Pesticide Spraying and Reduced Cholinesterase Activity among Hill Tribe Farmers in Thailand

Background. Farming is an important occupation in Thai hill tribe communities, which are often remote, and lack other economic opportunities along with basic educational, health care, and occupational health and safety services. Additionally, these communities have a unique culture and language. Objectives. The present study was conducted in northern Thailand to evaluate pesticide exposures and associated health impacts among hill tribe farmers, and to compare them to Thai farmers. Methods. Lahu hill tribe farmers in a mountain community were recruited by public health hospital staff, along with a reference group of lowland Thai farmers. Participants completed a survey on demographic factors and work practices, and blood and urine samples were collected by a trained nurse. Acetylcholinesterase activity (AChE) was quantified to assess pesticide exposure, whereas liver and kidney functions were evaluated using clinical biomarkers. Results. A large fraction (nearly 50%) of Lahu farmers were illiterate and could not speak Thai. Thai farmers worked fewer hours per week (39.4) than did Lahu farmers. Among Lahu farmers, AChE levels were significantly lower (worse) than those of Thai farmers. However, other health outcomes in these populations were similar. Formal education and language skills were not associated with pesticide exposures or health outcomes. Pesticide spraying was found to be a significant predictor of reduced AChE (OR=8.5, 95% CI 1.1–69.6). Conclusions. Pesticide exposures are a significant occupational health hazard among Thai hill tribe farmers. Training, potentially delivered by community health volunteers, is needed to communicate safe pesticide work practices to these farmers. Participant Consent. Obtained Ethics Approval. The study protocol was approved by the Institutional Review Board of Mae Fah Luang University (REH-61080). Competing Interests. The authors declare no competing financial interests.


Introduction
Farming is a major occupation and source of family income in Thailand. The northern region of Thailand has excellent conditions for growing high quality crops and has the highest rate of corn cultivation among all regions of Thailand. A total of 793 333 hectares are harvested for corn in upland areas of the region for corn cultivation between May and October. 1 Pesticides are often used to enhance agricultural production in Thailand; 110 000 tons of pesticides were imported into the country in 2007, which increased to 172 000 tons in 2013. 2 Most Thai farmers are self-employed or work informally, and pesticide use among informal workers is not regularly monitored. 3 Although the Thai government has provided guidance to raise awareness of workplace health and safety among self-employed and informal workers, no administrative structure to provide occupational safety and health services to these workers has been established. 4 Given these circumstances, pesticide use is an ongoing potential occupational risk among farmers.

Acetylcholinesterase activity (AChE)
is a marker to indicate exposures to organophosphates (OP) and carbamates (CA), which are the most commonly used families of pesticides. 5 Lower AChE levels have been associated with OP and CA pesticide exposures. 5 Measurement of AChE activity can indicate OP and CA exposure before permanent health outcomes manifest, 6 and Research can be used to produce estimates of chronic exposure. This biomarker has been used previously in a number of populations, including Thai farmers, a group in which associations were observed between AChE levels and health outcomes. 7 An important advantage of this biomarker is that it can be processed quickly with basic laboratory equipment, which makes field-based pesticide exposure screening more feasible.
Chiang Rai is a province in northern Thailand located close to the border of Myanmar and Laos. Migrants from these two bordering countries, known as hill tribes, settled in Chiang Rai a century ago. 8 There are six main hill tribe groups in Chiang Rai: Akha, Lahu, Hmong, Yao, Lisu, and Karen. The Lahu tribe is the second most populous population among hill tribe minorities of Thailand. 9 The Lahu divide themselves into subgroups, such as the Lahu Na (Black Lahu), Lahu Nyi (Red Lahu), Lahu Hpu (White Lahu), and Lahu Shi (Yellow Lahu); these subgroup names refer to the colors of each subgroup's traditional clothing. 10 Lahu hill tribes have a unique and distinct culture, and their main occupation is farming (primarily corn, rice, and flowers) in mountainous areas. 9 This activity provides financial support and can be done locally, without the need to travel outside hill tribe villages. However, these villages are commonly located in remote areas, with limited facilities for education, electricity, clean water and health care services. Pesticides are commonly used by Lahu farmers to increase crop yields. The companies selling these pesticides provide manuals and/or guideline labels, but this information is not used by most farmers due to literacy barriers. Limited literacy and lack of formal education may increase pesticide exposure risks, 11 and awareness of occupational hazards appears to be low in this population.
Given that pesticide exposure may represent a significant health hazard among informal workers and minority groups with limited access to education and basic services in Thailand, the present study was conducted to assess health status and the relationship between pesticide exposures and agriculture practices among Lahu farmers in Chiang Rai, and to compare their results to those from a group of Thai farmers.

