Abstract

Chronic human exposure to pesticide mixtures, even at low doses, is widespread but remains poorly characterized in conventional risk assessment, which largely focuses on single compounds. This systematic review synthesizes current evidence on the health effects of long-term exposure to pesticide mixtures. Bibliographic searches were conducted in PubMed Central, ScienceDirect, and Google Scholar using predefined PICO criteria and structured search equations. After screening 608 records and applying eligibility criteria, 63 experimental and epidemiological studies addressing chronic, low-dose exposure to pesticide mixtures were included. Across diverse designs and biological models, the reviewed studies consistently reported multi-system toxicity. Documented outcomes included metabolic disturbances (glucose and lipid dysregulation, hepatic steatosis, diabetes risk), enzymatic alterations (CYP induction, AChE/BChE inhibition), neurological and neurobehavioral effects, oxidative stress, genotoxicity, epigenetic alterations, endocrine disruption, reproductive toxicity, respiratory impairment, and histopathological damage in hepatic, renal, hematopoietic, and reproductive tissues. Several studies also suggested emerging multidrug resistance phenomena following chronic pesticide-mixture exposure. Mechanistically, oxidative stress, mitochondrial dysfunction, endocrine and immune dysregulation, DNA damage, and persistent epigenetic reprogramming emerged as recurrent pathways, often amplified by additive, synergistic, or non-linear interactions within mixtures. Methodological heterogeneity, incomplete exposure characterization, and limited multigenerational data remain important constraints, particularly for vulnerable populations such as agricultural workers, children, and pregnant women. Nevertheless, the overall convergence of findings underscores that chronic exposure to pesticide mixtures cannot be considered innocuous, even at doses individually deemed acceptable. These results highlight the urgent need to integrate mixture toxicity and cumulative risk assessment into regulatory frameworks, strengthen environmental and occupational monitoring, and promote safer pest-management strategies to better protect public and environmental health. Despite the overall convergence of findings, the interpretation of results remains constrained by methodological heterogeneity, variable exposure assessment, and limited multigenerational and longitudinal data.

Keywords

Pesticide Mixtures, Chronic Low-Dose Exposure, Genotoxicity, Endocrine Disruption, Oxidative Stress

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1. Introduction

1.1. General Context of the Topic

Pesticides, chemical substances designed to prevent, destroy, or control harmful organisms, play a central role in modern agricultural practices. Their use increases yields, secures food production, and ensures crop quality [1]. Beyond agriculture, these products are also applied in public health, forestry, fisheries, and urban areas, particularly for vector control and the management of domestic pests [2].

However, the widespread presence of pesticides raises major concerns regarding their potential impacts on human health and the environment [3]. Intensive and sometimes inappropriate use of these substances leads to their dissemination in soil, air, water, and food, thereby exposing the entire population to low-dose residues [4].

1.2. Scientific and Socio-Economic Importance of the Topic

From a socio-economic perspective, pesticides remain essential tools for global food security and the fight against agricultural losses, especially in developing countries where production systems still heavily rely on their use [5]. Nevertheless, their contribution to sustainable development is increasingly questioned in light of growing scientific evidence of their adverse effects on human health and biodiversity.

Pesticides have been associated with a wide range of harmful effects, including endocrine disruption, respiratory and cancerous diseases, and neurological, immune, and reproductive disorders [6] [7]. However, these effects are often non-specific and difficult to attribute to a single substance, highlighting the complexity of actual chemical exposures. These effects vary according to dose, duration, route of exposure, and individual characteristics such as age, sex, and pre-existing health conditions. Understanding these mechanisms is essential to developing more protective prevention and regulatory strategies.

1.3. Key Previous Studies

Research on the health impacts of pesticides has historically focused on evaluating the individual effects of isolated molecules. Such approaches have led to the identification of major pesticide classes (organochlorines, organophosphates, carbamates, pyrethroids) and their mechanisms of action, particularly on the nervous and endocrine systems [8] [9].

Epidemiological and toxicological studies have demonstrated associations between chronic pesticide exposure and increased risks of neurodegenerative diseases, cancers, and hormonal disorders [10] [11]. However, most of these studies examine single substances, while mixture exposures are multiple and simultaneous [10] [12] [13].

