Development of a specific and sensitive method for the detection of glyphosate pesticide and its metabolite in tea using dummy molecularly imprinted solid-phase extraction coupled with liquid chromatography-tandem quadrupole mass spectrometry.

Glyphosate, a widely used herbicide, and its primary metabolite aminomethyl phosphonic acid have been found to cause environmental and ecological issues and threaten human health. The conventional pretreatment method was insufficient for the extraction, concentration, and enrichment of trace substances, resulting in poor specificity. Thus, our objective was to develop a method for glyphosate pesticide detection using dummy molecularly imprinted solid-phase extraction (DMI-SPE) combined with liquid chromatography-tandem quadrupole mass spectrometry (DMI-SPE-LC/MS/MS). The sol-gel method was used to prepare the molecularly imprinted material, using glyphosine as the dummy template molecule, to achieve specific adsorption to glyphosate and reduce costs. The optimized polymerization conditions achieved maximum adsorption of 28.6 µg/mg glyphosate by the molecularly imprinted material. The established DMI-SPE-LC/MS/MS method was used to detect glyphosate and its metabolite (aminomethyl)phosphonic acid in tea. The concentration ranges of glyphosate and (aminomethyl)phosphonic acid (from 0.05 to 4 µg/mL) were linear with correlation coefficients of 0.999 and 0.991, respectively. The recoveries of (aminomethyl)phosphonic acid at three spiked levels ranged from 79.95% to 83.74%, with RSDs between 6.40% and 7.45%, while the recoveries of glyphosate ranged from 98.69% to 106.26%, with RSDs between 0.91% and 1.18%. Our results demonstrate that the developed DMI-SPE-LC/MS/MS method achieves high sensitivity and specific detection of glyphosate and its metabolite (aminomethyl)phosphonic acid in tea matrices.

[Investigation on health status of workers exposed to glyphosate].

Objective: To analyze the concentrations of glyphosate and its metabolites in occupational exposed workers and their possible effects on human health, so as to provide a reference for improving the safe use of glyphosate and toxicity research. Methods: From April to December 2020, 247 workers directly exposed to glyphosate in 5 enterprises were selected as the contact group, and 237 workers who were not exposed to glyphosate and other pesticides in the same enterprise were selected as the control group. Questionnaire survey and occupational health examination were conducted on objects, and the concentrations of glyphosate and its metabolites in the air of workplaces and biological samples were detected. The correlation between the concentrations and the difference of health examination between the two groups were analyzed. Results: The urine glyphosate concentration (0.022-47.668 mg/L), the rate of exceeding the standard (60.32%, 149/247) and the urine aminomethyl phosphonic acid concentration (<0.010-1.624 mg/L) in the contact group were higher than those in the control group [urine glyphosate concentration (<0.020-4.482 mg/L), the rate of exceeding the standard (2.53%, 6/237) and the urine aminomethyl phosphonic acid concentration (<0.010-0.524 mg/L) ], respectively (P<0.001). The exceeding standard rate of glyphosate concentration in the workplace was 33.67% (33/98). The concentration of glyphosate in the workplace was positively correlated with the concentrations of glyphosate and aminomethylphosphonic acid in urine (r(s)=0.804, 0.238, P<0.001), and the concentration of glyphosate in urine was positively correlated with the concentration of aminomethylphosphonic acid in urine (r(s)=0.549, P<0.001). The alanine aminotransferase (ALT), white cell ratio, creatinine, uric acid, the abnormal rates of ALT and total protein (TP) in the contact group were higher than those in the control group, and TP was lower than that in the control group, the differences were statistically different (P<0.05). The abnormal rates of overall liver function, overall renal function, blood routine test, urine routine test, electrocardiogram, liver B ultrasound and blood lipid in the contact group were significantly higher than those in the control group (P<0.05) . Conclusion: The concentration of glyphosate in the workplace is related to the concentrations of glyphosate and aminomethyl phosphonic acid in the urine of workers, and exposure to glyphosate may have some harmful effects on human health.

