Critical Review of "Effect of oral administration of chlorine dioxide on hematological, physiological parameters and intestinal microbiota in a murine model"

The paper by Juárez-Trujillo et al. (2024) investigates the effects of oral chlorine dioxide (ClO₂, referred to as CD in the paper) administration on hematological, physiological, and intestinal microbiota parameters in a murine model. While the study addresses a relevant topic given ClO₂’s controversial use as a disinfectant and purported therapeutic agent, it is marred by significant methodological, analytical, and interpretive deficiencies. The omission of Oxidation-Reduction Potential (ORP) and redox chemistry considerations, vague toxicity assessments, inadequate reporting of experimental details, and other critical flaws severely undermine the study’s scientific rigor and conclusions. Below, I outline these issues in detail, emphasizing the specified areas of concern.

1. Omission of Oxidation-Reduction Potential (ORP) and Redox Chemistry

A fundamental flaw in the paper is the complete absence of any discussion or measurement of ORP, which is critical for understanding ClO₂’s oxidative mechanisms. ClO₂ is a potent oxidant, and its antimicrobial and toxicological effects are driven by its ability to alter redox environments in biological systems, including microbial cells and host tissues. ORP quantifies the electron transfer potential that governs ClO₂’s interactions with cellular components such as amino acids, nucleotides, and gut microbiota, as noted in the paper’s mention of chemical oxidation via electron transfer. However, the authors fail to measure or discuss ORP, leaving a significant gap in contextualizing ClO₂’s effects. For instance, the paper reports changes in gut microbiota composition (e.g., increased Firmicutes, decreased Bacteroidetes) but does not explore how ClO₂’s oxidative stress, modulated by redox potential, might selectively affect microbial taxa with varying redox tolerances. Similarly, the reported hematological toxicity (e.g., methemoglobinemia) is inherently redox-driven, as ClO₂ oxidizes hemoglobin to methemoglobin, yet the paper does not discuss the redox conditions required for this reaction. Without ORP data, claims about ClO₂’s mechanisms and toxicity are speculative and lack mechanistic grounding, significantly weakening the study’s scientific validity.

2. Vague and Unsupported Toxicity Claims

The paper’s assessment of ClO₂’s toxicity is alarmingly vague, lacking precise quantitative data and detailed methodologies. The authors report that ClO₂ at doses of 2, 4, 8, and 10 mg/kg body weight caused “toxicity in hematological parameters,” colon and cecum damage, and reduced weight gain in rats. However, they provide no specific data on the nature or severity of hematological changes (e.g., percentage decrease in red blood cell counts, hemoglobin levels, or methemoglobin concentrations). Toxicity is dose-dependent, and without reporting exact measurements, exposure durations, or thresholds for adverse effects, these claims are scientifically inadequate. For example, the paper mentions methemoglobinemia as a potential effect but does not quantify methemoglobin levels or compare them to established toxicological benchmarks. Similarly, the claim of “colon and cecum damage” is supported only by unspecified histological observations, with no description of lesion severity, extent, or statistical significance. The assertion that “even low doses” of ClO₂ are harmful is not substantiated, as the lowest dose tested (2 mg/kg) is not contextualized against regulatory limits (e.g., EPA’s 0.8 mg/L for drinking water, equivalent to ~0.08 mg/kg/day for humans). The lack of dose-response relationships or comparisons to control groups further undermines the toxicity conclusions.

3. Inadequate Methodological Detail

The methodology section is woefully incomplete, lacking critical details necessary for reproducibility and evaluation. The paper states that ClO₂ was produced at 16,578.62 mg/L (16,578 ppm) using sodium chlorite and citric acid, but it does not describe the preparation protocol, reaction conditions (e.g., pH, temperature), or quality control measures to ensure purity and stability. The reported concentration is extraordinarily high compared to typical disinfectant levels (e.g., 0.1–50 ppm), raising questions about its relevance to real-world applications, yet this is not justified. The dosing regimen (2, 4, 8, 10 mg/kg for 90 days) is described, but the method of oral administration (e.g., gavage, drinking water) is not specified, nor is the volume or frequency of doses. The paper also fails to report the number of rats per group, the statistical power of the study, or the specific hematological and physiological assays used. For microbiota analysis, the authors note changes in Firmicutes and Bacteroidetes percentages but do not describe the sequencing platform, depth of sequencing, or bioinformatics pipeline, making it impossible to assess the reliability of these findings. Such omissions erode confidence in the data’s validity and the study’s conclusions.

4. Inconsistent and Confusing Reporting of ClO₂ Concentrations

The paper’s reporting of ClO₂ concentrations is inconsistent and confusing, undermining its clarity and credibility. The initial ClO₂ concentration is reported as 16,578.62 mg/L, which is orders of magnitude higher than concentrations cited in referenced studies (e.g., 25–50 ppm for nasal irrigation, Cao et al., 2022). The authors do not explain how this stock solution was diluted to achieve doses of 2–10 mg/kg or whether the administered solution was stabilized to prevent degradation. The paper also uses “ppm” and “mg/L” interchangeably without clarifying conversions, and it fails to account for ClO₂’s volatility and reactivity, which could affect actual exposure levels. For example, the claim that ClO₂ remained stable for four weeks is contradicted by a “significant decrease” in week five, yet no quantitative data (e.g., percentage loss) or stability assays are provided. This lack of precision in reporting concentrations is particularly problematic given ClO₂’s dose-dependent toxicity.

