Exposure and risk assessment for agricultural workers during chlorothalonil and flubendiamide treatments in pepper fields

Pesticides are indispensable tools in modern agriculture for enhancing crop productivity. However, the inherent toxicity of pesticides raises significant concerns regarding human exposure, particularly among agricultural workers. This study investigated the exposure and associated risks of two commonly used pesticides in open-field pepper cultivation, namely, chlorothalonil and flubendiamide, in the Republic of Korea. We used a comprehensive approach, encompassing dermal and inhalation exposure measurements in agricultural workers during two critical scenarios: mixing/loading and application. Results revealed that during mixing/loading, dermal exposure to chlorothalonil was 3.33 mg (0.0002% of the total active ingredient [a.i.]), while flubendiamide exposure amounted to 0.173 mg (0.0001% of the a.i.). Conversely, dermal exposure increased significantly during application to 648 mg (chlorothalonil) and 93.1 mg (flubendiamide), representing 0.037% and 0.065% of the total a.i., respectively. Inhalation exposure was also evident, with chlorothalonil and flubendiamide exposure levels varying across scenarios. Notably, the risk assessment using the Risk Index (RI) indicated acceptable risk of exposure during mixing/loading but raised concerns during application, where all RIs exceeded 1, signifying potential risk. We suggest implementing additional personal protective equipment (PPE) during pesticide application, such as gowns and lower-body PPE, to mitigate these risks.


Exposure characteristics of chlorothalonil during mixing/loading and application
In this study, we employed the WBD method to assess dermal exposure and the distribution patterns throughout the entire body of agricultural workers during the mixing/loading and application phases 8 .Table 1 and Table S3 presents data on dermal and inhalation exposure and distribution specifically for chlorothalonil.During the mixing/loading process, the dermal exposure on the entire clothing surface amounted to 3.33 mg, which equates to a mere 0.0002% of the total active ingredient (a.i.) present during mixing/loading.These findings are consistent with reported ratios of dermal exposure to total a.i.during mixing/loading, ranging from 0.0003 to 0.59% in previous studies 14,16,17,27,[41][42][43][44][45] .Distribution analysis of worker exposure by body part during mixing and loading revealed that the highest exposure occurred on the hands, accounting for 50% of the total exposure.The chest and stomach followed at 11%, with the back at 10% (see Fig. 1).This pattern of exposure can be attributed to the direct contact of workers with the pesticide while tearing the pouch and pouring the wettable powder (WP) formulation into the mixing reservoir to create a suspension 10,27,46 .Similar distribution patterns have been reported in previous studies for various pesticides, ranging from 19.0 to 90.4% exposure on the hands 10,17,27,41,42 .Additionally, exposure distribution to the upper body (excluding the hands) accounted for 33% of the total exposure (refer to Table 1).This can be attributed to wind dispersal of the WP powder during mixing/loading, as previously documented 27,44 .Given that inner clothing is in direct contact with the skin, measuring the quantities of pesticide on innerwear is crucial as it represents a potential major dermal exposure route 9,16 .In this study, dermal exposure on inner clothing during mixing/loading was low, ranging from 0.003 to 0.02 mg.Determining the penetration rates for clothing and gloves aimed to identify body parts most susceptible to contamination due to frequent  www.nature.com/scientificreports/contact between outer and inner clothing 41 .Clothing penetration rates varied by body part, ranging from 0.6 to 8.9%, while the penetration rate to the hand was determined to be 3.4%.With regard to WBD, it was established that the dermal absorption of pesticides (both solid and liquid) during mixing and application is 10% 47,48 .During the mixing and loading process, inhalation exposure was measured at 0.001 mg, constituting a mere 0.0002% of the total exposure and an exceptionally low 6.9 × 10 −8 % of the total a.i.present during mixing/loading.During the application phase, the total dermal exposure on the entire clothing surface was quantified at 648 mg, accounting for 0.037% of the total a.i.present during application.This percentage aligns with the range of 0.003% to 0.066% reported in previous studies 17,27,41,44,45 .The distribution of worker exposure by body part during application indicated that the highest exposure occurred on the pelvis, constituting 32% of the total exposure, followed by the thigh at 24%, shin at 15%, and buttocks at 12% (see Fig. 1).Exposure to the hands was relatively lower at 0.2% compared to that at other body parts.This distribution pattern for chlorothalonil during application closely resembled findings reported by Choi and Kim in their research on thiophanate-methyl in pepper fields 44 .In pepper fields, characterized by high plant density and closely spaced rows, workers frequently interact with crops.This pattern is consistent with previous research on Korean cabbage, rice, and pepper, where body parts at the same height as the crops showed higher exposure distribution 8,25,41,44,49 .Dermal exposure on inner clothing during application ranged from 0.025 to 4.763 mg.The penetration rate to the hand was found to be 1.4%, whereas clothing penetration rates varied across body parts, ranging from 0.9 to 14%.Notably, the left upper arm (14%) and left forearm (12%) exhibited higher clothing penetration rates, in line with the commonly used default value of 10% 17,47,48 .In terms of inhalation exposure during application, the recorded amount was 0.001 mg (refer to Table 1).This inhalation exposure represented a mere 0.002% of the total exposure and an exceedingly low 6.9 × 10 −8 % of the total a.i.during application.These figures are consistent with findings from previous studies, where inhalation exposure ranged from 0.001 to 0.115% of the total exposure and from 7.6 × 10 −7 to 4.9 × 10 −5 % of the total a.i.during application 10,27,41,42 .

