Research ArticleExperimental Studies
Open Access

Nutritional Assessment of Pesticide-associated Metabolic Stress in Plant-based Diets

RAMONA ALINA TOMUȚA, MARIA FLAVIA GÎTEA, MARC CRISTIAN GHITEA, EVELIN CLAUDIA GHITEA, TIMEA CLAUDIA GHITEA and FLORIN BANICA
In Vivo January 2026, 40 (1) 136-150; DOI: https://doi.org/10.21873/invivo.14179

Abstract

Background/Aim: Chronic low-dose pesticide exposure through high fruit and vegetable consumption is an underappreciated risk factor for metabolic dysfunction. While plant-based diets provide antioxidants and polyphenols, co-exposure to pesticide residues and heavy metals may induce subtle but clinically relevant biochemical disruptions.

Materials and Methods: We analyzed the detailed metabolomic organic acid profiles from 26 individuals reporting high intake of conventionally grown fruits and vegetables. Dietary modeling was performed to estimate daily polyphenol intake, while metabolomic data were evaluated for markers of detoxification stress, oxidative damage, mitochondrial function, gut dysbiosis, and heavy metal burden.

Results: Both profiles revealed reproducible patterns of metabolic disturbance, including elevated methylmalonic acid, formiminoglutamic acid, and xanthurenic acid (suggestive of methylation and B-vitamin deficits); increased lipid peroxides and 8-OHdG (indicative of systemic oxidative stress); raised Krebs cycle intermediates and β-hydroxybutyrate (suggesting mitochondrial dysfunction); mild to moderate dysbiosis markers and evidence of fungal overgrowth; and elevated mercury levels exceeding reference thresholds. Despite estimated high polyphenol intake (2.5-3.5 g/day), antioxidant biomarkers remained elevated, supporting the hypothesis of pesticide-induced oxidative burden.

Conclusion: These findings suggest that chronic dietary pesticide exposure – even at regulatory-compliant levels – may produce a consistent metabolomic signature, particularly when at least five different pesticide, herbicide, or fungicide residues are simultaneously detected, highlighting the potential for cumulative biological effects characterized by oxidative stress, detoxification pathway strain, gut microbiome disruption, and mitochondrial impairment. This underscores the need for integrated dietary strategies to reduce contaminant intake and highlights the importance of further cohort studies to clarify health impacts and guide nutritional interventions.

Keywords:

Introduction

Modern agriculture relies heavily on pesticides, herbicides, and fungicides to maximize yield. While regulatory thresholds aim to ensure safety, chronic low-dose exposure through high intake of conventionally farmed fruits and vegetables may affect human metabolism in subtle but clinically significant ways. Such exposures are often cumulative and can involve mixtures of pesticide residues, their metabolites, and even heavy metals introduced through soil amendments or historical contamination (1-5).

Pesticides are designed to be biologically active, often targeting essential enzymatic systems in pests, but their mechanisms can overlap with human metabolic pathways. For example, organophosphates and carbamates inhibit cholinesterase activity but can also generate reactive oxygen species (ROS) during biotransformation via cytochrome P450 enzymes. This biotransformation can overload hepatic Phase I and Phase II detoxification systems, including methylation and glutathione conjugation pathways, especially when nutritional cofactor reserves (e.g., folate, B12, B6, NAC) are marginal (6-8).

Furthermore, emerging evidence indicates that pesticides and certain fungicides can disrupt the gut microbiome, reducing beneficial taxa such as Lactobacillus and Bifidobacterium, while promoting overgrowth of opportunists like Candida spp. (9, 10). Such dysbiosis may further compromise nutrient absorption, immune balance, and even enterohepatic detoxification cycles. Heavy metals such as mercury and arsenic, historically used in agriculture, can also persist in soils and enter the food chain through plant uptake, contributing to cumulative toxic burden (11-13).

