Despite being the most widely used herbicide in the world, the long-term health effects of glyphosate remain unknown. Air, water, and soil have been polluted by its widespread use, and human and animal health problems have been linked to it. The case of glyphosate illustrates the difficulty involved in evaluating, testing, assessing, and regulating chemicals. The implementation of human studies is challenging; subsequent studies on safety and risk assessment are lacking; environmental impacts are not well understood; industry participation in assessment and approval systems is extensive; and these evaluations have significant legal implications. In addition, there are also ethical issues such as informed consent, confidentiality, and protection of vulnerable populations. Furthermore, there are also concerns about potential conflicts of interest that may arise from industry involvement in these evaluations. All of these factors make it difficult to accurately assess the risks of such pesticides and take appropriate action to mitigate them prior to their widespread use. Without proper regulation, these products can have devastating consequences. It is essential to create and enforce regulations that limit the use of high risk pesticides and ensure the safety of the public and the environment. Governments should also invest in research to better understand the potential risks of these chemicals and develop better strategies for managing them in the future. Although EPSP does not exist in humans, glyphosate has relatively low acute toxicity. Adjuvants are used in pesticides to make the active ingredients more effective. However, they can also increase the toxicity of the pesticide, making it more likely to cause negative health effects. A similar scenario could occur with glyphosate [44].

Glyphosate’s carcinogenic effect has been investigated several times since its introduction for weed control in different crops. In the last few decades, various agencies have reported different results regarding glyphosate’s carcinogenicity. Some have found that glyphosate is a probable human carcinogen, while others conclude that it is non-carcinogenic. The International Agency for Research on Cancer (IARC) classified glyphosate as probably carcinogenic in 2014 [29]. This classification has caused wide-spread concern among farmers and environmental groups. As a result, some countries have imposed restrictions on the use of glyphosate in agriculture. Fig. 2 illustrates the critical results of these agencies regarding glyphosate’s carcinogenicity.

Glyphosate may interfere with hormones because of its endocrine-disrupting effects, according to several studies [45]. These effects could potentially impact reproductive health, development, and other physiological processes. However, the evidence on glyphosate’s endocrine-disrupting potential is inconclusive, and further research is needed to establish a clear link. An overview of some studies suggests that glyphosate is capable of disrupting the endocrine system. Thongprakaisang et al. [46] performed in vitro experiments using human breast cancer cells and found that glyphosate and its commercial formulations could accelerate the growth of hormone-dependent breast cancer cells. Their hypothesis was that glyphosate might disrupt normal hormonal signals because of its estrogenic properties. Gasnier et al. [47] investigated the effects of glyphosate-based herbicides on human placental cells. In their study, herbicides triggered estrogenic responses, suggesting potential endocrine disruption. In this study, herbicides were used at very high concentrations, which may not reflect real-world exposures. Paganelli et al. [48] assessed the influence of glyphosate on amphibian larvae’s endocrine system. The researchers observed that glyphosate exposure led to developmental abnormalities and disruption of the hormonal system in amphibians. Further research is needed to determine whether these findings are relevant to human health. Clair et al. [49] evaluated glyphosate’s effects on reproductive health in rats. Reproductive parameters, hormonal profiles, and gene expression related to the reproductive system were altered. There was evidence that these effects could cause endocrine disruption, however, the study’s design and methodology were criticized. It’s critical to note that while these studies suggest potential endocrine-disrupting effects of glyphosate, other studies have not found similar results, and regulatory agencies have not universally accepted these findings. Regulatory agencies such as the EPA (Environmental Protection Agency) and EFSA (European Food Safety Authority) have concluded that glyphosate is unlikely to have any endocrine-disrupting effects when used as directed.

In animal studies, high doses of glyphosate have been associated with acute toxicity and organ damage. These effects are primarily triggered by exposures far higher than those typically found in the environment or in food. Despite this, long-term health effects associated with chronic low-dose exposure to glyphosate residues are still being researched and debated. Glyphosate formulations were found to induce cell death and cause oxidative stress in liver, kidney, and placental cells, according to Mesnage et al. [50]. Their study suggests that commercial glyphosate formulations’ adjuvants and other ingredients might contribute to the observed results. Despite this, it should be noted that the study used concentrations higher than those commonly found in the environment or dietary sources. In Koller et al.’s [51] study, rats were exposed to glyphosate and its effects were studied. Oxidative stress and changes in liver and kidney function were found to be caused by glyphosate. This research, however, used higher doses than normal for human exposure, similar to other studies. As reported by Cattani et al. [52], rats exposed to glyphosate during development had altered neurobehavioral parameters, oxidative stress, and neuropathological changes.