Investigation on health status of workers exposed to glyphosate / 中华劳动卫生职业病杂志

Objective: To analyze the concentrations of glyphosate and its metabolites in occupational exposed workers and their possible effects on human health, so as to provide a reference for improving the safe use of glyphosate and toxicity research. Methods: From April to December 2020, 247 workers directly exposed to glyphosate in 5 enterprises were selected as the contact group, and 237 workers who were not exposed to glyphosate and other pesticides in the same enterprise were selected as the control group. Questionnaire survey and occupational health examination were conducted on objects, and the concentrations of glyphosate and its metabolites in the air of workplaces and biological samples were detected. The correlation between the concentrations and the difference of health examination between the two groups were analyzed. Results: The urine glyphosate concentration (0.022-47.668 mg/L), the rate of exceeding the standard (60.32%, 149/247) and the urine aminomethyl phosphonic acid concentration (<0.010-1.624 mg/L) in the contact group were higher than those in the control group [urine glyphosate concentration (<0.020-4.482 mg/L), the rate of exceeding the standard (2.53%, 6/237) and the urine aminomethyl phosphonic acid concentration (<0.010-0.524 mg/L) ], respectively (P<0.001). The exceeding standard rate of glyphosate concentration in the workplace was 33.67% (33/98). The concentration of glyphosate in the workplace was positively correlated with the concentrations of glyphosate and aminomethylphosphonic acid in urine (r(s)=0.804, 0.238, P<0.001), and the concentration of glyphosate in urine was positively correlated with the concentration of aminomethylphosphonic acid in urine (r(s)=0.549, P<0.001). The alanine aminotransferase (ALT), white cell ratio, creatinine, uric acid, the abnormal rates of ALT and total protein (TP) in the contact group were higher than those in the control group, and TP was lower than that in the control group, the differences were statistically different (P<0.05). The abnormal rates of overall liver function, overall renal function, blood routine test, urine routine test, electrocardiogram, liver B ultrasound and blood lipid in the contact group were significantly higher than those in the control group (P<0.05) . Conclusion: The concentration of glyphosate in the workplace is related to the concentrations of glyphosate and aminomethyl phosphonic acid in the urine of workers, and exposure to glyphosate may have some harmful effects on human health.

Higher proportion of agricultural land use around the residence is associated with higher urinary concentrations of AMPA, a glyphosate metabolite.

INTRODUCTION: Pesticides, including herbicides, are widely used for agricultural and sanitary reasons and concerns have been raised about their various health effects. Little research has been done into the extent to which agricultural land use in the residential surroundings contributes to (internal) exposure of pesticides. OBJECTIVES: We investigated the associations between the proportion of agricultural land use around the residence and the exposure to pesticides in adolescents in Flanders (Belgium). MATERIAL AND METHODS: We included 424 adolescents participating in the fourth Flemish Environment and Health Study (FLEHS IV) between 2016 and 2020. The residential address of all participants was geocoded and the proportion of agricultural land use around the residence was estimated in several buffers (300 m, 500 m, 1000 m and 2000 m). The concentrations of the following biomarkers of pesticides were measured in urine and adjusted for the specific gravity: glyphosate and its metabolite, aminomethyl-phosphonic acid (AMPA); 3-phenoxybenzoic acid (3-PBA); 3,5,6-trichloro-2-pyridinol (TCPy) and 2,4-dichlophenoxy-acetic acid (2,4-D). We categorized the pesticide biomarkers in three categories according to the exposure levels and used ordinal logistic regression models adjusted for sex, season and household education to estimate the odds ratio for an increase in an interquartile range (IQR) of proportion of agricultural land use. We also used binary logistic regression models in which the category of highest exposure was compared to the category of lowest exposure. In addition, we explored potential effect modification by sex and season. RESULTS: We found a significant association between the proportion of agricultural land use in a buffer of 2000 m around the residence and the levels of urinary AMPA divided into three categories (OR = 1.35 for an IQR increase in the proportion of agricultural land use around residence; 95% CI: 1.00-1.83). This association was less pronounced and not statistically significant for the other studied pesticides (OR ranging between 0.95 and 1.16). Stratified analysis showed the strongest association of the proportion of agricultural land use within 2000 m buffers for AMPA among boys (OR = 1.89; 95% CI: 1.19-3.04). Results using smaller buffers were comparable, but did not reach statistical significance. CONCLUSION: Our findings suggest that a higher proportion of agricultural land use around the residence might increase exposure to AMPA.

Effects of low-concentration glyphosate and aminomethyl phosphonic acid on zebrafish embryo development.