While plant-based diets are widely recommended for their high nutrient density, fiber, and antioxidant content, including polyphenols that can reach several grams per day in high consumers, these benefits may be attenuated or complicated by co-exposure to agricultural chemicals. Paradoxically, ROS generated by pesticide metabolism may deplete these dietary antioxidants, exacerbating systemic oxidative stress rather than mitigating it (14-16). Organic acids serve as sensitive indicators of key metabolic processes, including mitochondrial energy production, detoxification capacity, oxidative damage, and microbiota-derived metabolites. Their urinary excretion reflects systemic biochemical status and provides early markers of metabolic stress (17).

This study integrates dietary modeling with organic acid urinary metabolomic profiling to evaluate whether high fruit and vegetable intake–estimated to provide substantial polyphenol exposure–correlates with biochemical markers of oxidative stress, detoxification pathway stress, gut dysbiosis, and mitochondrial dysfunction. By examining these interconnected metabolic pathways, we aim to clarify potential health implications of chronic, low-level multi-residue pesticide exposure, emphasizing that the accumulation of at least five different substances–pesticides, fungicides, or herbicides–even at levels below maximum residue limits (MRLs), may cumulatively affect metabolism and overall health.

Materials and Methods

Data sources. This study integrated two major data sets. First, we analyzed a series of official analysis bulletins issued by accredited laboratories in the network of the National Veterinary Sanitary and Food Safety Authority (ANSVSA) of Romania, covering the period January 2024 to March 2025. These documents present the results of national surveillance and veterinary food safety controls on non-animal food products, mainly vegetables, fruits and cereals.

Each food sample was analyzed as part of a routine surveillance batch (3-5 subsamples per product), sourced directly from retail outlets and stored refrigerated (4°C) before transport to the testing facility. Samples were analyzed in their raw, unwashed form to reflect realistic consumer exposure. Pesticide residue analysis followed ISO/IEC 17025 accredited protocols (15).

Second, we included metabolome profiles of individuals consuming predominantly plant-based diets, analyzed by a certified urine and blood testing protocol, in 26 adult patients, and we compared it with the database. These results provided information on metabolic and nutritional status by profiling urinary organic acids, oxidative stress markers, mitochondrial imbalances, intestinal dysbiosis, heavy metal exposure, and essential micronutrient requirements.

The study population comprised 26 adult volunteers (age=23-60, 54% female), residing in northwestern Romania, all of whom reported daily fruit and vegetable consumption >2.5 kg and were free of diagnosed chronic disease. All subjects were non-smokers and did not report supplement or medication use.

Sample types analyzed. Metabolomic profiles were retrieved retrospectively from patients referred to the Echo Laboratoare Oradea for personalized nutrition consultation between January 2024 and March 2025. Eligible participants were adults (>18 years), reporting daily fruit and vegetable intake >1 kg/day and no known chronic illness, selected consecutively. Metabolomic testing was performed using the Metabolomix+ protocol (Genova Diagnostics, Asheville, NC, USA), processed by accredited laboratories following standardized handling and analysis procedures. Urine samples (first-morning voids, 10-20 ml) were collected in sterile containers and immediately stored at 4°C, then frozen at −20°C within 4 h. Samples were shipped under cold chain to Genova Diagnostics within 48 hours and processed within 7 days according to ISO 15189 standards.

Food products analyzed for pesticide testing. The analysis included a variety of commonly consumed plant-based food items. Vegetables tested comprised tomatoes, cucumbers, potatoes, white beans, and garlic. Among fruits, apples were selected due to their frequent presence in the participants’ diets. Cereal products included wheat and white flour, while additional plant-based items such as seeds and canned goods were also examined for potential pesticide residues.

Human samples for metabolomic testing. For the assessment of metabolic profiles, the Metabolomix+® test (Genova Diagnostics) was utilized. This comprehensive urine organic acid assay evaluates multiple metabolic pathways, including the Krebs cycle, amino acid metabolism, B vitamin status, gut dysbiosis markers, oxidative stress, mitochondrial function, and exposure to heavy metals. The integrated nature of this test offers a broad overview of each participant’s nutritional and functional health status. All individuals included in the metabolomic analysis were considered apparently healthy, although they reported mild to moderate gastrointestinal symptoms such as bloating, flatulence, constipation, diarrhea, or postprandial fatigue within the six months preceding testing.