Exposure characteristics of flubendiamide during mixing/loading and application
The dermal exposure amount of flubendiamide on whole clothing during mixing/loading was 0.173 mg (refer to Table 2 and Table S4), which was 0.0001% of the total a.i. in mixing/loading.The ratios of dermal exposure to the total a.i. in mixing/loading were lower than those reported in previous studies (0.0003-0.59%) 14,16,17,28,4 1-45 .Previous studies using liquid formulations (emulsifiable and soluble concentrates) showed large variations in dermal exposure rates relative to the total a.i. of mixing and loading, ranging from 0.0007 to 0.59%.In the current study, it was 0.0001% (suspension concentrate, SC) 44 .Dermal exposure during mixing/loading before spraying is influenced by field conditions rather than crop differences and mainly affects the hands.Therefore, wearing protective gloves during mixing and loading is recommended.The distribution of worker exposure by body part during mixing/loading was the highest for hands (82%) (see Fig. 2).The distribution pattern on the hands for chlorothalonil (see Fig. 1) was similar to that of previous studies 9,10,28,41,43 .This trend can be attributed to direct contamination during mixing and loading.Therefore, we recommended that agricultural work should wear protective gloves while mixing and loading to minimize dermal exposure, particularly on the hands 28,43,44 .Dermal exposure to the inner clothing during mixing/loading was below LOQ (< 0.005 mg kg −1 ); the penetration rates to various body parts and hands were below 2.5%.The amount of inhalation exposure during mixing and loading was 0.00027 mg, representing 0.2% of the total exposure and 1.9 × 10 −7 % of the total a.i.during mixing/ loading.The inhalation exposure amount was approximately 100-fold lower than that of chlorothalonil (refer to Tables 1 and 2).This could be related to the dispersion caused by the formulation during mixing/loading.The dermal exposure on whole clothing during application was 93.1 mg (refer to Table 2), representing 0.065% of the total a.i.during application.This percentage is similar to the previously reported range of 0.003-0.066% 17,27,41,44,45.The distribution of worker exposure by body part during application showed the highest exposure on the thigh (40%), followed by the shin (26%), chest and stomach (7.6%), and hand (7.3%) (see Fig. 2).The distribution on lower body parts (thigh, shin, pelvis, and buttocks) for flubendiamide during application was 72% (refer to Table 2), which was similar to the distribution on lower body parts for chlorothalonil (83%, Table 1).This similarity may be linked to field conditions, such as crop height and the degree of contact between workers and crops.Crop height in the flubendiamide field was lower than that in the chlorothalonil field (refer to Table S5), which may explain the high exposure of the thigh to flubendiamide 25,44 .During application, dermal exposure on inner clothing ranged from 0.003 to 4.205 mg.Clothing penetration rates of the upper body ranged from 9.2 to 69%, whereas the penetration rate to the hand was 0.04%, and lower body penetration ranged from 4 to 32%.The field conditions for flubendiamide, including field area and spray volume, were more extensive than those for chlorothalonil (refer to Table S5).Therefore, the increased frequency of contact between workers and crops may result in a higher rate of flubendiamide penetration.The right arm demonstrated the highest penetration (upper arm 32%, forearm 69%) since the applicator handles the nozzle with the right arm, leading to more contact with the crop during application 20,43 .Applicators may use additional PPE such as arm sleeves and gowns to decrease arm exposure.The inhalation exposure amount during application was 0.066 mg (refer to Table 2), which was 0.1% of the total exposure and 4.7 × 10 % of the total a.i. of during application.These inhalation exposure rates were higher than those reported in previous studies (0.001-0.115% of total exposure and 7.6 × 10 −7 to 4.9 × 10 −5 % of the total a.i.during application 10,27,41,42 .