Methods
The study protocol was approved by the Institutional Review Board of Mae Fah Luang University (REH-61080). All study participants were 21 years and older and resided in Chiang Rai Province. Staff from the Mae Chedi Mai Health Promoting Hospital helped recruit Lahu farmers in the survey area. The Lahu study community was located nearly 100 km from Chiang Rai in a mountainous area (770 m about sea level) (Figure 1). Family information on record at the Mae Chedi Mai Health Promoting Hospital from 2018 was used to identify Lahu farmers to approach for participation; 146 Lahu farmers were identified in this way. One hundred Lahu farmers agree to participate, and Mae Chedi Mai Health Promoting hospital staff then scheduled data and sample collection. A Lahu language translator from the Lahu community was used to interview participants who were not able to speak Thai. A reference group of Thai farmers was recruited by Tha Khao Plueak Health Promoting Hospital staff using the same methods; 86 Thai farmers were identified in this way and approached to participate. The Thai farming community was located more than 130 km away from the Lahu study community in a flat lowland area (mean elevation 388 m above sea level).

Sample collection
Farmers in the two communities were provided with an overview of the study, and the 100 interested individuals provided informed consent and were enrolled into the study. Individuals unable to provide written consent indicated consent with a thumbprint. All subjects participated in work observations and provided urine and blood samples. To increase community engagement, a community leader and community health volunteer were invited to join the research team during data collection, following consent of the participants.
We assessed pesticide exposures and potential adverse health effects among participating farmers using a combination of survey data, biomarker collection and analysis, and environmental sampling. The survey used was based on a previous study of worker health in a marginalized community (Supplemental Material). 12 The survey was administered at the local community center in the two communities and took 35-45 minutes to complete. Survey Research questions addressed demographic factors, farm practice, pesticide use, self-reported health and presence of several pesticide-related neurological symptoms, including numbness, tingling, and trembling.
Trained nurses collected 10 mL of urine and 10 mL of venous blood from workers. Five ml of blood was collected in BD Vacutainer® Plus venous blood collection serum tubes, and another 5 ml was collected in BD Vacutainer® EDTA tubes. Serum tubes were left at room temperature for 2 hours to allow blood clotting and then centrifuged at 4 500 rpm for 15 minutes. Serum was separated into cryo-vials. All blood and urine samples were stored at -80°C for further analysis.

Blood and urine analysis
Urinary creatinine, urinary calcium, serum creatinine, serum calcium, aspartate aminotransferase (AST), and complete blood counts were measured by Meng Rai Laboratory in Chiang Rai, which is accredited by the Medical Technology Council of Thailand. Analyses were completed with an XN-550 Hematology Analyzer (Sysmex Corporation, Kobe, Japan) and COBAS INTEGRA® 400 Plus Analyzer (Roche, Basil, Switzerland). Levels of AChE were analyzed from whole blood using the Ellman method. 13 Levels <8 200 units/liter (U/L) were

-Survey area for Lahu farmers in Ban Huai Muang, Wiang Pa Pao District and Thai farmers in Ban Phan Suek, Mae Chan District
Research classified as reduced AChE activity and presumed high pesticide exposure. Levels >8 200 U/L were classified as normal AChE activity. 14

