Neuropathy caused by pesticide exposure on farmers in Ngablak District, Magelang, Central Java, Indonesia: An electroneuromyography study

Uncontrolled and unsafe use of pesticides can lead to acute and chronic toxicity in farmers, with neuropathy being one of the most common symptoms of chronic toxicity. However, the effects of this toxicity on farmers' electroneuromyography (ENMG) are still unclear. To address this, we conducted a cross-sectional study from July to October 2017 in Ngablak District, Magelang, Central Java, Indonesia. Eligible farmers who were exposed to pesticides underwent electrophysiology examinations, as well as additional tests such as physical examination and laboratory testing. We collected general information such as age and work history by interview. In total, 64 farmers were included in this study. Out of these, 44 farmers were found to have polyneuropathy, with 41 of them having motor polyneuropathy and 19 of them having sensory polyneuropathy. Our findings showed that low blood cholinesterase was associated with distal latency prolongation (p-value: 0.014). The group exposed to organophosphate/carbamate pesticides was also significantly associated with prolonged distal latency (p-value: 0.012). However, motor polyneuropathy was significantly associated with chronic exposure to organophosphate/carbamate pesticides (p-value: 0.009) and not with low blood cholinesterase levels (p-value: 0.454). The study concludes that chronic exposure to organophosphate or carbamate pesticides could result in polyneuropathy disease, particularly in the motor system.


Introduction
Pesticides have been used widely in agricultural fields to increase crop and food production, especially in major agricultural nations such as Indonesia. Farmers are routinely using pesticides to control pests and diseases of the crops. In the last three decades, the widespread use of pesticides has rapidly increased [1]. This overuse could lead to several serious medical problems. Chronic exposure to organophosphates, one category of pesticides, has been linked to many debilitating diseases, including diabetes mellitus [2], Alzheimer's disease [3], breast cancer, and thyroid cancer [4]. In neurological systems, chronic exposure could induce neuropsychological impairment in cognitive function, memory, attention, coordination, and many more functional aspects [5]. Carbamate, another cholinesterase inhibitor pesticides, could cause psychomotor and neuropsychiatric problems in its long-term effects [6].
Pesticide poisoning could be influenced by internal and external risk factors. Internal risk factors include age, sex, nutritional status, hemoglobin status, health condition, and education. On the other hand, external aspects of pesticide poisoning include pesticide dose, environmental temperature, wind direction, concentration and potency of pesticides, duration of pesticide exposure, duration of spraying, frequency of spraying, protective equipment use, and type of pesticides [7,8].
Organophosphate Induced Delayed Neuropathy (OPIDN) is a disease caused by organophosphate pesticides poisoning. OPIDN is characterized by a progressive distal axonopathy of peripheral nerves and spinal cord, usually induced by acute high-dose intoxication [9]. A chronic low-dose exposure (such as occupational exposure) effect on peripheral nerve or causing OPIDN is relatively new, and the evidence is limited. Some articles have studied the pesticide effect on peripheral nerves using electrophysiology study. The results were mixed; Some showed prolonged distal latency, reduced motor and sensory nerve conduction velocity, and reduced motoric amplitude in the exposed group. Other studies showed low-dose organophosphate pesticides exposure is not always associated with peripheral neurophysiology impairment [10]. Based on these findings, this study was conducted to assess the electrophysiologic parameters of farmers chronically exposed to pesticides in Ngablak District, Magelang, Indonesia, and compared other risk factors that could influence those abnormalities.

Study sample
This cross-sectional analytic study was conducted from July to October 2017 in Ngablak District, Magelang, Central Java, Indonesia, and Sardjito Hospital, Yogyakarta. Seloprojo village was selected based on the principle of stratified random sampling. In total, 64 farmers were chosen based on inclusion and exclusion criteria. The inclusion criteria were: 1) Farmers who used pesticides in the last two weeks and have been using them for at least two weeks; and 2) willing to be brought to Dr. Sardjito Hospital for an electrophysiology examination. Farmers with other diseases that could induce peripheral neuropathy were excluded, such as diabetes mellitus, chronic alcohol use, amputation of extremities, and foot ulcer. All included farmers were informed about the study procedure and asked to sign the informed consent forms after they understood the process. This study was approved by the Institutional Ethics Committee of the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, with ethical clearance number KE/ FK/0340/EC/2017).