The reference (control) values for mitochondrial biomarkers were obtained from the standard laboratory intervals provided by Genova Diagnostics in the Metabolomix+® assay. The reported normal ranges were as follows: malic acid: 0-3.0 μmol/mmol creatinine, cis-aconitic acid: 0-1.4 μmol/mmol creatinine, and β-hydroxybutyrate: 0-4.0 μmol/mmol creatinine. These intervals represent expected values in a healthy population and were used as the control reference group in statistical comparisons.

Detection methods employed. Pesticide residue detection was performed using advanced chromatographic techniques. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was employed to identify a wide range of pesticides, including organochlorines, organophosphates, pyrethroids, and carbamates. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for less volatile, more systemic compounds that are commonly found in processed produce. Additionally, microscopic methods were applied to detect the presence of mycotoxins and biological contaminants such as ergot bodies and sclerotia. For all analyses, the limit of detection (LOD) and the limit of quantification (LOQ) were reported in accordance with regulatory standards, and results were annotated accordingly in the dataset.

Interpretation criteria. Pesticide results. The interpretation of pesticide residue findings was conducted in accordance with the criteria established by Regulation (EC) No 396/2005 concerning maximum residue limits (MRLs) for food products. Each sample tested was classified based on its regulatory compliance status as either compliant or non-compliant. The concentration of pesticide residues was categorized into three levels: “not detectable” (below the limit of detection, <LOD), “<LOQ” (detected but not quantifiable), and quantifiable concentrations reported in milligrams per kilogram (mg/kg) when values exceeded the LOQ.

Dietary intake estimation and polyphenol content modeling. Based on participant dietary recall and reported habitual energy intake averaging 2700 kcal/day, macronutrient distribution was estimated at 55% carbohydrates, 30% protein, and 15% fat. Approximately 50% of total daily caloric intake was reported to derive from plant-based sources, primarily vegetables and fruits. To estimate daily fruit and vegetable intake, an average caloric density of 30 kcal/100 g was used. This value reflects a conservative estimate accounting for mixed raw produce, including lower-calorie leafy greens and watery fruits. The resulting estimated daily intake of plant-based foods is (formula 1):

Embedded Image (1)

Participant self-reports suggested an average intake of 2.5-3.5 kg of fruits and vegetables per day, consistent with 50-60% of energy intake from low-calorie plant sources (e.g., cucumbers, leafy greens, tomatoes). Based on a conservative polyphenol content of 100 mg/100 g (18, 19), this corresponds to an estimated 2.5-3.5 g/day of polyphenols [formula (2)]:

Embedded Image (2)

This modeling approach reflects an upper-bound scenario for individuals consuming a very high proportion of calories from plant-based sources with minimal processing loss. Actual intake may be lower due to variation in produce type, agricultural practices, seasonal differences, and food preparation losses, which can reduce bioavailable polyphenols by 10-50%.

The food categories selected (tomatoes, apples, cucumbers, garlic, etc.) represent items most frequently consumed and tested under national surveillance programs. While not exhaustive, they reflect staples of a plant-based Romanian diet and are considered sentinel products for pesticide residue surveillance. Future studies should expand to include leafy greens, berries, and legumes for broader assessment.

Ethical considerations. This study was conducted in accordance with the principles of the Declaration of Helsinki and the General Data Protection Regulation (GDPR 2016/679). All participants included in the microbiome analysis gave informed consent prior to sample collection and data use. Personal data were anonymized and processed exclusively for scientific purposes. Since the microbiome samples were collected as part of routine clinical diagnostics and the study design was observational, no interventional procedures were applied. The study protocol was reviewed and approved by the local ethics committee affiliated with the coordinating institution.