Blood chemistry test
A battery of blood parameters was used to assess health impairment. Anemia was defined in men as hemoglobin (Hb) <13 mg% and in women as Hb <12 mg%. Liver injury was defined as AST >40 U/L. Glomerular filtration rate (GFR) was estimated from serum creatinine levels using the Modification of Diet in Renal Disease (MDRD) equation: GFR <90 ml/min/1.73 m 2 was defined as kidney impairment. 15 Fractional excretion of calcium (FECa), the percentage of calcium clearance as a fraction of creatinine clearance, was calculated according to Steward 2012, 16 and FECa >2% was interpreted as abnormally high excretion of Ca via urine. 17

Personal protective equipment use
Self-reported use of personal protective equipment (PPE) was used to group farmers into PPE categories. This method was applied from a 2020 study by Mueangkhiao et al. 18 The PPE-0 category was assigned to farmers who reported no use of PPE (i.e., reported PPE protection percentage = 0). The PPE-1 category was assigned to farmers who reported using face shields, fabric/leather gloves, or other protective clothing (protection percentage = 20). The PPE-2 category was assigned to farmers who reported using cartridge respirators, gas masks, or disposable outer clothing (protection percentage = 30), PPE-3 was assigned to farmers who reported using chemical resistant rubber gloves (protection percentage = 40), and PPE-4 was assigned to farmers who fell into both the PPE-1 and PPE-3 categories (protection percentage = 60).

Discussion
In 2012, there were 216 hill-tribe villages with a population of 48 835 in Chiang Rai province. 9 This population has limited access to education and other services due to their mountainous location and cultural differences, and these conditions result in increased health risks, including risk of exposure to occupational hazards. Most hill tribe farmers are informal workers who do not have the infrastructure and resources necessary to create healthy workplace environments.
Exposures to pesticides are a major occupational hazard for farmers, and can occur during storing, mixing and spraying activities. 19 Health outcomes associated with acute pesticide exposure include, dizziness, cramps, nausea, vomiting, abdominal pain, numbness, fatigue, headache, excessive salivation, respiratory problems and blurred vision. 20 Chronic health outcomes resulting from pesticide exposure include kidney and liver dysfunction and increased cancer risk. 18 In Thailand, the rate of pesticide-related morbidity increased from 2.5 to 9.0 per 100 000 population from 2010 to 2015, with 70.8% of cases occurring in individuals between the ages of 15 and 59. 21 This situation suggests that safe practical guidelines are needed to reduce pesticide exposures among farmers in Thailand.

Research
The present study evaluated pesticiderelated activities and exposures as well as health outcomes in Lahu farmers compared to Thai farmers. While poverty and poor health are often more common in marginalized communities, roughly 10% of participating Thai and Lahu farmers had expenses that exceeded their income, and the majority of farmers in both groups reported good health. Despite these similarities, differences in blood chemistry were found between the two groups. In Thai farmers, AChE levels were significantly higher than Lahu farmers, reflecting differences in pesticide exposure levels. Two measures of kidney function, FECa and GFR, were significantly different between the Thai and Lahu farmers. Age may be one explanation for this finding, given the significantly higher mean age of the Thai farmers, therefore logistic regression models were used to adjust for age in the present study. We did not find differences in the prevalence of renal and liver dysfunction, evaluated as presence increased AST, increased FECa, or decreased GFR among participants with reduced AChE compared to those with normal AChE. This indicates that pesticide exposures did not appear to contribute to more health impairments. A similar result was reported in a study by Aroonvilairat et al., 7 in which the prevalence of health impairment did not differ significantly between pesticide-exposed and non-exposed farmers. On the other hand, reduced AChE prevalence was significantly higher (adjusted OR of 8.55, Table 4) in pesticide spraying farmers compare to non-spraying farmers, showing that AChE reflected pesticide exposure more than health impairment.
Farmers can be exposed to pesticides via inhalation, skin absorption, and ingestion resulting from three basic activities: mixing and loading, spraying, and storing. In the present study, only pesticide spraying was significantly related to reduced AChE. The prevalence of AChE <8 200 U/L (indicating reduced AChE) was six times higher (25.6%) for farmers who sprayed pesticides compared to those who did not (4.0%). There was an 8.5 (95% CI 1.05-69.6) OR for reduced AChE among farmers who sprayed pesticides, adjusting for age, ethnicity, PPE category, working hours per week, and education status. Spraying may result in a longer exposure duration compared to other pesticide-related activities, which may result in greater doses of pesticides. 22 Other studies have reported a substantial body burden of pesticides among pesticide sprayers. 23 Pesticide spraying increases pesticide exposure via inhalation and dermal absorption. Dermal absorption may occur as a result of splashes, spills, or spray drift, whereas small droplets can be inhaled. 23 Kokkinaki et al. 24 showed that pesticide metabolite levels in urine samples from pesticide sprayers were higher than in a control group. This indicates a higher pesticide exposure risk among sprayers. Contributing factors for pesticide exposure from spraying include spraying duration and frequency, smoking, wind direction, temperature, pesticide type, 25 spray nozzle type, 26 spraying equipment cleaning, and PPE use. 27 Roughly half of Thai and Lahu farmers in this study were assessed to have minimal use of PPE (i.e., categories PPE 0 or PPE 1). Personal protective equipment use in this population, and particularly among farmers in the remote Lahu community, may be limited due to factors such as discomfort, weight, and restriction of movement, 28 in addition to potentially reduced access to PPE due to lack of Nambunmee et al