Subject characteristics, pesticide exposure, personal protective equipment, and knowledge
Subjects' characteristics were assessed from interviews (age, length of work as a farmer) and physical examination (Body Mass Index). The research team developed a questionnaire to determine the exposure to pesticides. The questionnaire covers various aspects of pesticide usage, such as time, duration, and frequency of spraying, the brand and type of pesticides used, methods for pesticide preparation, and more. The type of pesticide used by farmers was determined from the reported brand, and the active ingredients were identified through the pesticide label, Material Safety Data Sheet (MSDS), and data from the National Institute for Occupational Safety and Health Institute (NIOSH). Personal Protective Equipment (PPE) scores were assessed by asking if they used each of the 8 PPE equipment the World Health Organization recommended [11]. For every component used, subjects got 5 points, and the scoring range was 0 -40. Pesticide knowledge was determined using a questionnaire developed by the research team, as shown in the Appendices.

Blood cholinesterase levels
Blood samples were collected and stored in EDTA vacutainer tubes. The tubes were transported immediately to a private laboratory using wet ice to be analyzed. Ellman's method was used to determine cholinesterase levels [12]. Normal blood cholinesterase levels range from 9.572 to 15.031 IU/mL. A level below 9.572 is considered a Low Blood Cholinesterase Level (LBCL) [13].

Nerve conduction study (NCS)
All subjects were brought to Dr. Sardjito Hospital in Yogyakarta. This study used Nihon Kohden Neuropack X1 EMG/EP Measuring System MEB-2300K for NCS examination. All four extremities were studied. In the upper extremities, Compound Muscle Action Potential (CMAP) and Sensory Nerve Action Potential (SNAP) in median nerves and ulnar nerves were recorded; for lower extremities, CMAP was recorded from tibial nerves and SNAP was recorded from sural nerves.
The results were shown in numeric data and compared to other variables using categorical data. We divided the NCS results into two groups, normal and abnormal, based on Liveson's normal results [14]. Distal latency (DL) abnormality was diagnosed if the results were higher than the upper limit of normal values. Nerve conduction velocity (NCV) abnormality was diagnosed if the results were lower than the lower limit of the normal value. For distal amplitude (DA) and proximal amplitude (PA), the abnormality was diagnosed if the results were lower than the lower limit of the normal value. To conclude abnormal results in motor DL, the subjects must have abnormal distal latency results in all three nerves (median, ulnar, and tibial nerve). This algorithm is also used for nerve conduction velocity (NCV), distal amplitude (DA), and proximal amplitude (PA). This algorithm was used to eliminate the possibility of subclinical mononeuropathy caused by ergonomic problems in farmers' daily activity, such as carpal tunnel syndrome [15]. For sensory DL and DA, there must be abnormal results in all three nerves (median, ulnar, and sural). Motor polyneuropathy was diagnosed if the subject had abnormal results in all four motor nerve markers: DL, NCV, DA, and PA. In contrast, sensory polyneuropathy was diagnosed if the subjects had abnormal sensory DL and DA results. If the subjects had either motor or sensory abnormal results, the subject was said to have polyneuropathy. We categorize the results into demyelination or axonal degeneration to understand the possible mechanism. Demyelination was diagnosed if there was slower NCV (< 75% than the lower limit of normal scores), prolonged DL (> 130% than the upper limit of normal scores), or both. In contrast, axonal degeneration was diagnosed if CMAP or SNAP amplitude was lower than the lower limit of normal scores.

Statistical analysis
Descriptive data was used to present results, which were then categorized into Normal and Low Blood Cholinesterase Levels (LBCL) groups based on cholinesterase examination. The two pesticide exposure groups were distinguished based on the type of pesticide exposure: chronic exposure to organophosphate/carbamate pesticides or exposure to other pesticide types. Shapiro-Wilk testing was used to determine the normality of data distribution. Data with normal distribution were shown using mean ± standard deviation (SD), whereas the median (minimum-maximum) was used for abnormal distribution. Next, we used an independent t-test (parametric) or Mann-Whitney (non-parametric) to test categorical-numerical association and a chi-square test to test categorical-categorical association. Last, to exclude age, sex, body mass index (BMI), length of work, and farmers' knowledge, we used Binomial Logistic Regression to find any association with the NCS results.

