Pesticides are widely and extensively used worldwide in agriculture for controlling pests such as insects, rodents, and disease organisms (1). Insecticides are most frequently used in agriculture and the one of most common groups used insecticides is organophosphates (OP) (2, 3). Farmers and agricultural workers are at high risk of being exposed to these chemicals, the main routes of exposure being dermal contact, ingestion, and inhalation (4, 5). Exposure to these chemicals can cause both acute and chronic health consequences (4). Several studies have shown that exposure to OP was associated with chronic diseases such as asthma, rheumatoid arthritis, cancers, Alzheimer's disease, Parkinson's disease, and cognitive impairment (6–9).

The main mechanism contributing to the OP toxicity is that they inhibit red blood cells and the enzyme plasma cholinesterase (AChE) in the nervous system, which is responsible for the degradation of acetylcholine. When inhibition of AChE occurs, acetylcholine builds up in the neuro muscular and synaptic gaps, leading to an overabundance of acetylcholine within them (10). Therefore, neurological health problems demonstrate the main effects of OP toxicity. Furthermore, OP may have an effect on cognitive performance through cholinergic stimulation, oxidative stress, inflammation induction, and mitochondrial dysfunction in the nervous system (11, 12). Previously available studies found that long-term exposure to OP was associated with neurological disorders such as Alzheimer's disease, dementia, Parkinson's disease, and cognitive impairment (8, 9). Mild cognitive impairment is an early stage of cognitive impairment that leads to Alzheimer's disease and dementia. A study by Kim et al. (13) investigated the relationship between pesticide exposure and cognitive decline in a rural South Korean population, and they found that exposure to pesticides was associated with cognitive impairment. A study by Ramirez-Santana et al. (14) also investigated the effect of chronic exposure to OP on the neurobehavioral performance among agricultural workers and rural inhabitants, and they suggested that occupational exposure to OP resulted in a significantly lower neurobehavioral performance. Exposure to OP in farmworkers had also an effect on executive function, language, memory, attention, processing speed, visual-spatial function, and coordination (15). However, previously available studies had limitations such as those associated with study design, indirect measurement of OP exposure (questionnaire or measurement of AChE inhibition) (13–15). Although there is evidence that low-level exposure to OP were linked with cognitive decline and impaired neurobehavioral function, the potential effects of long-term low-level exposure to OP are unclear (16, 17).

In Thailand, agriculture employs more than 30% of the workforce and accounts for a major economic sector of the gross domestic product of the country. Pesticide use has risen over the past decade, and OP are some of the most used insecticides for controlling pests in agriculture (18, 19). Thai farmers are at high risk of adverse health effects from pesticide exposure due to lack of pesticide knowledge, unsafe occupational practices, and improper use of PPE (19). In this study, we hypothesized that improper use of PPE among pesticide applicators may increase urinary OP levels, and subsequently increase risk of cognitive impairment. Therefore, this study aims to: (a) compare urinary OP metabolite levels and cognitive performance between pre-and post-pesticide application seasons; (b) PPE use, and factors of PPE use associated with increased urinary OP metabolite levels during pesticide application; and (c) the association between urinary OP metabolite levels and cognitive performance.

Our findings show that the DEP metabolite was the major metabolite in urine samples as DEP had the highest detected and the highest levels when compared to other metabolites. It is possible that the DEP and DMP metabolites has different toxicokinetic characteristics. Diethyl OP are more lipophilic than dimethyl OP, therefore they can be stored in fatty tissues for longer periods of time, but dimethyl OP are immediately eliminated into the urine (26). Another possibility is that the most common OP used in this study area are triazophos (88.6%), followed by chlorpyrifos (15.2%). These two OP insecticides were classified as having diethyl groups; therefore, DEP metabolites were found as the major metabolite in urine samples.