Statistical analysis. Descriptive statistics were used to summarize participant characteristics and metabolomic biomarker data derived from the Metabolomix+® organic acid testing reports. Biomarker values were interpreted relative to reference ranges provided by the testing laboratory (Genova Diagnostics). For detoxification pathway markers, oxidative stress indicators, mitochondrial dysfunction scores, and gut dysbiosis markers, results were categorized as within or above reference thresholds. Counts and percentages of elevated or abnormal biomarker values were calculated to characterize patterns of metabolic stress in the participants.

Estimated dietary polyphenol intake was modeled based on reported energy intake and the proportion of calories from plant-based sources, using standard caloric density and polyphenol content assumptions. Simple correlations between estimated polyphenol intake and oxidative stress markers (e.g., lipid peroxides, 8-OHdG) were explored using scatterplots to evaluate potential relationships. Due to the small sample size and the exploratory nature of the analysis, formal statistical tests were not emphasized. All data collation and visualization were performed using Microsoft Excel (version 2304, Microsoft Corporation, Redmond, WA, USA) and IBM SPSS Statistics (version 30.0, IBM Corp., Armonk, NY, USA). Pesticide-linked metabolic stress in high plant-based diets is presented in Figure 1.

Figure 1.
Figure 1.

Conceptual model linking plant-based diets with metabolic stress biomarkers. The figure illustrates the potential pathway from high fruit and vegetable intake–despite high polyphenol content (~5–10 g/day)–to low-dose pesticide and heavy metal exposure. Metabolomix+® testing revealed biomarkers of detoxification strain (↑MMA, FIGLU), oxidative stress (↑lipid peroxides), mitochondrial dysfunction (↑Krebs intermediates), dysbiosis (↑D-arabinitol), and elevated mercury. These alterations may contribute to subclinical health risks, including gut imbalance, impaired detoxification, and metabolic stress.

Results

Results of pesticide residue and contaminant analysis. Table I presents a summary of the results obtained for six categories of plant-based food products, including vegetables (tomatoes, cucumbers, potatoes, white beans, garlic), fruits (apples) and cereals (wheat and white flour). The products come from both domestic sources (Romania) and international imports (mainly Turkey and Poland). Each sample was analyzed for a diverse range of active substances, including organophosphates (e.g., chlorpyrifos, fosthiazate), pyrethroids (e.g., fenpropathrin), neonicotinoids (e.g., imidacloprid), as well as ergot alkaloids in cereals. The number of substances tested varied considerably, with more extensive analyses (up to 100 substances) performed for imported vegetables, such as potatoes and cucumbers.

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Table I.

Description of demographic characteristics.

In all cases, pesticide residues were either below the LOD or below the LOQ, with one notable exception: imidacloprid was detected in tomatoes, but at concentrations compliant with the MRLs established by EC No. 396/2005. No samples exceeded the MRLs, and all products tested were within current EU food safety standards.

Figure 2 provides an integrated classification of the six food product categories analyzed, indicating for each the pesticide residue detection status and compliance with EU MRLs. This diagram highlights both the number of compounds analyzed per category (higher in the case of garlic and apples, suggesting increased regulatory oversight) and the distribution of results according to regulatory thresholds. The majority of products had no detectable residues, while the remainder had levels either below the LOQ or MRL, confirming general compliance but underlining the need for continued monitoring. This approach is particularly essential in the context of the potential for long-term cumulative exposure to pesticides with endocrine-disrupting or neurotoxic properties, underlining the importance of further food safety investigations along the supply chain.

Figure 2.
Figure 2.