1.4. Identified Gaps in the Literature

One of the major challenges in environmental toxicology lies in the complexity of pesticide mixtures to which individuals are exposed. In real-world conditions, agricultural and non-agricultural populations encounter cocktails of residues resulting from the combined or successive application of multiple products [14] [15].

These mixtures can interact synergistically or additively, altering overall toxicity and making health outcomes difficult to predict [4]. Yet, most regulatory and scientific assessments continue to rely on single-substance analyses, without integrating the cumulative dimension of exposure.

Furthermore, current regulatory frameworks, whether international (Codex Alimentarius, Stockholm, Rotterdam, and Basel Conventions) or national (chemical management plans, Maximum Residue Limits), are still poorly adapted to assessing the risks related to low-dose multiple exposures [16] [17]. This limitation reduces the ability to anticipate emerging risks and effectively protect public health.

In addition, emerging frameworks such as cumulative risk assessment and exposome-based approaches remain insufficiently integrated into current pesticide risk evaluation strategies.

1.5. Purpose and Objectives of the Review

In light of these observations, the present review aims to synthesize current knowledge on the health impacts of chronic exposure to low doses of pesticide mixtures. Specifically, it seeks to:

• examine exposure pathways and health effects associated with long-term exposure;

• analyze toxic interactions among compounds within mixtures;

• identify existing methodological and regulatory limitations; and

• propose directions for future research and integrated risk management.

This review ultimately aims to contribute to a broader and more critical understanding of the combined effects of pesticides, thereby supporting stronger prevention and risk assessment policies for sustainable public health protection.

2. Methodology

2.1. Objective of the Study

The objective of this review is to conduct a systematic synthesis of the existing literature on the health effects of chronic, low-dose exposure to pesticide mixtures.

The review serves as the central methodological approach, allowing for the rigorous and comprehensive aggregation, evaluation, and analysis of results drawn from a wide range of scientific publications.

This method provides a coherent overview of the available data and highlights emerging trends related to the health impacts of pesticide mixtures.

2.2. Methodological Approach

The reproducibility of this research is ensured through a transparent and systematic approach, documenting each stage of the process, from the literature search to the synthesis of data, so that other researchers can trace and evaluate the methodology with full transparency [18].

Among the various methods available for literature synthesis, a systematic review was selected because of its ability to combine the results of multiple primary studies addressing the same research topic.

This study was conducted following six main stages:

1) Formulation of the research question;

2) Search and identification of relevant literature;

3) Selection of studies according to predefined eligibility criteria;

4) Data extraction;

5) Synthesis and analysis of results;

6) Interpretation and discussion of findings.

This sequence ensures methodological rigor, validity of results, and reproducibility of the overall process. The review was conducted in accordance with established reporting guidelines for systematic reviews, including the PRISMA framework.

Due to substantial heterogeneity in study designs, exposure metrics, and outcome measurements, a formal meta-analysis was not feasible. Therefore, a qualitative synthesis approach was adopted.

2.3. Selection Criteria

The selection of studies included was based on specific eligibility criteria, as follows:

• Observational or experimental studies;

• Research explicitly addressing the health impacts of pesticide mixtures;

• Studies assessing chronic exposure rather than acute exposure;

• Original research articles published in peer-reviewed scientific journals;

• Availability of the full text;

• Presence of sufficient data for extraction and analysis.

Review articles, editorials, short communications, and studies focusing exclusively on individual pesticides without consideration of mixtures were excluded from the analysis.

For the purpose of this review, “low-dose” exposure was strictly defined according to toxicological safety standards. It refers to pesticide concentrations that are:

• Below or equal to established No Observed Adverse Effect Levels (NOAELs) in experimental models;

• Within the range of Acceptable Daily Intakes (ADIs) or Maximum Residue Limits (MRLs) for human populations;

• Reflective of real-world environmental background exposure, this excludes cases of acute poisoning or high-dose accidental spills.

In epidemiological studies, this includes chronic, long-term exposure to residual levels found in diet, water, or ambient air, as well as chronic occupational exposure where health outcomes result from prolonged low-level accumulation rather than acute toxicity.

This definition excludes acute poisoning scenarios and high-dose accidental exposures. However, some occupational studies were retained when they reflected chronic, repeated exposure patterns with biomarker or health outcomes indicative of cumulative low-dose effects rather than acute toxicity.

