Determination of Glyphosate in human urine from farmers in Mato Grosso-BR

This study was conducted based on the evaluation of glyphosate levels present in rural workers in the region of Nova Mutum-MT. We analyzed 90 urine samples from farmers between 2017 and 2018. The samples were analyzed based on the development of the high-performance liquid chromatography (HPLC-FL) method. The results showed that 12% of the farmers presented glyphosate levels, but that the highest concentration determined by the method used was not above the limits allowed by Brazilian regulators. The HPLC-FL method proved to be practical and accurate for the determination of glyphosate in urine samples with limits of detection and quantification of 0.34 and 1.15ng/mL, respectively. These data show the importance of evaluating the occupational exposure of farmers to adopt strategies for the biomonitoring of this region, considering that casual exposures to pesticides can generate health risks, as well as cases of intoxication. The samples were removed from the freezer, allowing them to reach the room temperature of their homogenization. We pipetted 2mL of urine and added 0.4mL of the standard working solution and 0.6mL of acetonitrile. The samples were centrifuged at 3000rpm for 10 minutes and passed in SPE C18 pre-conditioned cartridges with 2mL of acidified methanol with 100µL of 0.1% formic acid (solution of deaerated methanol) and eluted with 3mL of acetonitrile. The calibration curve was evaluated from 1.15 to 120.00ng/mL. All calibration points were reproduced in triplicate. For the derivatization reaction, a concentration of 1.6µL/mL of Fmoc-Cl and 500µL of borate buffer solution pH 9 for each 1mL of sample was added, so that the proportion of glyphosate and Fmoc-Cl was 1:4. The derivatization reaction was performed in an ultrasonic cleaner for 15 minutes at room temperature.

Pre-Publication Release of Accepted Article DOI: https://doi.org/10.31005/iajmh.v3i0.124 occurring in Brazil are due to the lack of public policies for the correct use of pesticides; low level of schooling of farmers and little understanding of the use of these agents during a continuous and long-term exposure. Moreover, the country lacks biological monitoring to evaluate the exposure levels of pesticides in rural workers [11].
Given this context, we can affirm the Brazilian official data do not show the country's reality about pesticide intoxication [11]. Therefore, it became essential to evaluate the populations exposed to pesticides to measure the levels of contamination present in the exposed individuals; provide for diagnoses of changes in their health status and propose stricter public safety measures to control this exposure.
Pre-Publication Release of Accepted Article DOI: https://doi.org/10. 31005/iajmh.v3i0.124 in 1974 by Monsanto with the trade name Roundup ® [13]. In 1978, it was sold for the first time in Brazil by importation and, only in 1984, it began to be produced in the country [14]. It is a non-selective, systemic, post-emergent agent and has high efficiency in weed elimination [12].
Its wide use can be justified, as this herbicide has generated one of the greatest advances in agriculture from the environmental perspective. This is because, in the past, the biggest problem in crop regions was the erosion caused by the techniques of ploughing and harrowing, used for combating weeds. With the emergence of glyphosate, it was possible to extinguish these techniques, which increased the use of no-tillage and drastically reduced the occurrence of erosions in the crop areas [15]. In Figure 1, we can observe the distribution of the amount of glyphosate marketed in Brazil between 2009 and 2017 [13].
A study on the distribution of pesticide use in Brazil by Pignati, 2017, showed that, between 2012 and 2016, glyphosate was among the 20 most used active ingredients in Brazil. In addition, the authors reported that of the many cultures in which this herbicide is applied, the main ones are soybean, sugarcane and maize.
The authors of the study also showed that transgenic monoculture can increase the use of pesticides, since these crops are generally tolerant to glyphosate and, therefore, cause the emergence of more resistant pests, increasing thus the use of this herbicide [4].
Currently, glyphosate has been undergoing a toxicological revaluation due to suspicions of presenting possible mutagenic, teratogenic and carcinogenic characteristics. In this context, ANVISA reassessed the parameters for the risk assessment of glyphosate by establishing new levels for: Acceptable daily intake (ADI) = 0.5mg/kg/day; acute reference dose (ARfD) = 0.5mg/Kg/day and acceptable operator exposure level (AOEL) = 0.1mg/Kg/day [13]. In the United States, regulatory bodies consider an ADI of 1.75mg/kg/day [16]. In the European Due to the facts mentioned, it is necessary to monitor biological and reliable biomarkers to ensure safety regarding the exposure of humans to pesticides.

Sample collection region
The state of Mato Grosso is the Brazilian state with the highest soybean production. According to Brasil, 2010 Therefore, it is important to analyze the relationship between occupational exposure and socioeconomic data of rural workers, as they may be directly related to cases of pesticide intoxication.

Study design
It is a study on the impacts of exposure and direct contact in the long term, that is, more than 1 year, of glyphosate to the rural worker in the region of Nova

Sample preparation
The stock solutions of the glyphosate pesticide were prepared by the dissolution in water of 10mg of standard dose for a final volume of 10mL. We prepared 100mL of a working solution of concentration 6.0ng/mL, containing the glyphosate pesticide. Automatic sampler temperature: 24°C; Detector: wavelength emission and excitation: 263nm and 317nm, respectively.

Results
The validation was performed following the parameters recommended by the The F test performed in the repeatability test and intermediate precision showed that no statistically significant differences were found between the precisions on different days, since the values were in the range of 1.11 and 2.74, less than the tabulated (5.05). The applicability of the method was verified through the analysis of a fortified matrix with glyphosate at the levels described in Table 2.
Although glyphosate residues were present in some of the samples analyzed, none of the cases exceeded the values allowed, established at national and international levels, since the highest level found was 7.13ng/mL. The highest value found in the samples was 0.007mg/kg. This value represents an external dose of 0.0011mg/kg that was determined based on the studies carried out by Niemann [15] and   The results give an initial idea that the workers are exposed to glyphosate and the direct contact seems to be the main source of exposure. However, a more detailed study is needed to distinguish between different exposure situations, in addition to direct contact. Thus, the analytical method developed fulfills the validation requirements and is suitable for the determination of glyphosate residues in urine.

Conclusion
Numerous factors can influence changes in the individual's health beyond direct contact with the crop protection product. Therefore, assessing the exposure of rural workers to pesticides is still quite complex. Likewise, it is necessary to diagnose possible health events caused by these agents.
The results presented indicate that on the day of glyphosate exposure, detectable levels of this pesticide were found in urine samples, but during the period before and after exposure and no direct contact. These data corroborate international studies in the literature.
Although these levels indicate a low occupational risk, biomonitoring studies such as this need to be expanded to support more scientifically based regulatory actions.