Integrated classification of the six food product categories (tomatoes, cucumbers, garlic, white beans, potatoes, apples) according to the detection of pesticide residues and compliance with EU maximum residue limits (MRLs) (2024-2025). The figure illustrates the percentage of plant-based food samples with pesticide residue levels below detection (LOD) or quantification (LOQ) limits, as determined by validated GC-MS/MS and LC-MS/MS methods. The percentages represent the proportion of samples from each food type with undetectable or non-quantifiable pesticide residues. For example, 79% of tomato samples, 90% of white bean samples, and 95-100% of cucumber and garlic samples had residue levels that fell below the analytical thresholds. These percentages reflect sample-level compliance with safety benchmarks and provide insight into the variability of pesticide contamination among different plant-based foods. For each category, 10 samples were analyzed. The average results are presented in the study. Error bars represent standard deviation (SD) for each food category.

Pesticides and their impact on Phase II detoxification pathways (methylation and conjugation). Pesticides such as organophosphates, carbamates, and fungicides are primarily detoxified in the liver through Phase II pathways, including methylation and glutathione conjugation. Methylation uses SAMe, which depends on adequate folate (B9) and B12 levels, while glutathione conjugation neutralizes electrophilic pesticide metabolites via GST enzymes (18). Chronic pesticide exposure may deplete these nutrients and reduce methylation efficiency, as indicated by lower sarcosine levels and GSH demand (Table II). Additionally, vitamins B2 (riboflavin) and B6 (pyridoxal-5′-phosphate) act as essential enzyme cofactors for multiple steps in both methylation and transsulfuration pathways, linking methylation to glutathione synthesis. Pesticide metabolism can thus indirectly strain these vitamin-dependent processes. In our Metabolomix+ data, several biomarkers strongly indicated stress on Phase II detoxification pathways. Elevated methylmalonic acid suggested a functional deficiency of vitamin B12, while increased formiminoglutamic acid pointed to folate depletion. Borderline or elevated levels of xanthurenic acid, a sensitive indicator of vitamin B6 insufficiency, were also observed. Collectively, these findings contributed to a high Methylation Imbalance Score, reflecting systemic pressure on the body’s methylation capacity (Table III). These findings support the hypothesis that chronic dietary exposure to pesticide residues can contribute to functional depletion of methyl donors and conjugation capacity. Over time, this may reduce the body’s ability to neutralize not only pesticides themselves but also other endogenous and exogenous toxins, contributing to cumulative toxic burden and oxidative stress (Figure 3).

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Table II.

Summary of pesticide and contaminant residue analysis in plant-based food products (2024–2025).

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Table III.

Biomarkers of methylation pathway stress with interpretations and nutrient dependencies.

Figure 3.
Figure 3.

Conceptual model of Phase II hepatic detoxification of pesticides via methylation and glutathione conjugation pathways, highlighting key nutrient dependencies.

Pesticides and oxidative stress. Pesticide exposure increases oxidative stress by generating ROS during liver metabolism. This leads to lipid peroxidation, inflammation, and DNA damage, reflected by elevated markers like 8-OHdG, indicating disrupted redox balance. In the Metabolomix+ results analyzed here, clear evidence of oxidative stress was observed, i.e., elevated lipid peroxides, consistent with ongoing lipid membrane damage and peroxidation, as well as increased 8-OHdG, indicating DNA oxidative lesions. These findings support the hypothesis that dietary pesticide exposure contributes to elevated oxidative stress, overwhelming endogenous antioxidant systems such as glutathione peroxidase, superoxide dismutase, and catalase. Over time, this imbalance can disrupt cellular signaling, promote chronic inflammation, and increase the risk of metabolic and degenerative diseases (Table IV).

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Table IV.

Biomarkers of oxidative stress observed in organic acid testing.

Pesticides and intestinal dysbiosis. In the Metabolomix+ results analyzed here, several findings support a pattern of mild to moderate dysbiosis potentially linked to chronic dietary pesticide exposure. Elevated markers of bacterial imbalance, including benzoic acid, hippuric acid, and other aromatic metabolites, were observed. Additionally, the prior detection of D-arabinitol suggested possible Candida or other fungal overgrowth. A high Probiotic Support Score further indicated an increased need for probiotic repletion to help restore microbial balance. These results collectively suggest that regular consumption of conventionally farmed produce with pesticide residues may contribute to an altered gut microbiome profile, characterized by loss of beneficial taxa, expansion of pathobionts or opportunists, and increased production of metabolites detectable via organic acid testing. Such dysbiosis can impair nutrient absorption, promote intestinal permeability, and modulate systemic immune and inflammatory responses (Table V).