2.4. Formulation of the Research Question

The research question was developed using the PICO framework (Population - Intervention/Exposure - Comparison - Outcome), a method commonly applied in systematic reviews to structure the research problem and define inclusion parameters.

Based on this conceptual framework, the research question was formulated as follows:

What health risks are associated with chronic, low-dose exposure to pesticide mixtures?

This formulation served as the foundation for constructing the search equation, which was based on carefully selected keywords in both English and French to ensure comprehensive coverage across international databases.

2.5. Information Sources and Search Equations

The bibliographic data were collected from three major electronic databases:

• PubMed Central (PMC)

• ScienceDirect

• Google Scholar

Each database was queried using tailored search equations, combining selected keywords through Boolean operators (AND, OR).

These search strategies were designed to maximize the retrieval of relevant publications while maintaining specificity to studies addressing the health impacts of chronic, low-dose exposure to pesticide mixtures.

The details of structuring the research question according to the PICO method, as well as the search equations applied to each database, are summarised in Table 1.

Table 1. PICO method and search equations.

2.6. Study Selection Process

The study selection process is illustrated in Figure 1, which presents the PRISMA flow diagram (Preferred Reporting Items for Systematic Reviews and Meta-Analyses). This diagram summarizes the various steps of study identification, screening, and inclusion undertaken in this review.

A total of 608 potential records were identified across the three databases searched:

• Google Scholar (n = 266)

• PubMed Central (PMC) (n = 188)

• ScienceDirect (n = 154)

After removing 42 duplicates, 566 unique articles remained for title and abstract screening. Following this initial screening, 124 studies were selected for full-text evaluation to determine their eligibility according to the predefined inclusion criteria.

At this stage, 63 studies met all eligibility requirements and were retained for qualitative synthesis, while 61 were excluded for reasons such as:

1) single-compound focus,

2) acute rather than chronic exposure,

3) insufficient health outcome data, or

4) non-original publication type (e.g., reviews or editorials).

Figure 1. PRISMA flow diagram of the study selection process.

The flowchart depicts the sequential steps of identification, screening, eligibility assessment, and inclusion of the studies considered in the systematic review.

3. Results

3.1. Description of Included Studies

A total of 63 studies met the inclusion criteria and were included in this review. These investigations were conducted across diverse geographical regions and encompass a wide range of methodological designs, reflecting the complexity of assessing chronic low-dose exposure to pesticide mixtures.

Overall, the dataset comprises:

• 29 experimental studies, including 27 in vivo models (conducted on mammals, fish, and insects) and 2 in vitro assays.

• 34 epidemiological studies, consisting of 17 cross-sectional studies, 5 cohort studies, 4 case-control studies, 3 longitudinal studies, and 5 other observational or field designs.

The selected studies address a broad spectrum of health outcomes, including reproductive toxicity, genotoxicity, metabolic disturbances, oxidative stress, endocrine disruption, respiratory effects, neurobehavioral impairments, and mechanisms of antimicrobial or drug resistance.

This thematic diversity highlights both the complexity of pesticide mixture toxicology and the heterogeneity of research approaches used to investigate their health effects.

A complete list of the 63 included studies, including authors, study titles, and study designs, is provided in Supplementary Table S1 (General Overview of Included Studies).

3.2. Characteristics of the Included Studies

The 63 studies retained for this review encompass a broad range of experimental and epidemiological designs, reflecting the complexity of assessing chronic low-dose exposure to pesticide mixtures. The evidence base includes in vivo studies (n = 27) that examined toxicological mechanisms and physiological alterations in animal models, in vitro investigations (n = 2) focused on cellular and biochemical responses, and epidemiological studies (n = 34) composed of cross-sectional, cohort, longitudinal, and case-control designs.

Across these studies, a wide spectrum of health outcomes was documented. Overall, most epidemiological studies were rated as having a moderate risk of bias, primarily due to exposure misclassification and reliance on self-reported data, while experimental studies showed variable quality depending on study design and reporting completeness.