Glyphosate (GLY) is the most widely used broad-spectrum, non-selective herbicide in the world, whose main degradation product is aminomethyl phosphonic acid (AMPA). Because of long-term and large-scale use, residual GLY and AMPA in the environment pose great environmental and human health threats. The purpose of this study is to evaluate the effects and mechanism of residual low-concentrations of GLY and AMPA in the environment on the development of zebrafish embryos. Zebrafish embryos were exposed to 0, 1, 10, 100, and 700 ng·mL-1 GLY and AMPA for 72 h (from 2 to 74 h post-fertilization). With increasing exposure dose, heart rates of both embryos and larvae showed a rising trend and obvious arrhythmia appeared. Defects in cardiac development and function of zebrafish juveniles may be related to altered transcription levels of cardiac development genes (TBX5, NKX2.5, BMP4) and apoptosis genes (Bcl-2, Bax). In addition, pericardial edema and bone deformation of zebrafish embryos may be caused by inhibition of Na+/K+-ATPase and Ca2+-ATPase after exposure to GLY and AMPA. The present results demonstrated that at typical environmental residual concentrations of GLY and AMPA had similar developmental toxicity in zebrafish embryos.

Bioconcentration of glyphosate in wetland biofilms.

Wetland biofilms were exposed to the herbicide glyphosate via in situ field exposures and controlled microcosm experiments to measure bioconcentration and metabolism of glyphosate by biofilm organisms. Concentrations of glyphosate in biofilms were two to four orders of magnitude higher than the surrounding water, bioconcentration factors averaged 835 and 199 L·kg-1 in field- and lab-exposed biofilms, respectively. Glyphosate in water where it had been detected in biofilms at field-exposed sites ranged from below detection (<1 µg·L-1) up to 130 µg·L-1. Bioconcentration of glyphosate in biofilms was inversely proportional to concentrations in the surrounding water, and the retention kinetics were similar to both adsorption and enzymatic models. Microorganisms present in both the water and biofilms metabolized glyphosate to its primary breakdown product aminomethyl phosphonic acid (AMPA), with increased rates of breakdown in and around the biofilms. Photosynthetic efficiency of the algae within the biofilms was not affected by 24 h controlled glyphosate exposures. Our results demonstrate the role of biofilms in improving wetland water quality by removing contaminants like glyphosate, but also as a potential exposure route to higher trophic levels via consumption. Due to bioconcentration of pesticides, exposure risk to organisms consuming or living in biofilms may be much higher than indicated by concentrations in ambient water samples.

Determination of glyphosate and AMPA in oat products for the selection of candidate reference materials.

The use of reference materials (RMs) is critical for validating and testing the accuracy of analytical protocols. The National Institute of Standards and Technology (NIST) is in initial stages of developing a glyphosate in oats RM. The first aim of this study was to optimize and validate a robust method for the extraction and analysis of glyphosate and aminomethylphosphonic acid (AMPA). The optimized method was used to screen thirteen commercially available oat products to identify candidate RMs. Glyphosate was detected in all samples, with the highest glyphosate mass fraction of 1100 ng/g; lower levels were measured in grains from organic agriculture. AMPA was quantified in nine samples up to 40 ng/g. The findings of this study led to the identification of candidate RMs, with "high" and "low" glyphosate levels. A preliminary stability study determined that glyphosate is stable in oat material at room temperature for six months.

Dissolved organic matter does not promote glyphosate degradation in auto-heterotrophic aquatic microbial communities.

Environmental dissolved organic matter (DOM) has been proved to increase microbial population sizes and stimulate the degradation of some pesticide molecules. Among these molecules, the present study investigated the biodegradation of the herbicide glyphosate depending on photoautotrophs DOM supply in a microbial consortium isolated from river biofilms. Degradation experiments in the laboratory were performed in dark and light conditions, as well as after antibiotic supply, in order to characterize the eventual interactions between photoautotrophs and heterotrophs activity during glyphosate degradation. Fifty percent of the initial concentration of glyphosate (0.6 mM) was transformed into aminomethyl phosphonic acid (AMPA) after 9 days in presence or absence of light. Accordingly, the photoautotrophic DOM supply was not stimulating glyphosate degradation by microbial heterotrophs. This lack of response was probably explained by the low net primary production values and weak dissolved organic carbon production recorded in light treatments. The supply of the antibiotic drastically stopped glyphosate transformation demonstrating the central role of bacteria in the biodegradation of the herbicide. Glyphosate also modified the structure of prokaryotes assemblages in the consortium by increasing the relative abundances of Alphaproteobacteria and slightly decreasing those of Gammaproteobacteria. The chemoorganotrophic bacteria Phenylobacterium sp. (Alphaproteobacteria) was related to the transformation of glyphosate in our microbial consortium. The present study highlights the complexity of microbial interactions between photoautotrophs and heterotrophs in microbial assemblages that can contribute to the degradation of pesticides present in aquatic environments.