Risk assessment of chlorothalonil and flubendiamide
In this study, we calculated RIs as part of the risk assessment for chlorothalonil and flubendiamide among agricultural workers 29,41 .RIs greater than 1 indicate the presence of a potential risk.We considered exposure to inner clothing as actual dermal exposure (ADE), with the default dermal adsorption rate of 10% 48 .Similarly, inhalation exposure was also considered ADE.The AOELs for chlorothalonil and flubendiamide were established at 0.009 mg kg bw −1 day −1 and 0.006 mg kg bw −1 day −1 , respectively 47 .Based on these values, the RIs for chlorothalonil and flubendiamide were computed as follows: 4.3 × 10 −2 and 0.7 × 10 −3 for mixing/loading, and 4.6 and 5.8 for application, respectively (refer to Table 3).Agricultural workers involved in mixing/loading can generally be www.nature.com/scientificreports/considered acceptable risk, as the RIs for both pesticides were well below 1.However, it is important to emphasize the significance of wearing protective gloves to protect the hands, given the high exposure observed (see Figs. 1  and 2).Chlorothalonil and flubendiamide may pose a risk to applicators, as their respective RIs exceed 1.If a single agricultural worker is responsible for both mixing/loading and application, the RIs for chlorothalonil and flubendiamide are 4.7 and 6.0, respectively.This approach to pesticide application greatly increases a worker's risk of exposure.In a previous study, the combination of polyester/cotton coveralls and body gowns demonstrated a high level of protection (98.7%), indicating its effectiveness in safeguarding the body 28 .Therefore, to mitigate the risk associated with chlorothalonil and flubendiamide for applicators, additional PPE such as gowns or lower body protection could be employed during application to shield workers from sprayed pesticides and potential contact with crops 28,29 .In present study, we assessed agricultural workers' dermal and inhalation exposure.However, research on the internal pesticide exposure of agricultural workers during various farming activities remains limited, which warrants further investigation.

Conclusions
This study employed the WBD method to evaluate the exposure and risk of chlorothalonil and flubendiamide among agricultural workers in pepper fields in the Republic of Korea.The assessment considered two distinct scenarios: mixing/loading and application.In the mixing/loading scenario, the hands had the highest exposure to chlorothalonil and flubendiamide.Conversely, in the application scenario, agricultural workers experienced higher exposure to these pesticides in the lower part of their bodies, including the pelvis and thighs.Importantly, inhalation exposure in both scenarios remained below 1% of the total exposure for agricultural workers.RIs were calculated for dermal and inhalation exposure, and the results indicated that for both chlorothalonil and flubendiamide, the RIs during mixing/loading were below 1, signifying the acceptable risk of this phase for workers.However, the RIs for applying chlorothalonil and flubendiamide to pepper crops exceeded 1, suggesting a potential risk to workers.To mitigate this risk, we recommend protective measures.Agricultural workers should safeguard their hands by wearing gloves during mixing/loading, where direct hand exposure is prominent.Additionally, to protect workers from potential pesticide exposure and prevent skin contact with the crops, using additional lower-body PPE during application in pepper fields is advisable.This study primarily evaluated inhalation and dermal pesticide exposures in agricultural workers.To comprehensively assess pesticide exposures in this population, further studies involving biomonitoring with urine and blood samples taken before and after work are needed, which will provide a more comprehensive understanding of the health risks associated with pesticide exposure among agricultural workers.