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Table V.

Markers of intestinal dysbiosis detected via organic acid testing.

Pesticides and heavy metal co-exposure. In the Metabolomix+ results analyzed here, several findings indicate elevated heavy metal exposure potentially linked to dietary or environmental sources. Mercury levels were markedly elevated at 16 μg/g, well above reference thresholds, suggesting significant systemic accumulation. Arsenic concentrations were at the upper limit of the reference range, pointing to possible chronic low-level intake, while antimony levels approached the threshold, raising concerns about cumulative exposure. These findings underscore that diets high in conventionally farmed fruits and vegetables, particularly those sourced from regions with intensive pesticide and fertilizer use, may represent a plausible source of ongoing low-level heavy metal exposure. Such co-exposure is clinically relevant because heavy metals can disrupt multiple metabolic pathways, induce oxidative stress, impair detoxification enzymes (including those responsible for pesticide metabolism), and accumulate in sensitive tissues over time (Table VI and Figure 4).

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Table VI.

Heavy metal levels detected in Metabolomix+ testing and potential dietary sources.

Figure 4.
Figure 4.

Boxplot comparing concentrations of heavy metals (antimony, arsenic, mercury) between the Study group (n=26) and Control group (n=26). The box represents the interquartile range (IQR), the line within each box denotes the median, whiskers show standard deviation (SD), and the black dot indicates the group mean. Statistical analysis was performed using the unpaired two-tailed Student’s t-test; p-Values are reported for group comparisons. Significant differences (p<0.05) were observed for mercury.

Pesticides and mitochondrial dysfunction. The Metabolomix+ results analyzed here support evidence of mitochondrial dysfunction potentially linked to metabolic stress. An elevated Mitochondrial Dysfunction Score suggests systemic strain on mitochondrial energy production. Increased levels of Krebs cycle intermediates, such as malic acid and cis-aconitic acid, indicate reduced efficiency of the TCA cycle. Additionally, elevated β-hydroxybutyrate points to a compensatory shift toward ketogenesis, likely reflecting impaired oxidative phosphorylation and decreased glucose utilization. Collectively, these findings suggest that chronic low-level pesticide exposure may impose sustained metabolic pressure on mitochondrial function, prompting compensatory shifts in energy metabolism. Over time, this imbalance can contribute to fatigue, reduced metabolic flexibility, and increased vulnerability to metabolic and degenerative diseases, presented in Table VII and Figure 5.

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Table VII.

Biomarkers of mitochondrial dysfunction detected via organic acid testing.

Figure 5.
Figure 5.

Mean values with 95% confidence intervals (CI) for selected mitochondrial biomarkers: β-hydroxybutyrate, cis-aconitic acid, and malic acid (A), and mitochondrial dysfunction score (B) in the Study group (n=26) versus the Control Reference group. Error bars represent 95% confidence intervals (CI). Study group values are based on measured patient data, while Reference group values reflect standard laboratory intervals provided by Genova Diagnostics. Statistical comparisons were performed using unpaired two-tailed Student’s t-tests. All biomarkers showed significant differences (p<0.05), with the mitochondrial dysfunction score markedly elevated in the study group.

Scatter plots (Figure 6) illustrate a paradoxical positive correlation between estimated daily polyphenol intake (from high fruit/vegetable consumption) and urinary markers of oxidative stress (lipid peroxides, 8-OHdG). This supports the hypothesis that pesticide contamination may overwhelm antioxidant defense, resulting in elevated oxidative biomarkers despite high polyphenol intake.

Figure 6.
Figure 6.