It has been suggested that glyphosate could disrupt the gut microbiome, which is crucial to human health. Several studies have shown that glyphosate exposure alters microbial composition and function. Nevertheless, more research is needed to fully understand how these changes will impact human health. The effect of glyphosate on the gut microbiota of poultry was investigated by Shehata et al. [53]. They discovered that glyphosate exposure decreased the amount of beneficial bacteria and increased the amount of potentially harmful bacteria in the gut microbiome. There was a potential disruption of gut microbial balance as a result of these changes. According to Motta et al. [54], glyphosate altered the diversity and composition of honey bee gut microbiota. It was found that these changes led to increased susceptibility to opportunistic pathogens and an impaired immune system. Despite these studies suggesting glyphosate may disrupt microbiomes, further research is necessary to fully comprehend the extent and significance of these effects. There are many factors that can affect glyphosate’s impact on the gut microbiome, such as dose, exposure duration, and particular microbial communities. Several studies report conflicting findings regarding glyphosate’s effects on gut microbiota, with some observing no significant effects. Further investigation is needed to determine the significance of the observed changes in the microbiome for human health.

Geographical disparities in residue levels further complicate global risk management. U.S. honey samples exhibiting 59% exceedance rates above 15 ppb, compared to European honey samples, where 21% exceeded the 50 μg kg⁻¹ maximum residue limit (MRL) [17,82]. These variations reflect differences in agricultural practices, regulatory stringency, and environmental persistence, particularly in regions with high soil organic matter content that enhances glyphosate adsorption [34]. In Canada, glyphosate concentrations in mushrooms and juice concentrates ranged from 0.0052 to 0.21 ppm and 0.0042 to 0.038 ppm, respectively, well below national MRLs [83]. In contrast, Uruguayan honey samples exceeded the European MRL of 50 μg kg⁻¹ in 50% of cases, reflecting lax regulatory enforcement and intensive glyphosate use in soybean cultivation [84]. These findings emphasize the need for harmonized global monitoring programs, particularly in low- and middle-income countries where regulatory infrastructure is underdeveloped.

Variability in limits of detection (LOD) and quantification (LOQ) complicates cross-study comparisons. Adams et al. [85] used ion chromatography-mass spectrometry (IC-MS) with an LOD of 0.01 mg kg⁻¹, whereas Xu et al. [86] achieved 0.03 mg kg⁻¹ via HPLC-MS/MS. Such differences may underestimate or overestimate residue prevalence. Studies like Kolakowski et al. [83] analysed Canadian retail foods (2015–2017) but lacked longitudinal data to assess temporal trends. Similarly, Pareja et al. [84] focused on Uruguayan honey without considering seasonal application patterns.

While glyphosate itself is often deemed low-risk, commercial formulations (e.g., Roundup) contain adjuvants like polyethoxylated tallow amine (POEA), which amplify toxicity. Hao et al. [56] attributed DNA damage in A549 cells to POEA, yet regulatory assessments frequently overlook adjuvant synergism. The ubiquity of glyphosate residues underscores the need for harmonized global monitoring programs. Regulatory frameworks must integrate adjuvant toxicity and chronic exposure risks, particularly for vulnerable populations (e.g., infants consuming cereal-based formulas).

Chromatographic techniques dominated residue determination, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) emerging as the most sensitive and reliable method. Santilio et al. [19] achieved a limit of detection (LOD) of 0.002 mg kg⁻¹ for glyphosate in rice using LC-MS/MS, while Goscinny et al. [95] reported a limit of quantification (LOQ) of 0.02 mg kg⁻¹ for cereals in Belgium via ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