Recurrent endpoints include metabolic disorders, neurological and neurobehavioral impairments, genotoxic and oxidative stress responses, endocrine disruption, reproductive and developmental toxicity, respiratory disturbances, and early indications of antimicrobial or multidrug resistance mechanisms. The thematic distribution of results highlights that pesticide mixtures are linked to multi-systemic effects, often more pronounced than those observed for individual compounds, suggesting additive, synergistic, or non-linear interactions.

A series of detailed tables (Tables 2-11) synthesizes the findings by major health outcome categories, each presenting the mixtures investigated, the biological models, Methodological approaches, and the main toxicological or clinical effects observed.

Summary of Observed Effects

A summary of the main categories is provided in Table 12.

Table 2. Metabolic effects of chronic low-dose exposure to pesticide mixtures.

Table 3. Enzymatic effects associated with chronic exposure to pesticide mixtures.

Table 4. Neurological and neurobehavioral effects.

Table 5. Oxidative stress effects.

Table 6. Genotoxic effects.

Table 7. Endocrine effects.

Table 8. Reproductive effects.

Table 9. Respiratory disorders.

Table 10. Histological effects.

Table 11. Drug resistance effects.

Table 12. Summary of health effects reported across included studies.

4. Discussion

The issue of pesticide exposure has been extensively investigated, yet it remains a critical public health and environmental concern in the contemporary context [77]. Pesticides today play a major role across multiple sectors: industrial production [78] [79], agriculture, public health interventions [80], and even domestic settings. Their widespread use leads to diffuse contamination of ecosystems and food resources [81], resulting in chronic and often unnoticed human exposure to complex mixtures of pesticides [82]. The health effects arising from such exposures are particularly difficult to assess because they involve cumulative, interactive, and potentially synergistic mechanisms rather than the toxicity of individual compounds alone [83].

This systematic review aims to synthesize the available scientific evidence to improve understanding of the potential health risks associated with chronic low-dose exposure to pesticide mixtures. By analyzing studies across different designs and methodological approaches, it offers a comprehensive perspective on the wide range of adverse outcomes reported in various biological systems and human populations. While several studies included in this review (see Table S1) conclude that synergistic interactions occur between pesticides, these findings should be interpreted with caution. In many cases, the term “synergy” is used by authors to describe a potentiation of effect. From a strict toxicological perspective, most of these combined actions likely follow the Concentration Addition (CA) model, where the total effect is the sum of individual toxicities. Few studies applied formal mathematical interaction models to rigorously distinguish between additive and synergistic effects. True synergy, which implies an effect significantly greater than the predicted additive sum, remains sparingly demonstrated through formal mathematical modeling in the current literature.

The comparison between experimental and epidemiological findings should be interpreted with caution. While experimental studies provide mechanistic insights under controlled conditions, epidemiological studies are subject to multiple confounding factors, including co-exposure to other environmental toxicants, variability in personal protective equipment use, lifestyle factors, and socioeconomic conditions.

Despite these terminological nuances, the present review reveals a strong convergence of evidence. The data collectively show that chronic exposure to pesticide mixtures can induce a broad spectrum of adverse health effects, including metabolic disturbances, enzymatic alterations, oxidative stress, genetic and transgenerational abnormalities, epigenetic modifications, neurological and neurobehavioral impairments, endocrine disruption, reproductive toxicity, respiratory dysfunction, and histopathological damage. This diversity of outcomes reflects the multi-systemic and multifactorial nature of pesticide mixture toxicity, which is frequently more pronounced than the effects attributed to individual compounds.

4.1. Metabolic Disturbances

Recent studies demonstrate that chronic low-dose exposure to pesticide mixtures is associated with substantial metabolic disturbances in humans and experimental models. Epidemiological evidence from Pakistan, Cameroon, and China links long-term exposure to organophosphates and mixed organochlorine-herbicide formulations with dyslipidemia, hyperglycemia, hepatic dysfunction, and an increased risk of type 2 diabetes, particularly through insulin resistance and oxidative stress pathways [28] [29]. Experimental findings corroborate these associations. In rodents, chronic or perinatal exposure to pesticide mixtures produces hepatic steatosis, glucose intolerance, altered lipid metabolism, and sex-dependent obesogenic and diabetogenic effects, largely driven by mitochondrial impairment, oxidative stress, and endocrine disruption [23] [24].