[Rapid determination of glyphosate, aminomethyl phosphonic acid, glufosinate, and ethephon residues in environmental water by direct injection-ultra performance liquid chromatography-triple quadrupole mass spectrometry].

A simple method based on direct injection-ultra performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS/MS) was established for the rapid determination of glyphosate, aminomethyl phosphonic acid, glufosinate, and ethephon residues in environmental water. The water samples were filtered through a 0.22-µm filter membrane or frozen and centrifuged to remove impurities, and then, the filtrate was directly subjected to quantitative analysis without derivatization. The analytes were separated on a Metrosep A Supp 5 column (150 mm×4.0 mm, 5 µm), and gradient elution was carried out using an ammonium bicarbonate-ammonia solution as the mobile phase. The data were collected by positive electrospray ionization in the multiple reaction monitoring (MRM) mode. The results showed that the correlation coefficients (r) of the linear calibration curves were greater than 0.999 in the corresponding linear ranges (0.50-50.0 µg/L). The detection limits of the analytes were 0.05-0.09 µg/L. The recoveries of glyphosate, aminomethyl phosphonic acid, glufosinate, and ethephon were in the ranges 76.3%-108%, 83.0%-107%, and 87.0%-105% at low, medium, and high spiked levels, respectively. The corresponding relative standard deviations were in the ranges 2.0%-12.3%, 2.4%-5.6%, and 2.7%-6.8%. Using this method, 34 water samples collected from Hainan Province were analyzed, among which 30 drinking water sources were found to be free from the four pesticides. Glyphosate and aminomethyl phosphonic acid were detected in three water samples near a betel nut orchard, while glufosinate and aminomethyl phosphonic acid were detected in a water sample near a banana orchard. This method is advantageous over the traditional derivatization method because of its simple operation, good reproducibility, and high accuracy; furthermore, the matrix interference effect is absent. Thus, this method is suitable for analyzing glyphosate, aminomethyl phosphonic acid, glufosinate, and ethephon residues in environmental water samples.

The utilisation of ion chromatography and tandem mass spectrometry (IC-MS/MS) for the multi-residue simultaneous determination of highly polar anionic pesticides in fruit and vegetables.

A robust and sensitive method utilising a hybrid ion chromatography tandem mass spectrometry system (IC-MS/MS) for the simultaneous determination of nine (9) highly polar anionic pesticides (chlorate, ethephon, fosetyl aluminium, glufosinate, glyphosate, N-acetyl AMPA, N-acetyl glyphosate, perchlorate and phosphonic acid) in fruit and vegetables is described. Mean recoveries (n = 6) at two fortification levels ranged from 83 to 112% with %CVs in the range 3-14%. The linearity range was 0.005-0.4 mg kg-1 and R2 values were >0.99 and the sensitivity of the method allowed (20× or 30×) dilution of samples. Provision of qualitative determination of aminomethylphosphonic acid (AMPA) was also facilitated via minor modification of the chromatographic conditions. Compliance with method validation criteria, survey results from the statutory UK/EU Pesticide Residues in Food 2018 programmes i.e. pea, pineapple, melon and successful z-scores for a UK proficiency testing scheme sample (ethephon in pineapple) demonstrate successful application of this IC-MS/MS method.

Meta-analysis of glyphosate contamination in surface waters and dissipation by biofilms.

One consequence of the intensive use of glyphosate is the contamination of rivers by the active substance and its metabolites aminomethyl phosphonic acid (AMPA) and sarcosine, inducing river eutrophication. Biofilms are the predominant lifestyle for microorganisms in rivers, providing pivotal roles in ecosystem functioning and pollutant removal. The persistence of glyphosate in these ecosystems is suspected to be mostly influenced by microbial biodegradation processes. The present study aimed to investigate the tripartite relationship among biofilms, phosphorus and glyphosate in rivers. The first part consists of a co-occurrence analysis among glyphosate, AMPA and phosphorus using an extensive dataset of measurements (n = 56,198) from French surface waters between 2013 and 2017. The second part investigated the capacity of natural river biofilms to dissipate glyphosate, depending on phosphorus availability and the exposure history of the biofilm, in a microcosm study. A strong co-occurrence among glyphosate, AMPA and phosphorus was found in surface waters. More than two-thirds of samples contained phosphorous with glyphosate, AMPA or both compounds. Seasonal fluctuations in glyphosate, AMPA and phosphorus concentrations were correlated, peaking in spring/summer shortly after pesticide spreading. Laboratory experiments revealed that natural river biofilms can degrade glyphosate. However, phosphorus availability negatively influenced the biodegradation of glyphosate and induced the accumulation of AMPA in water. An increase in alkaline phosphatase activity and phosphorus uptake was observed in glyphosate-degrading biofilms, evidencing the tight link between phosphorus limitation and glyphosate degradation by biofilms. The results of the present study show that phosphorus not only is a key driver of river eutrophication but also can reduce complete glyphosate degradation by biofilms and favour the accumulation of AMPA in river water. The predominant role of biofilms and the trophic status of rivers must therefore be considered in order to better assess the fate and persistence of glyphosate.