Pesticide residues consumed in quantity and type must be considered when assessing possible adverse effects of pesticide residue intake, along with pesticide acceptable daily intake (ADI) Xu et al. [96]. The ADI is the maximum level of a pesticide that can be considered safe for human consumption. It is important to consider the type of pesticide, the type of food, and the quantity consumed when assessing the possible effects of pesticide residue intake. To assess glyphosate residue levels in soy-based infant formulas from the Brazilian market, de Souza et al. [97] used HPLC with fluorescence detection. Infant formula samples detected glyphosate concentrations ranging from none to 1.08 mg kg⁻¹. According to this study, participants’ exposure to glyphosate is not significant when compared to the acceptable daily intake (ADI) recommended by the appropriate authorities. Therefore, it can be concluded that soy-based infant formulas in Brazil are safe for consumption. The presence of glyphosate residues in yam was assessed by LC-MS/MS with a detection limit of 0.04 mg kg⁻¹, and a quantification limit of 0.12 mg kg⁻¹ Wumbei et al. [98]. Twenty-one percent of field experiments and market samples contained glyphosate, although at levels below the quantification limit. The study found that glyphosate residues are present at very low levels in yam, where they may not pose a health risk. The authors concluded that glyphosate residues are not a significant health concern for consumers of yam. However, they recommended that further research be conducted to determine the long-term effects of glyphosate residues on yam. Swiss market foods were evaluated using LC/MS/MS chromatographic technique for glyphosate residues by Zoller et al. [99]. Glyphosate has been detected in pasta, wine, fruit juice and honey samples at low concentrations by quantification limits 0.0005 to 0.0025 mg kg⁻¹. Despite this, the highest residue concentration did not approach the Acceptable Daily Intake (ADI). Therefore, glyphosate residues are not a major health issue in Switzerland’s foodstuffs.

Generally, the high solubility in water, ionic nature, low mass, high polarity, complex evaporation, low solubility in organic solvents, and low volatility are the most challenging factors in glyphosate residue analysis using simple analytical techniques Valle et al. [100]. Common chromatographic techniques, including high-performance liquid chromatography (HPLC) and gas chromatography (GC) using a wide range of detectors, are primary methods for determining glyphosate in food and the environment Ulrich and Ferguson, [101]. GC and HPLC could be connected with mass spectrometry (MS) to isolate complex compounds, quantify analytes, determinate unfamiliar peaks and detect trace contamination residues in different matrices Almeida [102]. In other words, mass spectrometry could play a vital role in improving the efficiency and accuracy of residue detection. In every residue determination method, the critical factors such as detection efficiency, recovery percentage, the limits of detection and quantification depend on extraction and purification techniques and the origin of the sample used for residue analysis Kapsi et al. [103].

The losses caused by consuming food contaminated with herbicide residues are much more significant than the cost of measures in the residue determination process Sharma et al. [104]. Analysis and identification of glyphosate residues are costly and time-consuming; therefore, the simple simultaneously analyzing methods cannot detect this herbicide and its metabolites in different matrices Kim et al. [105]. On the other hand, as the most used pesticide globally, glyphosate is not continuously monitored. Accordingly, no systematic data is associated with glyphosate and its metabolites residues in the environment, crops, and food. A basic chromatographic determination of glyphosate residues in crops and food can be seen in Fig. 4.

Despite advancements, methodological heterogeneity posed challenges. For example, Adams et al. [85] utilized ion chromatography-mass spectrometry (IC-MS) with an LOD of 0.01 mg kg⁻¹ for cereal-based infant foods, whereas Xu et al. [86] employed HPLC-MS/MS for Wuyi Rock tea, achieving an LOD of 0.03 mg kg⁻¹. Such discrepancies complicate cross-study comparisons, particularly when analyzing complex matrices like soy protein isolate or bovine muscle, where glyphosate binds to proteins or lipids, necessitating matrix-specific adaptations [106,107].

Validation gaps is another challenge regarding this subject. Few studies validated their methods against international guidelines (e.g., SANTE/11813/2017). Ehling and Reddy [106] demonstrated robust validation for soy protein isolate but excluded milk, raising concerns about extrapolation to other matrices. Disparities in extraction and derivatization methods (e.g., dansyl chloride vs. FMOC-Cl) hinder data comparability. For example, Khrolenko and Wieczorek [108] reported 0.025 mg L⁻¹ in juices using p-toluenesulphonyl chloride, whereas Rubio et al. [17] used alkaline hydrolysis for honey, yielding higher variability.