Alterations of the gut-liver axis and microbiota dysbiosis emerge as central mechanisms. Studies in zebrafish and other models reveal increased intestinal inflammation, disruption of tight-junction integrity, modulation of apoptotic pathways, and significant shifts in bacterial communities that affect bile acid turnover and lipid processing [21] [30] [31]. Synergistic effects within pesticide mixtures further disrupt hormone biosynthesis and fatty-acid metabolism [19] [22].

Collectively, these findings show that pesticide mixtures, even at low environmental doses, produce coherent metabolic toxicity patterns potentially linked to cardiometabolic diseases such as atherosclerosis [59].

Beyond a qualitative description, the magnitude of these metabolic disruptions is noteworthy. Across the included studies, chronic exposure to pesticide mixtures frequently led to measurable reductions in body weight gain and significant alterations in insulin secretion [20] [32]. In human cohorts, these biological shifts translated into clinical relevance, with reported increases in odds ratios for metabolic syndrome and type-2 diabetes [28] [29]. This confirms that the impact of these mixtures is not only present but possesses substantial clinical potency even at doses individually deemed safe.

4.2. Enzymatic Effects

Chronic exposure to pesticide mixtures causes significant disruption of enzymatic function, affecting key pathways involved in metabolism and detoxification. In vivo findings from [33] indicate strong induction of hepatic cytochrome P450 isoenzymes (CYP1A1, CYP2B1, CYP3A1) in rats exposed to triazole fungicide mixtures, reflecting enhanced biotransformation activity and increased metabolic burden. Similarly, [32] observed elevated hepatic enzyme levels in rats treated with insecticide mixtures, suggesting cumulative activation of detoxification pathways under combined exposures.

Conversely, inhibitory effects on essential enzymes have also been documented. Dopavogui et al. [72], in a longitudinal study, reported significant reductions in blood cholinesterase activity following chronic exposure to mixed pesticides, consistent with the synergistic neurotoxic actions of organophosphates, carbamates, and related compounds.

Overall, pesticide mixtures induce both up- and down-regulation of phase I and phase II enzymes, highlighting their cumulative impact on metabolic homeostasis, xenobiotic processing, and biochemical signaling, even at environmentally relevant doses.

4.3. Neurological and Neurobehavioral Effects

Chronic exposure to pesticide mixtures consistently leads to inhibition of acetylcholinesterase (AChE), a central biomarker of neurotoxicity, and produces widespread neurological and neurobehavioral disturbances. Experimental studies demonstrate that combined exposures disrupt neurotransmitter balance, promote oxidative stress, and trigger neuroinflammatory responses. Gasmi (2020) [50] reported that a deltamethrin-acetamiprid mixture reduced dopamine and serotonin while increasing glutamate, resulting in anxiety-like behavior and cognitive deficits. Sergievich et al. (2020) [40] further described dose-dependent behavioral impairments under chronic exposure to complex mixtures. Tsatsakis et al. [13] showed that exposure to a six-pesticide mixture at ADI-equivalent doses impaired memory, locomotion, and anxiety regulation, with greater severity under vitamin-deficient conditions.

Human studies corroborate these effects. Kumar et al. [34] and Dhalla and Sharma (2013) [38] observed AChE inhibition and associated neurobehavioral symptoms among agricultural workers. Cytogenetic alterations reported by Valencia-Quintana et al. (2021) [35] and oxidative imbalance described by Silvério et al. (2017) [36] further support multifactorial neurotoxicity driven by combined pesticide exposures.

A major limitation across studies lies in the complexity and variability of real-life exposure scenarios. Human exposure to pesticide mixtures is dynamic, involving fluctuating combinations of compounds over time, which are rarely captured by single-point measurements. In addition, the frequent reliance on self-reported exposure data or indirect environmental indicators introduces uncertainty and potential misclassification bias.

4.4. Oxidative Stress

Chronic exposure to pesticide mixtures is consistently linked to oxidative stress, reflecting a disruption of redox homeostasis through excessive reactive oxygen species (ROS) generation and depletion of antioxidant defenses. Among Spanish agricultural workers, Lozano-Paniagua et al. [41] reported increased TBARS levels, indicating lipid peroxidation, accompanied by compensatory activation of antioxidant systems such as FRAS and paraoxonase-1 (PON1). Reduced acetylcholinesterase (AChE) activity was also observed, likely mediated by oxidative mechanisms. In Thailand, Intayoung et al. [59] found decreased total antioxidant capacity and elevated urinary 8-isoprostane among farmers exposed to glyphosate–paraquat mixtures. Experimental studies corroborate these findings: Bouabdallah et al. [55] showed reduced glutathione (GSH) and increased malondialdehyde (MDA) in rats exposed to cypermethrin, mancozeb, and metalaxyl.