A remarkable finding was that urinary OP metabolite levels post-pesticide application were significantly higher than those pre-pesticide application. Furthermore, the results also showed that the applicators who used motorized tank sprayers had higher total DEP levels than the applicators who used motorized knapsack sprayers. It is therefore likely that the type of spraying equipment was the primary factor influencing the risks of pesticide exposure. Hand and motorized knapsack sprayers had a maximum volume of 20 liters, and took about 1–2 h to empty whilst spraying pesticides on their field. On the other hand, motorized tank sprayers had a maximum capacity of 100 liters and took more than 2 h to spray the total contents onto the field. Therefore, the applicators who used motorized tank sprayers were more likely to be exposed to pesticides for longer periods of time than those who used other sprayers.

Improper use of PPE was also an important factor influencing pesticide exposure. Our findings revealed that the most regularly used PPE were long-sleeve shirt, and long-sleeve trousers, and boots, while goggles, gloves, and hat were worn the least frequently. The findings are consistent with a systematic review by Sapbamrer and Thammachai (27), which found that the most PPE worn among pesticide handlers in all world regions was long-sleeve shirt (66.1%), and long-sleeve trousers (71.1%), and the least common PPE worn were an apron (8.6%), goggles (24.3%), and gloves (40.5%). Theoretically, agricultural workers should wear all items of PPE to protect themselves from pesticide exposure via skin and inhalation while applying pesticides. However, with regard to protective clothing, agricultural workers should wear long-sleeve shirts and long-sleeve trousers made of waterproof cloth, according to the Department of Disease Control, Ministry of Public Health, Thailand (28). Although most Thai farmers always wore a long-sleeve shirt and long-sleeve trousers when applying pesticides, these PPE were usually made of woven fabrics. The main reasons for this are comfort and affordable price (15). According to Sapbamrer et al. (29), the efficiency of chlorpyrifos protection for woven clothing (76.5%) was much lower than shown by a Tychem^® coverall (90.7%). Also, our findings stated that goggles, gloves, and a hat were the least frequently used while applying pesticides. This could be because of a lack of practicality when working on their field. As a result, some chemicals from the pesticides can penetrate through the skin (19). A study by Aprea et al. (30) which investigated skin and respiratory exposure during spraying of lufenuron and re-entry in ornamental plant greenhouses suggested that the dose absorbed ranged from 0.144–0.171 and 0.005–0.124 μg/kg body weight during spraying and stapling, respectively, and the respiratory dose ranged from 68.7–74.6 and 0.022–0.636% of the total real dose during spraying and stapling, respectively. They also suggested that appropriate use of PPE and equipment was the fundamental aspect in reducing pesticide exposure.

Our findings also showed that increased urinary total DEP levels were associated with not wearing gloves while mixing pesticides and not wearing a mask while spraying pesticides, after adjusting for covariates. These results support the findings of Aprea et al. (5), who found that the highest levels of urinary OP metabolites in agricultural workers were linked to a lack of respiratory and hand protection. Measurement of urinary OP metabolites is used as a biomarker for monitoring of exposure to OP (31). As a result, the increased urinary OP metabolite levels may refer to the lack of PPE use during pesticide application. Agricultural workers had a risk of exposure to pesticides throughout any step of the pesticide application process, including mixing pesticides, spraying pesticides, and cleaning of sprayer equipment. Dermal contact and inhalation were the main routes of exposure (4). Agricultural workers can be exposed to pesticides by spills and splashes of concentrated and diluted pesticides in the mixing of the pesticides, therefore, not wearing gloves during this operation may increase the risk of being exposed to pesticide drips and splashes from both concentrated and diluted (4, 32, 33).