The exclusion of co-formulants in analytical protocols further limited risk assessments. Woźniak et al. [62] demonstrated that Roundup 360 PLUS, a commercial formulation containing poly-ethoxylated tallow amine (POEA), induced greater DNA damage in human peripheral blood mononuclear cells than glyphosate alone, yet analytical studies rarely quantified adjuvant residues. This oversight underscores the need for harmonized protocols that integrate multi-residue analysis of glyphosate, its primary metabolite aminomethylphosphonic acid (AMPA), and adjuvant compounds to reflect real-world exposure scenarios.

Carbon-based adsorbents (e.g., woody biochar, graphene oxide) achieved >90% removal efficiency in aqueous systems [26,117]. Mayakaduwa et al. [117] reported a maximum adsorption capacity of 44 mg g⁻¹ for woody biochar at pH 5–6, attributed to hydrogen bonding and electrostatic interactions, while Santos et al. [26] achieved a 92% removal rate using graphene oxide modified with iron oxide nanoparticles. However, field applications were hindered by soil matrix interference and production costs, particularly for energy-intensive biochar pyrolysis.

The process of adsorption is one of the most commonly used methods for removing chemicals from water solutions. It involves the use of adsorbents, which are materials with a higher affinity for the pollutants than the water. The adsorbents bind to the pollutants and are then removed, leaving the water free from contaminants Tian et al. [118]. Adsorption is a cost-effective and environmentally friendly method of water treatment, as it requires minimal energy input. Furthermore, it is safe to use for both humans and animals. It has been demonstrated in several laboratory studies that an adsorption approach is effective for the removal of pesticide residues from aquatic environments Odoemelam et al. [119]. A hierarchically porous metal-organic framework was reported to be an effective adsorbent for glyphosate removal from water Naghdi et al. [120]. According to Diel et al. [121] glyphosate was removed from an aqueous matrix using carbon nanotubes with multiple walls modified with metal nanoparticles. Various removal levels have been achieved, ranging from 40.33% to 68.38%. According to Sen et al. [122], forest soil shows 87.8 percent removal efficacy for glyphosate from aqueous solutions at pH 12. This suggests that forest soil could be a promising low-cost adsorbent for the removal of glyphosate from aqueous solutions. The pH of the forest soil was found to be optimal for glyphosate adsorption, which suggests that the forest soil contains compounds that can enhance glyphosate adsorption. Furthermore, the low-cost adsorbent nature of the forest soil could make it an attractive option for industrial and agricultural applications. In aqueous solutions, Eucalyptus camaldulensis bark activated char was tested as an adsorbent and 98 percent glyphosate removal was achieved at pH 10 Sen et al. [123]. As a result of the bark activated char binding with glyphosate molecules in the aqueous solution, less glyphosate is released into the environment.

Bioremediation strategies utilizing microbial and plant-based systems demonstrated variable efficacy. Burkholderia spp. and Pseudomonas aeruginosa degraded 76–85% of soil-bound glyphosate by cleaving the C–P bond via the glyA gene pathway [27,124], while Phytoremediation with Festuca arundinacea [125] and Salvinia biloba [126] achieved 87–100% efficiency, utilizing plant-microbe synergies. Adsorption and microbial activity are pH-sensitive. Hottes et al. [127] reported 90% glyphosate removal by Mg₂Al-layered double oxides at pH 10, whereas acidic conditions favored biochar efficacy. Such variability complicates field applications.

Herbicides reach the soil environment at high concentrations following application for weed control in agroecosystems. This is because herbicides are sprayed in large amounts onto crops and can be washed away by rain into the soil where they can spread and accumulate Mehdizadeh et al. [128]. This can have a detrimental effect on the ecosystem, as the herbicides can damage beneficial organisms like earthworms and other soil invertebrates. This can cause changes in the structure of the soil, leading to a decrease in soil fertility and a decrease in the growth of plants Ruuskanen et al. [129]. Additionally, herbicides can also have a negative effect on the water supply as they can leach into groundwater. There are a number of factors that affect herbicide fate in the environment, including soil physiochemical properties, soil microorganism populations, and climate conditions Hassaan et al. [130]. A rainfall rate of 1 mm/min for 1 hour carried 14% of sprayed glyphosate by runoff and suspended load from clay loam soil. This indicates that the amount of glyphosate removed from the soil surface was directly proportional to the rain intensity. Higher rain intensity resulted in more glyphosate being transported away from the soil surface Yang et al. [131]. Soil acts as a filter for herbicides, and its properties can affect the rate at which they are absorbed or degraded. Soil microorganisms can also break down herbicides, while climate conditions can influence how quickly they evaporate, runoff or leach into groundwater. Soil pH and organic matter content can positively influence the fate of herbicides, as well as the presence of other chemicals such as metal ions, which can bind the herbicide and reduce its availability for degradation van Hall et al. [132].