These alterations have genotoxic implications. Ledda et al. [54] reported elevated 8-oxo-dG, a key biomarker of oxidative DNA damage, among exposed workers. Overall, oxidative stress emerges as a central mechanism linking pesticide mixtures to DNA instability, mutagenesis, and long-term disease risks.

These findings are consistent with the concept of non-monotonic dose–response relationships, frequently observed with endocrine-disrupting chemicals, where low-dose effects cannot be predicted from high-dose toxicity data.

4.5. Genetic Abnormalities

Genetic abnormalities linked to chronic exposure to pesticide mixtures are well documented in both experimental and epidemiological studies. In Mexico, Valencia-Quintana et al. [35] reported significant DNA damage among agricultural workers exposed to mixtures of organophosphates, carbamates, organochlorines, and pyrethroids. Notably, these genotoxic effects occurred despite the absence of acetylcholinesterase (AChE) inhibition, suggesting a distinct dose-response relationship for genotoxicity or the involvement of additional chemical classes. Similarly, Brazilian workers exposed to comparable mixtures exhibited cholinesterase inhibition, chromatin condensation, budding cell abnormalities, and increased karyolysis, indicating broad genomic instability [36].

Unsafe agricultural practices exacerbate these effects. Increased chromosomal breaks and micronuclei were observed in Pakistani agricultural workers, who showed a nearly threefold increase in comet tail length after pesticide spraying compared with controls [63]. Serbian pesticide-factory workers exposed to complex mixtures likewise demonstrated chromosomal aberrations [61]. Numerous studies confirm that prolonged exposure and a lack of personal protective equipment amplify genotoxic risk [37] [67], with biomarkers such as micronuclei and chromosomal breaks serving as reliable indicators [70].

Evidence from Hazarika and Deka [60] and Ledda et al. [54] further shows that mixtures containing organophosphates, carbamates, and fungicides significantly increase DNA strand breaks and oxidative lesions, including 8-oxo-2’-deoxyguanosine (8-oxo-dG). Importantly, pesticide-related genetic damage is not limited to occupational settings: Panis et al. [66] reported an increased cancer risk in women exposed exclusively to domestic pesticides.

Experimental studies parallel these findings. Garaj-Vrhovac and Zeljezic [62] observed persistent chromosomal abnormalities in exposed workers, while Ambreen and [69] and Weeks Santos et al. [44] documented cumulative DNA damage and upregulation of repair mechanisms in fish models exposed to binary or ternary mixtures. In bees, acetamiprid-difenoconazole disrupted detoxification and immune genes, with effects exceeding those of single pesticides [21]. Crépet et al. [84] confirmed mixture-specific genotoxicity in human hepatocytes, showing that some combinations induced comet-assay DNA damage or γ-H2AX phosphorylation, while others had no detectable effect.

Finally, many pesticide mixtures are associated with stable epigenetic modifications, including altered DNA methylation, histone changes, and microRNA dysregulation, which may persist across generations (F1 - F3), contributing to long-term disease susceptibility and developmental abnormalities.

4.6. Epigenetic Effects

Transgenerational evidence shows that chronic exposure to pesticide mixtures can induce heritable epigenetic alterations affecting DNA methylation, histone structure, and microRNA profiles without modifying the underlying DNA sequence. In vivo work by Yu et al. [68] demonstrated that gestational exposure to organophosphate mixtures caused reproductive abnormalities and reduced fertility in F1 offspring, indicating persistent endocrine-related epimutations. Similarly, Jacobsen et al. [51] found that mixtures of endocrine-disrupting pesticides altered sexual and neurological development from early life to maturity, consistent with synergistic, mixture-driven reprogramming during sensitive developmental windows. Strong evidence of true epigenetic inheritance was provided by Thorson et al. [64], who observed stable sperm-DNA methylation changes and transgenerational testicular and renal diseases across F0 - F3 generations following permethrin-DEET exposure.