Glyphosate analysis using sensors and electromigration separation techniques as alternatives to gas or liquid chromatography.

Since its introduction in 1974, the herbicide glyphosate has experienced a tremendous increase in use, with about one million tons used annually today. This review focuses on sensors and electromigration separation techniques as alternatives to chromatographic methods for the analysis of glyphosate and its metabolite aminomethyl phosphonic acid. Even with the large number of studies published, glyphosate analysis remains challenging. With its polar and depending on pH even ionic functional groups lacking a chromophore, it is difficult to analyze with chromatographic techniques. Its analysis is mostly achieved after derivatization. Its purification from food and environmental samples inevitably results incoextraction of ionic matrix components, with a further impact on analysis derivatization. Its purification from food and environmental samples inevitably results in coextraction of ionic matrix components, with a further impact on analysis and also derivatization reactions. Its ability to form chelates with metal cations is another obstacle for precise quantification. Lastly, the low limits of detection required by legislation have to be met. These challenges preclude glyphosate from being analyzed together with many other pesticides in common multiresidue (chromatographic) methods. For better monitoring of glyphosate in environmental and food samples, further fast and robust methods are required. In this review, analytical methods are summarized and discussed from the perspective of biosensors and various formats of electromigration separation techniques, including modes such as capillary electrophoresis and micellar electrokinetic chromatography, combined with various detection techniques. These methods are critically discussed with regard to matrix tolerance, limits of detection reached, and selectivity.

Uptake, Translocation, Metabolism, and Distribution of Glyphosate in Nontarget Tea Plant (Camellia sinensis L.).

The uptake, translocation, metabolism, and distribution behavior of glyphosate in nontarget tea plant were investigated. The negative effects appeared to grown tea saplings when the nutrient solution contained glyphosate above 200 mg L-1. Glyphosate was highest in the roots of the tea plant, where it was also metabolized to aminomethyl phosphonic acid (AMPA). The glyphosate and AMPA in the roots were transported through the xylem or phloem to the stems and leaves. The amount of AMPA in the entire tea plant was less than 6.0% of the amount of glyphosate. The glyphosate level in fresh tea shoots was less than that in mature leaves at each day. These results indicated that free glyphosate in the soil can be continuously absorbed by, metabolized in, and transported from the roots of the tea tree into edible leaves, and therefore, free glyphosate residues in the soil should be controlled to produce teas free of glyphosate.

Determination of Glyphosate, Aminomethyl Phosphonic Acid and Glufosinate in Different Teas by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry / 分析化学

An efficient technique for the determination of multiclass pesticide residues ( glyphosate, aminomethyl phosphonic acid ( AMPA) and glufosinate) in four kinds of tea ( green tea, black tea, Oolong tea and Puer tea) was developed based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were extracted with 0. 05 mol/L NaOH solution, regulated pH value with HCl, followed by purification by Oasis HLB column, and then precolumn-derived with 9-fluorenylmethyl chloroformate ( FMOC-CL ) in borate buffer. All pesticide residues studied showed good linearity with correlation coefficient ( R2 ) greater than 0. 99 in the concentration range of 5-1000 μg/L. Limits of quantitation (LOQs) of three pesticide compounds ranged from 0. 03 to 0. 08 mg/kg (S/N=10). Tea samples spiked with 0 . 1 , 0 . 4 and 4 mg/kg showed the recovery ranging from 72 . 1% to 109 . 9%, and RSDs from 0. 5% to 9. 8% (n=6). In addition, the validated method was applied to commercial samples, and all the detections were confirmed by acquiring transitions for each pesticide in samples.