Relationship between estimated polyphenol intake and lipid peroxides (A), and relationship between estimated polyphenol intake and 8-OHdG (B).

Discussion

Our findings suggest that chronic dietary exposure to low-dose pesticide residues, even within regulatory limits, can exert cumulative metabolic stress. This effect may be particularly relevant when at least five different substances are detected concurrently, supporting the concept that multi-residue accumulation, rather than single-compound exposure, contributes to the observed biochemical stress pattern, which is detectable through urinary organic acid profiling. Despite a high estimated intake of plant-derived polyphenols (2.5-3.5 g/day from fruit and vegetable consumption), participants showed elevated biomarkers of oxidative stress, including lipid peroxides and 8-OHdG. This paradox aligns with prior studies indicating that pesticide metabolism can generate ROS via cytochrome P450 activation, overwhelming both endogenous and dietary antioxidant defenses (19, 20).

This underscores the importance of dietary strategies rich in antioxidants (e.g., vitamins C and E, polyphenols) and nutrients supporting endogenous defense systems such as glutathione synthesis (N-acetylcysteine, glycine). While antioxidant-rich diets are generally protective, our results support the hypothesis that in the presence of chronic pesticide exposure, dietary antioxidants may be consumed more rapidly, requiring increased nutritional support or careful sourcing of produce to minimize contamination (21-23). Moreover, pesticide detoxification relies heavily on hepatic Phase II conjugation pathways –including methylation and glutathione conjugation– which depend on adequate intake of folate, vitamin B12, vitamin B6, and sulfur-containing amino acids. Elevated methylmalonic acid, formiminoglutamic acid, and xanthurenic acid in our testing suggest functional deficiencies or increased demand in these pathways, corroborating previous reports linking pesticide exposure to methylation imbalance and glutathione depletion (23-27). This underscores the need for dietary strategies supporting these detoxification pathways as part of a broader approach to mitigating the metabolic impact of chronic pesticide exposure.

Gut microbiome integrity is another key factor potentially disrupted by pesticide residues. Our analysis indicated mild to moderate bacterial dysbiosis markers and evidence of fungal overgrowth (e.g., D-arabinitol elevation). Existing literature shows that certain pesticides can reduce beneficial taxa like Lactobacillus and Bifidobacterium while favoring opportunists such as Candida spp. (28, 29). This highlights the importance of dietary choices favoring reduced pesticide exposure –such as organic produce– and targeted nutritional or probiotic interventions to maintain a healthy gut microbiota.

Heavy metals remain a related but distinct concern. Our participants’ elevated mercury levels (16 μg/g) and borderline arsenic and antimony levels suggest cumulative exposure pathways potentially linked to contaminated soils, irrigation, or legacy pesticide use. Prior work confirms that such metals can persist in agricultural soils and accumulate in plant tissues (2, 30-33). This highlights the importance of sourcing produce from regions with rigorous soil contamination monitoring and, where possible, selecting organic or low-input cultivation to reduce cumulative heavy metal intake.

Finally, our data revealed markers of mitochondrial dysfunction, including elevated Krebs cycle intermediates and β-hydroxybutyrate, along with a high mitochondrial dysfunction score. Pesticide exposure has been shown to inhibit mitochondrial Complex I activity, impair β-oxidation, and increase ROS generation, leading to compensatory metabolic adaptations (34-37). These observations underscore the importance of assessing mitochondrial biomarkers in populations with high dietary pesticide exposure and highlight potential benefits of targeted nutritional support for mitochondrial function, such as coenzyme Q10, carnitine, and B vitamins (38-40).

Overall, our results support a holistic interpretation of diet quality, recognizing that while fruits and vegetables provide essential nutrients and antioxidants, their health benefits may be attenuated by contaminant exposure. Integrated strategies that combine dietary diversity, organic sourcing where feasible, and targeted nutritional support may help maximize the benefits of plant-rich diets while minimizing associated risks.