4.7. Endocrine Disorders and Reproductive Health

A substantial body of experimental and epidemiological evidence shows that chronic exposure to pesticide mixtures disrupts endocrine regulation, particularly affecting thyroid and gonadal pathways. These disruptions often translate into significant reproductive impairments and appear more pronounced than the effects induced by individual compounds.

Field observations by Bicho et al. [73] in lizards exposed to environmental mixtures revealed enlarged thyroid follicles and dysregulated hormone-receptor expression, directly impairing reproductive and thyroid function. In mammals, Dopavogui et al. [72] reported disrupted ovarian folliculogenesis and reduced progesterone levels in female mice exposed to multi-class mixtures, indicating impaired steroidogenesis. Similarly, Docea et al. [71] found that perinatal exposure to a mixture including glyphosate, dicamba, and 2,4-D produced dose-dependent thyroid-hormone alterations and renal dysfunction in dams and offspring.

Reproductive toxicity is consistently documented. Pascotto et al. [32] showed that combined dichlorvos-permethrin exposure disturbed estrous cyclicity, reduced estradiol, and induced ovarian abnormalities through hypothalamic-pituitary-ovarian axis disruption. In males, Valente et al. [74] reported exacerbated sperm defects and hormonal imbalance following glyphosate-2,4-D exposure, including germ-cell apoptosis and impaired blood-testis barrier integrity. Bouabdallah et al. [55] observed reduced sperm quality, low testosterone, oxidative stress, and testicular lesions in rats exposed to cypermethrin-mancozeb-metalaxyl mixtures.

At the molecular level, Crépet et al. [84] demonstrated that several mixtures strongly activated the human pregnane X receptor (PXR), inducing CYP3A4 expression and altering steroid and xenobiotic metabolism.

Collectively, these findings show that pesticide mixtures profoundly disturb endocrine signaling and reproductive function, highlighting the heightened vulnerability of hormonal systems to combined low-dose exposures.

4.8. Respiratory Disorders

Chronic exposure to pesticide mixtures, including highly toxic compounds such as paraquat, profenofos, and glyphosate, has been consistently linked to adverse respiratory outcomes in agricultural populations. In a cross-sectional study among Colombian farmers, Díaz-Criollo et al. [75] reported an increased prevalence of asthma, chronic bronchitis, allergic rhinitis, and chest pain in workers chronically exposed to these mixtures. Spirometry revealed reduced forced vital capacity (FVC) and forced expiratory volume in one second (FEV₁), indicating both obstructive and restrictive patterns.

Mechanistically, these effects are attributed to oxidative injury of alveolar membranes, excess reactive oxygen species, and chronic inflammatory responses. Paraquat, in particular, accumulates in lung tissue and generates superoxide radicals, promoting inflammation and fibrosis. Combined exposures may therefore exacerbate airway dysfunction, underscoring the elevated respiratory risks posed by pesticide mixtures.

4.9. Histological Effects

Histopathological and hematological alterations induced by pesticide mixtures are consistently documented in both experimental and epidemiological research. In a cross-sectional study of pesticide sprayers, Fareed et al. [45] reported significant hematological abnormalities, including reduced hemoglobin levels, decreased red blood cell counts, and pronounced cholinesterase inhibition—indicating subclinical yet biologically relevant impacts on hematopoietic and detoxification systems.

Experimental models reinforce these findings. In Helix aspersa, Zouaghi et al. [30] observed severe hepatic and renal lesions, cellular necrosis, hypertrophy, and metabolic disruption following subchronic exposure to neonicotinoid mixtures. Demur et al. [19] similarly reported altered signaling pathways and impaired hematopoiesis in bone-marrow progenitors of mice exposed to endosulfan, atrazine, and chlorpyrifos.

In vitro analyses reveal mixture-specific interactions: combinations such as M1 and M5 induced strong cellular responses exceeding additive expectations, whereas M2 and M6 showed minimal effects [84]. Overall, chronic exposure to pesticide mixtures can provoke substantial histological injury across hepatic, renal, and hematopoietic systems.