Results
There were 64 subjects included in this study based on inclusion and exclusion criteria. The sample population was 81% males and 19% females. The subjects' characteristics are shown in Table 1. The mean age ± SD in this study was 47.94 ± 10.96. The median length of work was 21.5 years. In the PPE score, the median was 20, with 5 as the lowest and 35 as the highest score. This meant nobody either used all the PPE or did not use any of the PPE. The knowledge median score was 17.5, ranging between 6 and 45. The BMI mean was 22.73 with 3.37 SD, and the blood cholinesterase level mean was 8.49 with 1.50 SD. In this study, 42.2% of farmers used organophosphate or carbamate pesticides types, and 57.8% did not use these two types. Table 2 displays the mean or median of each examination. In the case of the median nerve, the median of CMAP and SNAP DL were longer than the normal range, but their NCV median, CMAP DA, and CMAP PA mean were normal. The ulnar nerve's SNAP DL was below the normal range. However, the tibial and sural nerve's mean or median were within the normal range.
From blood cholinesterase levels examination, we found 45 subjects (70.3%) in the LBCL group and only 19 subjects (29.7%) in normal cholinesterase groups. When compared with the normal cholinesterase groups, we found slower NCV of tibial nerves, longer motor DL for median nerves and tibial nerves, longer sensory DL of ulnar nerves and sural nerves, and lower motor amplitude of median nerves, ulnar nerves, and tibial nerves in LBCL groups. Still, these differences were not statistically significant (Table 3).
When the subjects were divided into the organophosphate/carbamate pesticides exposed group and other pesticides group, subjects who were exposed to organophosphate or carbamate pesticides had prolonged median and ulnar CMAP DL, and lower median and tibial CMAP amplitude (Table 4). However, these differences were not statistically significant (p > 0.05).
From the bivariable analysis (Table 5), there were no significant differences between LCBL and normal cholinesterase groups in age, sex, BMI, length of work, PPE Scores, and Knowledge Scores (p > 0.05). These same results were found for the organophosphate/carbamate pesticides and other pesticide groups (p > 0.05) ( Table 6).
We could determine the amount of abnormality in the subjects ( Table 7). There were 44 subjects with polyneuropathy, of which 41 of them had motor abnormalities and 19 of them had sensory abnormalities. Based on the indices, we found the CMAP DL was the highest, with 36 farmers had abnormality in motor DL. There were only 5 farmers with NCV abnormality, 5 farmers with SNAP DL abnormality, and 9 farmers with SNAP amplitude abnormality.
Next, we divided the NCS results into normal and abnormal results and we compared them based on their cholinesterase levels and pesticide types (Table 8). Motor DL was the only one parameter that had significant association with LBLC groups (p = 0.014) with odds ratio (OR) of 5.046. There was no significant association between the other parameters with low blood cholinesterase levels. In this analysis, we did not analyze CMAP amplitude because of zero amplitude abnormalities.
Next, we found a significant association between organophosphate or carbamate pesticides chronic use with NCS abnormality (Table 9). Motor distal latency abnormality was found higher in the exposed group   The LBCL groups exposed to organophosphate or carbamate pesticides had a higher OR to have demyelination injury, but this was not significant (p = 0.522 and p = 0.814, respectively). There were also no significant associations with axonopathy mechanism (p = 0.106 and p = 0.090, respectively).