With regard to the spraying of pesticides, agricultural workers can be exposed to pesticides by direct contact with the spray. When pesticides are sprayed through nozzles, they produce very fine particles, therefore inhalation and skin contact are two main routes of exposure during this operation. Appropriate PPE, safety spraying equipment, and disposal methods for pesticide containers are important to reduce risks from pesticide exposure (34, 35). The recommendations by the Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO) (36) state that pesticide handlers should wear at least a respirator when handling pesticides. However, agricultural workers rarely comply with this recommendation. According to a systematic review by Sapbamrer and Thammachia (27), only 43.2% and 13.9% of pesticide handlers in all regions of the world wore masks and respirators, respectively, when working with pesticides. The main reasons given for non-compliance with the recommendations were thermal and mechanical discomfort, financial problems, and lack of availability (37). In Thailand, it was found that agricultural workers usually wore several types of masks made of woven fabrics, including cotton mask, robber mask, sun hat, and bandana. A study by Sapbamrer et al. (38) suggested that the insecticide filtration efficiency of respiratory protective equipment commonly worn in Thai farmers ranged from 64.9 to 95.4%, whereas half facepiece respirators were the most efficient in filtering insecticides, with a range of 96.5–98.9%. In the Sapbamrer study, the most common mask worn during spraying pesticides was a cloth mask (84.4%), followed by a surgical mask (13%), and an N95 (2.6%), respectively. As cloth and surgical masks have large pore sizes, they can only protect against large droplets, not against the fine mist used in the spraying of most pesticides. The surgical mask is designed for healthcare workers and infected people and some fine particles of OP can penetrate through the pores of these masks (36, 39). Subsequently, OP particles enter the respiratory system, accumulate in the body, and be excreted in urine in the form of OP metabolites.

Considering the effects of exposure to OP on cognitive performance, available previous studies report on evidence that exposure to pesticides and OP were associated with cognitive decline. Exposure to high levels of OP was significantly associated with the reduced scores of Mini-Mental State Exam (MMSE) (40–42). Exposure to high pesticide levels was also associated with the reduced MMSE and MoCA scores (13, 43, 44). In this study, the scores of MoCA-T, TMSE, and Mini-Cog test in post-pesticide application season were significantly lower than those pre-pesticide application season. However, no association was found between urinary OP metabolite levels and cognitive performance. It is possible that the cognitive decline found in this study might be due to other covariate factors. Another possibility is that exposure to OP at low levels and for short periods of exposure may not affect cognitive performance significantly. Table 6 presents average of urinary OP metabolite levels in other countries (45–51). Japanese farmers had the highest DAP levels (440.8 μg/g creatinine in summer), followed by floriculturists in Mexico (208.5 μg/g creatinine), and women farmworkers in South Africa (141.42 μg/g creatinine). However, pesticide applicators in Thailand (in the present study) had the lowest DAP levels (8.67 μg/g creatinine in summer). Therefore, long-term exposure and exposure to high levels of OP should be investigated further.

The longitudinal follow-up design is a strength of this study as this study design can control invariant (person-specific) confounding factors. However, there are some limitations to this study. First, the small sample size would reduce statistical power and decrease the flexibility of the effect size. To overcome this in future studies a higher sample size would be used and also across different areas to increase the transferability of the findings. Secondly, other pesticides were also used in potato fields although OP were the most common insecticides employed in general use. We only measured urinary OP metabolites as a biomarker for exposure to OP, while other pesticides were assessed by a questionnaire. Measurement of other pesticides in urine or other biological fluids is therefore warranted in future studies.

Our findings indicate that inappropriate PPE use during pesticide application was the major factor affecting urinary OP metabolite levels among pesticide applicators. Wearing gloves when mixing pesticides and wearing a mask when spraying pesticides could occupational exposure to pesticides. However, exposure to OP at low levels and for short periods of exposure may not be significant in causing neurological health problems. Long-term exposure and high levels of OP exposure should be investigated further. Continuous education and training regarding proper PPE use are vital in changing the behavior of agricultural workers toward PPE use.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Research Ethics Committee, Faculty of Medicine, Chiang Mai University (no. 219/2021). The patients/participants provided their written informed consent to participate in this study.

Conceptualization, methodology, and investigation: RS and JC. Validation, formal analysis, and resources: RS, JC, and SH. Data curation, writing original draft preparation, writing review and editing, supervision, project administration, and funding acquisition: RS. All authors have read and agreed to the published version of the manuscript.

This study was supported by the Faculty of Medicine, Chiang Mai University (Grant Number 003/2565).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.