4.10. Drug Resistance Effects

In vitro evidence indicates that chronic exposure to pesticide mixtures can promote multidrug resistance (MDR) in human cells. Doğanlar et al. [76] showed that repeated exposure to a mixture containing chlorpyrifos-ethyl, deltamethrin, metiram, and glyphosate induced strong and persistent resistance to chemotherapeutic agents—including cisplatin, 5-fluorouracil, and temozolomide—in U87 glioblastoma cells. This durable resistance was driven by marked overexpression of efflux transporters (P-gp/ABCB1, BCRP/ABCG2) and elevated glutathione-S-transferase (GST/M1) activity, enhancing xenobiotic clearance. These mechanisms impair drug accumulation and efficacy, raising concerns that environmental pesticide mixtures may contribute to reduced cancer treatment responsiveness.

5. Scope and Limitations of the Study

It should be noted that the distinction between chronic low-dose exposure and intermittent higher exposure peaks, particularly in occupational settings, remains a methodological challenge in environmental health research. This is particularly relevant for occupational settings, where intermittent high-exposure peaks may coexist with chronic low-level exposure, complicating the strict classification of such exposures as “low-dose”.

This review demonstrates the breadth and severity of health effects associated with chronic, low-dose exposure to pesticide mixtures. Across more than sixty studies, including in vivo, in vitro, and epidemiological evidence, consistent alterations were identified in metabolic, neurological, reproductive, endocrine, immunological, and genetic systems. Key mechanistic pathways such as oxidative stress, mitochondrial dysfunction, endocrine disruption, and DNA damage emerged repeatedly, underscoring the capacity of mixtures to elicit multi-system toxicity that often exceeds the effects of individual compounds. Frequent reports of synergistic or supra-additive effects, although often not confirmed by formal modeling, are noted. The observation of synergistic and cumulative interactions further challenges the long-standing hypothesis that low doses of isolated pesticides are biologically insignificant.

However, several limitations must be acknowledged. First, the substantial methodological heterogeneity across studies complicates direct comparison. Variations in mixture composition, exposure duration, administered doses, biological matrices, and analytical protocols introduce uncertainty in establishing consistent dose–response relationships (Table 2). Second, some studies do not report precise exposure levels, relying instead on estimated or self-reported data, which reduces toxicological accuracy. Third, many investigations focus on highly exposed populations, such as agricultural workers, which limits the generalizability of findings to the broader population. Finally, the mechanistic interactions between pesticides within mixtures, particularly concerning endocrine, epigenetic, mitochondrial, and immunological pathways, remain only partially understood, making it difficult to conduct a complete cumulative risk assessment.

Additional limitations include potential publication bias, language restrictions, and the absence of a quantitative meta-analysis, which may limit the generalizability and strength of the conclusions.

6. Conclusions

The analysis also highlights methodological and regulatory limitations. Most toxicological assessments still rely on single-compound testing, failing to reflect real-world exposure scenarios in which humans encounter complex and variable chemical mixtures. Significant gaps persist regarding long-term, multigenerational, and environmentally relevant exposure conditions, especially for vulnerable populations such as agricultural workers, children, and pregnant women.

These findings underscore the urgent need for updated regulatory frameworks that explicitly incorporate mixture toxicity, cumulative risk assessment, and mechanistic biomarkers of early effect. Strengthening environmental monitoring, improving occupational protection, and promoting integrated pest-management strategies are essential steps to reducing health risks. At the same time, research efforts must prioritize multidisciplinary approaches capable of capturing the complexity of mixture-driven toxicity, including systems biology, exposome-focused methods, and advanced computational modeling.

Overall, this review provides robust scientific evidence to support more protective public-health policies and to guide future research on the health risks associated with pesticide mixtures. These findings call for a paradigm shift in regulatory toxicology, moving from single-compound assessments toward an integrated evaluation of cumulative and mixture effects.

Addressing these challenges is critical for ensuring safer agricultural practices, preventing chronic diseases, and preserving long-term environmental and population health. Failing to account for mixture effects may lead to a systematic underestimation of real-world health risks.

Acknowledgements

This work was supported by the International Development Research Centre (IDRC) and the Ministry of Higher Education, Research, and Innovation (MESRI) through the FIRST program, within the SGCI funding framework.

Supplementary

Table S1. General overview of included studies.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References