Figure 3 -Thai farmer using personal protective equipment during pesticide spraying
Research availability or inability to afford, which have previously been documented as barriers to use by Satya et al. 29 Figure 3 shows common pesticide spraying behavior among both Thai and Lahu farmers, which include lack of chemical resistant gloves and use of a surgical mask with a cloth cover as PPE. This type of face covering is not recommended for handling pesticides, 30 and is not proper respiratory protection for pesticide application. Additionally, rather than relying on PPE for protection, the hierarchy of controls dictates that engineering and administrative controls should be implemented to reduce pesticide exposure. Such controls might include substitution with less toxic pesticides, improved designs for the control of spray direction and timing, and worker training. AChE represents a suitable biomarker to screen pesticide exposure, perhaps through the use of mobile clinics during the agricultural season. The biomarker only indicates exposure to organophosphate and carbamate insecticides, but can nevertheless provide very useful information to farmers, particularly given the prevalence of these pesticides in Thailand. The benefits of AChE include a short processing time and cost effective method of assessing pesticide exposure. Widespread use of this biomarker in Thai and hill tribe farming communities could identify cases of early, mild pesticide intoxication and guide both prevention and treatment of more severe intoxication.

Limitations
Language barriers were a major limitation of the present study; interviews took longer to conduct among Lahu farmers compared to Thai farmers. This could introduce differences in response accuracy between the two groups for questions regarding PPE use, overall health,

Research
perceptions of harm and neurological symptoms. We were not able to compare AChE levels before and after the spraying season; that approach would have allowed us to assess the impact of spraying on pesticide accumulation in farmers. Additionally, more details on spraying activities would have been useful, including frequency and duration of spraying, spraying nozzle type, spray timing, and spraying tool maintenance and cleaning methods.

Conclusions
The Lahu farmers in the present study were informal workers with limited access to public and occupational health services and experienced pesticide exposures. This study highlights the utility of mixed-method evaluation of pesticide-related farming activities, exposures, and health impacts, and the results can help guide practical recommendations for pesticide exposure reduction. Our results suggest that training is needed to support safe pesticide practices. Such training could be implemented at the beginning of the planting season through community health volunteers, who can effectively communicate this information to Lahu farmers in their native language. Any training should be complemented by health services to monitor pesticide exposures and promote healthy lifestyles. The research should be expanded to include other countries in Southeast Asia that have hill tribe communities to evaluate the degree to which pesticide exposure risks differ by country, and to assess whether similar pesticide exposure reduction strategies might be viable across these countries. Nevertheless, AChE is an acceptable biomarker that is inexpensive and readily available. Future research should evaluate pesticide exposures in a more controlled manner, with measurement immediately before and after spraying activities, which would allow for greater precision in assessing the association between exposures and health outcomes.