Microorganisms play a significant role in the degradation of pesticide compounds, since they are able to degrade the pesticide into simple or relatively non-harmful compounds. This process is known as bioremediation. Bioremediation is an effective and environmentally friendly way to remove pollutants from the environment and can be used to reduce pesticide residues in the environment Pande et al. [133]. There is no doubt that bacteria have the best detoxifying abilities among microbes. Bioremediation is also cost-effective and can be implemented with minimal disruption to the environment. Bacteria and other microbes are able to break down pollutants into smaller, more harmless molecules, which are then easily removed by natural processes such as evaporation and sedimentation Bala et al. [134]. The bioremediation of contaminated sites could be classified into two main categories, phytoremediation and microbial remediation. Phytoremediation uses plants to absorb and degrade pollutants, while microbial remediation relies on bacteria and other microbes to break down pollutants. Both methods have proven to be effective in reducing environmental contamination. Phytoremediation is usually used for heavy metals, while microbial remediation is recommended for organic pollutants. Both methods have advantages and disadvantages, and the most appropriate choice depends on the type of contamination and the resources available Shehata et al. [135].

Glyphosate can be degraded by native soil microbial communities that contain glyphosate-degrading microorganisms once it enters the soil environment. These microorganisms can rapidly degrade glyphosate, reducing the risk of environmental contamination and pollution. Therefore, management of soil pH and organic matter content can reduce herbicide contamination risk and promote glyphosate degradation Muskus et al. [136]. Nguyen et al. [25] investigated bacteria capable of degrading glyphosate and improved their degradation requirements for bioremediation. Using the strain Rhodococcus soli G41, it was found to have a high glyphosate degradation rate. It degraded 42.7% and 47.1% of glyphosate after 7 and 14 days, respectively. This can be attributed to the strain’s ability to produce enzymes that are capable of breaking down glyphosate into smaller substances that can then be further degraded into less toxic forms by bacteria. This suggests that the G41 strain is an efficient bacterium for bioremediation of glyphosate-contaminated soil.

Nevertheless, metabolite accumulation, particularly AMPA, remained a concern. Carranza et al. (2019) [137] documented 57% glyphosate degradation by Aspergillus oryzae in contaminated soil but did not quantify AMPA, a persistent byproduct with comparable toxicity. Additionally, Bioremediation often generates AMPA, a persistent metabolite. Carranza et al. [137] reported 57% glyphosate degradation by Aspergillus oryzae but did not quantify AMPA, risking secondary contamination. Similarly, urinary biomonitoring studies by Soukup et al. [138] detected glyphosate and AMPA in 99.6% of participants, with concentrations correlating positively with processed food consumption, yet regulatory thresholds do not account for cumulative dietary exposure.

However, field trials were notably scarce, with most studies confined to controlled laboratory conditions, raising questions about ecological trade-offs. For example, López-Chávez et al. [139] reported 87% glyphosate remediation using Panicum maximum, an invasive grass species that risks displacing native flora.

The review also highlighted significant discrepancies in glyphosate’s toxicological profile. While regulatory agencies such as the European Food Safety Authority (EFSA) and the U.S. Environmental Protection Agency (EPA) deemed glyphosate unlikely to pose carcinogenic risks at recommended doses, the International Agency for Research on Cancer (IARC) classified it as “probably carcinogenic” based on in vitro and animal studies [35, 69]. Epidemiological data further muddied the waters, Acquavella et al. [65] found no association between glyphosate and non-Hodgkin’s lymphoma in pooled cohort analyses. Endocrine disruption studies yielded similarly conflicting results. Gasnier et al. [47] observed estrogenic activity in human placental cells exposed to glyphosate-based herbicides. These inconsistencies underscore the influence of study design, exposure metrics, and adjuvant inclusion in toxicological assessments.