5. Overgeneralized and Unsupported Conclusions

The paper’s conclusions are overly broad and not fully supported by the data. The authors state that “even low doses of CD can negatively affect the microbiota, the morphology of the cecum and colon, and body weight,” yet the lowest dose tested (2 mg/kg) is not necessarily “low” compared to human exposure levels (e.g., ~0.08 mg/kg/day from drinking water). The claim that prolonged consumption should be avoided is reasonable but lacks specificity regarding safe exposure limits or durations. The paper also overgeneralizes ClO₂’s effects on gut microbiota, asserting “significant change” based on shifts in Firmicutes and Bacteroidetes without discussing functional implications or comparing these changes to other microbiota-disrupting agents. The conclusion that ClO₂ has a “significant antimicrobial effect” on dietary yeasts versus probiotics is not supported by detailed in vitro results, as the paper only briefly mentions this finding without quantitative data or statistical analysis.

6. Lack of Statistical Rigor

The paper’s statistical analysis is inadequate, further weakening its findings. The authors report “significant changes” in microbiota composition and hematological parameters but do not provide statistical tests (e.g., ANOVA, t-tests), p-values, or effect sizes to support these claims. Without statistical rigor, it is impossible to determine whether observed differences are meaningful or due to random variation. The microbiota data, showing Firmicutes at 81.9–87.1% in ClO₂ groups versus 63.05% in controls, and Bacteroidetes at 3.4–4.5% versus 22.5%, lack error bars or confidence intervals, making it difficult to assess variability or significance. The absence of statistical details for histological or weight loss findings further undermines the study’s credibility.

7. Neglect of Redox-Driven Microbiota Interactions

The paper’s discussion of gut microbiota changes is superficial and fails to consider redox-driven mechanisms. ClO₂’s oxidative properties likely influence microbial communities by altering the gut’s redox environment, yet the authors do not explore how these changes affect microbial metabolism, redox homeostasis, or interactions with host tissues. For example, Bacteroidetes, which decreased in ClO₂-treated rats, are known to thrive in specific redox niches, but the paper does not discuss whether their reduction is linked to oxidative stress. Similarly, the increase in Firmicutes is not contextualized with their redox tolerance or functional roles. The lack of ORP measurements or redox-sensitive assays (e.g., reactive oxygen species levels) limits the study’s ability to elucidate ClO₂’s microbiota effects mechanistically.

8. Poor Integration with Existing Literature

The paper fails to adequately situate its findings within the broader literature on ClO₂ toxicity and microbiota research. While it cites studies like Sciurba et al. (2021) and Sultan et al. (2014), it does not critically evaluate their methodologies or reconcile conflicting findings (e.g., Sciurba’s “minimal” microbiota changes versus the current study’s “significant” changes). The paper also overlooks key redox-based studies on disinfectants or microbiota-modulating agents, limiting its contribution to the field. For instance, it does not compare ClO₂’s effects to other oxidants (e.g., hydrogen peroxide) or discuss why ClO₂’s toxicity profile might differ. This insularity suggests a lack of scholarly diligence.

9. Formatting and Presentation Issues

The paper suffers from poor presentation, including typographical errors and unclear figures. The abstract refers to “than even low doses” (likely a typo for “that even low doses”), and the introduction contains awkward phrasing (e.g., “it is necessary to know that the effectiveness”). The graphical abstract is not described in the text, and Figure 1 (ClO₂ stability) is vaguely referenced without detailed data. The use of “CD” instead of ClO₂ is inconsistent with standard chemical nomenclature, and the paper does not define abbreviations like “DBP” (disinfection byproducts) upon first use. These issues hinder readability and professionalism.

Recommendations for Improvement

To address these deficiencies, the authors should:

• Measure and report ORP to contextualize ClO₂’s redox-driven effects on microbiota and host tissues.
• Provide precise quantitative data on hematological changes, histological damage, and weight loss, including dose-response relationships and statistical analyses.
• Detail experimental methods, including ClO₂ preparation, dosing protocols, sample sizes, and microbiota sequencing pipelines.
• Use consistent units for ClO₂ concentrations and clarify dilution procedures.
• Conduct robust statistical analyses, reporting tests, p-values, and effect sizes.
• Explore redox-driven microbiota interactions, including oxidative stress markers and microbial functional changes.
• Integrate findings with broader literature, critically evaluating prior studies.
• Correct typographical errors and improve figure clarity and nomenclature consistency.

Conclusion

The paper by Juárez-Trujillo et al. (2024) aims to evaluate ClO₂’s effects on a murine model but is severely undermined by critical flaws. The omission of ORP and redox considerations, vague toxicity claims, inadequate methodological detail, and lack of statistical rigor render the study scientifically inadequate. These shortcomings, combined with inconsistent reporting and poor literature integration, limit the paper’s credibility and utility. Significant revisions are needed to produce a robust, evidence-based study that meaningfully contributes to understanding ClO₂’s toxicological profile.