Although pesticide residues in food samples were below LOD or LOQ in most cases, this does not exclude chronic low-dose exposure, particularly in individuals with high-volume, daily consumption. Moreover, detection limits do not always reflect biological thresholds of effect. It is also possible that non-analyzed food items, storage containers, or cumulative environmental exposures contributed to the observed metabolomic patterns. The study therefore cannot establish causality but rather raises a hypothesis of biological stress in the context of high plant-based intake and potential exposure to undetected contaminants.

Study limitations. Firstly, dietary intake estimates were based on participant self-report and generalized caloric modeling, which may not precisely reflect actual food composition or portion sizes. The estimated polyphenol content also relies on average published values that vary significantly by produce type, season, cultivar, and preparation method. Secondly, while the organic acid testing provides useful surrogate biomarkers of metabolic stress, it cannot directly quantify pesticide or heavy metal residues in biological tissues. Heavy metal measurements were limited to a small panel, and no direct pesticide metabolite assays were performed. Thirdly, this analysis is observational and cross-sectional in nature, precluding causal inference. Confounding factors such as age, genetics, lifestyle, and environmental exposures beyond diet may also contribute to the observed metabolomic patterns. Future studies incorporating direct pesticide biomonitoring, more detailed dietary records, larger sample sizes, and longitudinal follow-up would strengthen causal interpretations and help refine nutritional recommendations.

The absence of a control group of healthy individuals without gastrointestinal symptoms or with low-pesticide dietary patterns significantly limits interpretability. Future studies should include matched control cohorts with defined dietary exposures to clarify associations and reduce confounding. All participants reported mild to moderate gastrointestinal complaints (e.g., bloating, flatulence). These symptoms may independently influence gut microbiota composition, oxidative stress, or nutrient absorption, acting as potential confounders. The exploratory nature of this pilot study does not allow separation of these effects from potential dietary contaminant-related mechanisms.

Conclusion

This integrative analysis reveals a paradox in plant-based nutrition: while diets high in fruits and vegetables offer antioxidant and polyphenol benefits, they may also introduce significant levels of pesticide residues and heavy metals. Despite an estimated polyphenol intake of ~5–10 g/day-typically associated with oxidative protection–Metabolomix+ results showed elevated markers of oxidative stress (lipid peroxides, 8-OHdG), detoxification strain (methylation imbalance), dysbiosis, and mitochondrial dysfunction.

These findings suggest that conventionally farmed produce, even in large quantities, can simultaneously nourish and expose the body to metabolic stressors. This highlights the need to consider both nutritional value and contaminant burden. Supporting antioxidant defenses, reducing exposure through organic sourcing, and enhancing detoxification pathways may be essential strategies for optimizing health in those consuming predominantly plant-based diets.

Acknowledgements

The Authors would like to thank the University of Oradea, for supporting the payment of the invoice, through an internal project.

Footnotes

  • Authors’ Contributions

    Conceptualization, R.A.T. and T.C.G.; methodology, M.F.G.; software, M.C.G.; validation, F.B.; formal analysis, T.C.G.; investigation, T.C.G.; resources, T.C.G.; data curation, T.C.G.; writing – original draft preparation, T.C.G.; writing – review and editing, T.C.G.; visualization, T.C.G.; supervision, T.C.G.; project administration, T.C.G.; funding acquisition, T.C.G. All Authors have read and agreed to the published version of the manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Funding

    The article processing charge was funded by University of Oradea, Oradea, Romania.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received August 16, 2025.
  • Revision received October 6, 2025.
  • Accepted October 20, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Nutritional Assessment of Pesticide-associated Metabolic Stress in Plant-based Diets
RAMONA ALINA TOMUȚA, MARIA FLAVIA GÎTEA, MARC CRISTIAN GHITEA, EVELIN CLAUDIA GHITEA, TIMEA CLAUDIA GHITEA, FLORIN BANICA
In Vivo Jan 2026, 40 (1) 136-150; DOI: 10.21873/invivo.14179
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