verbatim; this corresponds to roughly 2.9-fold and 3.4-fold reductions). CDw MDA peaked at day 15 (liver ~0.0029 µmol/g, kidney ~0.004 µmol/g) and declined thereafter. CDwZ MDA was significantly lower than CDw across days 15-45 (p < 0.05); CDwZ GSH was significantly higher than CDw across days 15-45 (p < 0.05) though it did not fully return to control levels by day 45. A strong inverse MDA-GSH correlation was reported (r = -0.947, p < 0.001).

Genotoxicity / micronuclei (Figure 7, MN per 1000 erythrocytes)

Values read from Figure 7. MN frequency rose significantly over time in CDw (p < 0.0001); CDwZ values remained close to control and Z-group levels with no significant elevation. Day-45 CDwZ vs CDw difference was statistically significant (p < 0.05).

Methods (brief)

Clinoptilolite preparation

Bulgarian clinoptilolite-rich tuff from the Beli Plast deposit (Eastern Rhodopes, related to first Rupelian acid Paleogene volcanic phase) was ground and sieved to the < 0.15 mm fraction. Na-exchange: 100 g natural material stirred with 1000 mL of 1 M NaNO3 (Merck Cat. No. 104711) at 500 rpm and 80 C in a closed bottle; NaNO3 solution replaced daily, procedure repeated 12 times; washed with deionised water and dried at 60 C. Material then mechanically tribo-activated (ball-mill) to reduce particle size and increase surface area.

Cd dosing and animal husbandry

Cd(NO3)2 0.00125 M (Merck Cat. No. 25154) in deionised water as drinking water for CDw and CDwZ. Standard pelleted rodent food (Cd-free for C and CDw; 12.5 wt.% Na-exchanged tribo-activated clinoptilolite for Z and CDwZ). Male ICR (CD-1) mice 6-8 weeks, 25-30 g, bred at the Institute of Neurobiology, Bulgarian Academy of Science vivarium, housed in individual ventilated cages (Tecniplast ULTEM, Cat. No. 02-934-122) with GOLDSPAN bedding (ISO 2000-accredited supplier, Brandenburgs Unternehmensgruppe). Block randomisation (block size 8) via Excel RAND; blinding on group allocation, outcome assessment, and data analysis. Protocols approved by Institute of Biodiversity and Ecosystem Research Ethical Committee (protocol 001/18.03.2024); EU Directive 2010/63/EU, Bulgarian Ordinance No. 20/01.11.2013, Animal Protection Act of 31 January 2008.

Clinoptilolite characterisation methods

• PXRD: PANalytical EMPYREAN diffractometer (Malvern Panalytical, Almelo, The Netherlands), 240 mm goniometer radius, 40 kV / 30 mA, CuKalpha, 3D pixel detector, 2-theta 3-70 deg, 0.013 deg / 80 s scan rate, room temperature.
• ICP-OES for major elements and LA-ICP-MS for trace elements: PerkinElmer ELAN DRC-e ICP-MS (PerkinElmer, Concord, Canada) with New Wave UP193-FX excimer laser (ATLEX-LR, Wermelskirchen, Germany); concentrations averaged from three lithium-tetraborate-pellet measurements at 1050 C.
• SEM/EDS: ZEISS EVO 25LS (Carl Zeiss SMT Ltd., Cambridge, UK) with EDAX Trident system at 16 kV / ~1 nA, with reference standards and EPMA.
• Laser diffraction particle sizing: Mastersizer 3000 (Malvern Panalytical, Almelo, The Netherlands) with wet dispersion (ISO 13320:2020); < 6 µm fraction sized by DLS on a Brookhaven Bi-90 plus (Brookhaven Instruments Corporation, New York, USA) at 657 nm, 90 deg, 25 C, triplicate measurements (one run = three 3-min cycles); data analysed with Particle Solutions v3.6.0.7136.
• BET surface area: Micromeritics 3Flex (Micromeritics, Norcross, GA, USA), N2 adsorption/desorption at -196 C, samples outgassed at 200 C.

Cd determination in tissues and feces

ICP-MS: Perkin Elmer SCIEX DRC-e with cross-flow nebuliser; RF, gas flow, and lens voltage optimised to minimise CeO+/Ce+ and Ba2+/Ba+ ratios. Cd working standards 0.01-500 µg/L prepared from single-element calibration solution (Merck KGaA, Darmstadt, 1000 mg/L stock). External calibration on isotopes 111, 112, 113, 114 Cd; calibration r >= 0.99. Performance evaluated against certified reference materials IAEA-H-8 horse kidney and NIES N6 mussel; results consistent with certified values.

Biological assays

• Body weight at every time point; organ index (%) = mean organ mass / mean body mass x 100.
• Hematology: tail-vein blood into 0.5 mL EDTA-K3 microtubes (Merck Cat. No. 65501-24-8), MindRay BC-30Vet automated veterinary haematology analyser; parameters RBC, WBC, Hb, Hct, MCV, MCH plus differential.
• MDA: TBARS test on liver and kidney homogenates, absorbance 532 and 600 nm, expressed as nmol MDA / g fresh weight.
• GSH: colorimetric, expressed as mmol GSH / g tissue.
• Micronucleus test: modified acridine orange staining (Merck Cat. No. 113000) with Sorensen’s sodium phosphate buffer (pH 7.4); OPTIKA B-383 FL fluorescence microscope at 400x, blue filter 460-490 nm excitation, 515 nm emission; MN per 2000 erythrocytes per animal, expressed per thousand.