Discussion
Blood cholinesterase levels had a normal distribution with mean of 8.49 ± 1.5. These results were below the normal range (9.572 U/L) [13]. There were 70.3% of subjects who had low cholinesterase levels, which is similar to a prior study [7] with 7.02% who had been poisoned. Organophosphate and organochlorine type of pesticides inhibit    pseudocholinesterase in plasma cholinesterase in red blood cells and in the synapse, which at a certain level cause poisoning. Carbamate also inhibits acetylcholinesterase but only causes reversible inhibition of butyrylcholinesterase [16]. Other pesticides, such as pyrethroid (which works in sodium and chloride channels) do not influence the cholinesterase levels [17]. This study did not find any significant association between blood cholinesterase and NCS results. Some studies showed similar results. In one study, there was no significant association found between decreasing blood cholinesterase before and after spraying with NCS [18]. Differing from organophosphate pesticides acute intoxication, the pathophysiology of polyneuropathy was not related to the inhibition of cholinesterase levels but was instead caused by the inhibition of neuropathy target esterase (NTE) [19]. As a result, this could explain why the severity of neuropathy was not correlated with the low cholinesterase levels.
In this study, we found a significant association between chronic exposure to organophosphate or carbamate pesticides with polyneuropathy, especially in the motor system, but not in the sensory system. Results on NCS were relatively mixed. One study did not find any difference in NCS results between baseline and after 1 year [9]. A study in Iran on chronic low-dose pesticide exposure showed a slower peroneal NCV, longer sural and radial SNAP latency, and smaller sural and radial SNAP amplitude [20]. The reason for these inconsistent results is due to the different study types, duration, and interpretation of NCS results. Farming and spraying are occupations that require immense energy expenditure each day and when a study showed abnormality in one nerve but not in the other, there was a possibility that subclinical mononeuropathy happened because of high work force [15]. Another problem caused by this was the difficulty obtaining a bigger picture and explanation of what happened if the lesion happened only in one nerve but not in another (i.e. why the NCV in peroneal is significantly slower but not in sural). Our study tried to conclude the result of each subject into a diagnosis (polyneuropathy), so we can be sure that pesticide was the cause, not muscle overuse. One prospective study [21] also tried to conclude all the results into a whole picture and that study showed there was significantly higher ratio of NCV abnormality but not in distal latency or amplitude. The study used the OR formula (an NCS index was abnormal if one or more parameters were out of normal range) while our study used the AND formula for each NCS marker (an NCS index was abnormal if all nerves in that index had abnormal results). Again, with the AND formula, we could exclude subclinical mononeuropathy lesions that are usually caused by nerve entrapment.
The neurotoxic effects of organophosphate or carbamate pesticides may be explained by their photodegradation properties. Organophosphate pesticides degrade relatively quickly in the environment [22]. However, their degradation metabolites are more toxic than the original compound. 3,5,6-trichloropyridinol (TCP) is one of the main metabolites of chlorpyrifos, one of the most commonly used organophosphate pesticides. TCP is even more toxic than chlorpyrifos itself [23]. This metabolite can permeate into the soil and water, leading to indirect exposure of farmers.
The mechanism of neuropathy by organophosphate and carbamate pesticides usually begins with axonal degeneration followed by secondary demyelination [24]. In this study, we could only find a higher OR to have demyelination injury in LBCL and organophosphate/carbamate pesticides exposed group, but this result was not significant. This finding could lead to another possible mechanism that induced demyelination without abnormal amplitude results. A prospective study [21] showed organophosphate pesticides did not produce a higher risk in SNAP and CMAP amplitude abnormality but it did produce abnormality in NCV.
This study has several limitations that should be considered when interpreting the results. Firstly, the study did not categorize the participants according to their exposure to pesticides, i.e. high dose versus low dose. This lack of categorization is important, as it is possible that a high dose of pesticide may lead to a different mechanism of toxicity, such as OPIDN, compared to a low dose. Secondly, there may have been some memory bias among the participants, as many of them had worked in farming for an average of 21.5 years. As a result, they may have only remembered the last pesticide that they used, and may have forgotten about previous exposure to organophosphate or carbamate pesticides. To mitigate this bias, the researchers identified all organophosphate and carbamate pesticides products in the area and asked the participants if they had used any of those products. If the participants answered "no", their response was categorized as "other pesticide type". Future studies All variables were adjusted with age, sex, BMI, length of work, PPE scores, and knowledge scores. CI, confidence interval; LBCL, Low Blood Cholinesterase Levels. * p-value < 0.05 was considered significant.

Table 9
The Association between organophosphate/carbamate pesticides chronic exposed group with abnormality in ENMG markers (n = 64). should consider examining metabolites of organophosphate pesticides such as diphenyl phosphate and bis(1,3-dichloro-2-propyl) phosphate [25]. Finally, environmental conditions such as temperature changes, precipitation, sunlight radiation, and air movement could affect the degradation of pesticides, which in turn could influence the toxicity mechanism of the pesticide [26]. Hence, these factors should be considered in future studies.

Conclusion
This study found that chronic exposure to organophosphate or carbamate pesticides could cause polyneuropathy, mainly in the motor system. However, this neuropathy was not associated with low blood cholinesterase levels.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
The data that has been used is confidential.