Statistics

A priori power F test for one-way ANOVA fixed effects (G*Power 3.1.9.4, Heinrich-Heine University Dusseldorf); effect size 0.4, alpha 0.05, power 0.95, n = 8 per time point per group. Analyses in Prism 9.0 (GraphPad Software, San Diego, CA). Normality (D’Agostino-Pearson) and variance homogeneity (Levene F-test) assessed; haematology by one-way ANOVA with Tukey post hoc; MN data non-normal, by Kruskal-Wallis with Dunn post hoc. Significance threshold p ⇐ 0.05.

Implications

Certification: Do not use this source in any food, infant-food, supplement, or cosmetic occurrence pool. It does not measure consumer-product Cd concentrations or document a reduction in a food matrix consumed by humans. It is relevant only as remediation-mechanism evidence for Na-exchanged tribo-activated clinoptilolite as an oral Cd sorbent in mammalian models, and as supporting context for the EU’s existing authorisation of sedimentary clinoptilolite as a feed additive under Commission Implementing Regulation (EU) No 651/2013 (referenced as [52] in the source) and feed-additive limits under Commission Regulation (EU) No 744/2012 (referenced as [51] in the source; 60 ppm Pb and 5 ppm Cd in the additive itself).

App: Context for upstream-mitigation and dietary-binder notes. Key takeaways: a 45-day dietary supplementation with Na-exchanged tribo-activated clinoptilolite from the Beli Plast (Bulgaria) deposit at 12.5 wt.% reduced ICP-MS-measured Cd burden in mouse liver by ~48%, kidney by ~61%, and increased faecal Cd output by ~36% relative to Cd-only controls, with concurrent rescue of hypochromic microcytic anaemia, normalisation of oxidative stress markers (MDA and GSH in liver and kidney), and prevention of micronucleus elevation. The Cd exposure (0.00125 M Cd(NO3)2 in drinking water) was framed by the authors as environmentally realistic for industrially polluted areas.

Courses: Useful for teaching the distinction between sorbent-bound and bioavailable metal in the gastrointestinal tract, the rationale for Na-exchange and tribo-activation modifications to natural zeolite to enhance Cd2+ ion-exchange capacity, the link between Cd-induced GSH depletion and lipid peroxidation, and the use of peripheral-blood micronucleus frequency as a genotoxicity readout in small-mammal models.

Wiki pages this source may touch

• cadmium
• remediation-evidence
• index

Verification notes

Page built from the full PDF including the abstract, Sections 2.1-2.9 (clinoptilolite source and sample collection, Na-exchange protocol, characterisation methods, experimental design and animal husbandry, heavy metal loading by ICP-MS, weight and organ index assays, hematology, oxidative stress markers, micronucleus test, statistics), Sections 3.1-3.7 (clinoptilolite characterisation, Cd bioaccumulation, growth rate, organ index, hematology, oxidative stress, micronucleus), Sections 4.1-4.6 (discussion), Section 5 (conclusions), Tables 1-4 and Figures 1-7 with references through [120].

products and ingredients frontmatter are intentionally empty: no food, ingredient, or consumer-product matrix was sampled. Clinoptilolite is regulated as a feed additive in the EU (Regulation 651/2013) but is not currently a slug in the wiki’s ingredients or products taxonomy snapshot. The matrices slugs (cd-spiked-drinking-water, mouse-liver-tissue, mouse-kidney-tissue, mouse-feces, clinoptilolite-feed-additive) are non-food-matrix proposals outside the project’s current controlled vocabulary; a future taxonomy refresh should decide whether to formalise an animal-model and remediation-matrix vocabulary or to route mammalian-model sources via a separate non-food register (consistent with the disposition recorded on pan2021-lignosulfonate-chitosan-pb-adsorbent). The ## Wiki pages this source may touch entries [[mitigation/remediation-evidence]] and [[testing/index]] point to wiki directories outside the four-section (ingredients/products/metals/regulations) taxonomy snapshot used for slug-vocabulary auditing; they are speculative routing leads pending taxonomy expansion.

The paper-internal GSH wording “falling by 185.71% and 235.04%” in the Section 3.6 Results is reproduced verbatim above with a parenthetical reframing (roughly 2.9-fold and 3.4-fold reductions) for clarity; this is not a wiki-side numerical correction.

Brand-firewall scan: the source does not name any consumer-product brands attached to contamination values. All vendor names retained in the page (PerkinElmer ELAN DRC-e, Perkin Elmer SCIEX DRC-e, PANalytical EMPYREAN, Malvern Panalytical, ZEISS EVO 25LS, EDAX Trident, Brookhaven Bi-90 plus, Mastersizer 3000, Micromeritics 3Flex, MindRay BC-30Vet, OPTIKA B-383 FL, Merck/Merck KGaA reagent catalogues, IAEA-H-8 and NIES N6 reference materials, GraphPad Prism, G*Power, GOLDSPAN bedding, Tecniplast ULTEM caging) refer to scientific instruments, reagents, reference materials, software, or husbandry materials and are permitted per Part 12 Exception 2 (scientific-method vendor/material names).

Audit subagent (2026-06-02) flagged two units concerns; both verified against PDF p. 13 Table 4 header. Applied: MO column units corrected from MO (10^9/L) to MO (10^8/L) to match the source header. Documented: the wiki’s WBC column units (10^9/L) are a silent normalisation of the source’s WBC (g/L) (clearly a source-side typo, since g/L is a protein-concentration unit and the other leucocyte columns in the same table use 10^x/L); the underlying numerical values are unchanged. The source also reports an anomalous RBC value for the C (control) group at day 45 of 0.9 +/- 2.6 (mean smaller than SD, and an order of magnitude below the C group’s day-30 RBC of 5.3 +/- 2.0); the wiki reproduces this verbatim because the auditor confirmed it is in the source as printed, but downstream synthesis passes should treat the C day-45 RBC datum as a likely source-side transcription error.

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.