Exposure matrices of agricultural workers
The study was approved by the Institutional Review Board (IRB) in the Republic of Korea and informed consent was obtained from all participants (i.e., agricultural workers in pepper cultivation).The field trials in North Gyeongsang Province, Republic of Korea, were permitted by an organization, the National Institute of Agricultural Sciences, certified by the Rural Development Administration of the Republic of Korea to carry out trials and collect samples.These trials were conducted in accordance with the relevant institutional, national, and international Good Laboratory Practice guidelines and legislation [48][49][50] .The dermal exposure of agricultural workers was measured using whole-body dosimetry (WBD).Dermal exposure of each body part was measured while wearing outer clothing (65/35, polyester/cotton, Uniseven, Seoul, Korea) and inner clothing (100% cotton, TRY®, Ssangbangwool, Seoul, Korea).Head and hand exposure were measured using gauze (10 × 10 cm) and nitrile gloves.To estimate the amount of pesticide, the head (face www.nature.com/scientificreports/and neck) was wiped with 0.01% Aerosol® OT-75 detergent-soaked gauze and then analyzed.The hands were thoroughly washed with a 0.01% aqueous detergent solution while wearing nitrile gloves.Subsequently, the gloves were removed, and the washing procedure was repeated.The washing solutions of gloves and hands were analyzed to estimate the amount of pesticide on the hands.Inhalation exposure was measured using a personal air pump (GilAir-3, Sensidyne, Clearwater, FL, USA) and an IOM sampler (SKC, Eighty Four, PA, USA) with a glass fiber filter (25 mm, SKC, Eighty Four, PA, USA).

Field trials and sampling
All field trials were conducted in the pepper-growing area located in North Gyeongsang Province in the Republic of Korea.Chlorothalonil 75% WP and flubendiamide 20% SC commercial products were diluted and mixed 600 and 2000 times, respectively 38 .A power sprayer was used to apply the pesticide solutions to pepper fields.The pesticides are sprayed using conventional pesticide application methods on pepper fields.The amount of active ingredient used per 1000 m 2 for chlorothalonil was 0.162-0.378kg a.i 1000 m −2 , while for flubendamide it was 0.013-0.033kg a.i 1000 m −2 (refer to Table S5).The pesticides were applied in July and September-the months when they are typically sprayed on pepper fields.Field trials were conducted for chlorothalonil and flubendiamide using two mixing/loading and application scenarios, each replicated ten times (see Fig. S1).The mixing/loading scenario involved one mixer/loader tested ten times, while the application scenario entailed ten applicators tested once.All agricultural workers wore personal protective equipment (PPE) such as outer and inner clothing, nitrile gloves, and inhalation exposure meters while mixing/loading and applying the pesticide.The air pump flow was calibrated and set to 2 L min −1 prior to testing.A mixing/loading worker mixed the pesticide suspension (1000 L) for 30 min to prepare the pesticide application.Applicators then applied the solutions to the pepper field using a hand-held sprayer with a hose and nozzle.Detailed information on the field, application, and climatic conditions is provided in the Supporting Information Table S5.After the mixing/loading and applying the pesticide, the gloves and hands were cleaned with 0.5 L of 0.01% detergent solution.The head (including face and neck) was wiped twice with gauze soaked in 4 mL of 0.01% detergent solution.All of the workers clothing (outer and inner) was taken off to avoid cross-contamination and divided into 11 pieces (see Fig. S1).After switching off the personal air pump, the fiberglass filter was removed from the IOM cassette.The sample was then stored at − 20 °C prior to sample extraction and instrumental analysis.

Analytical sample preparation and instrumental analysis
To analyze chlorothalonil residues on operator exposure matrices, inner and outer clothing were extracted with a 500 mL solution of 0.1% formic acid in acetonitrile.Gauze matrices were extracted with 50 mL, whereas glass fiber filters were treated with 10 mL of the same solution.The extract was shaken in a vertical shaker (SR-2DW, TAITEC, Koshigaya, Japan) at 300 rpm for 1 h and then filtered through a syringe filter (0.22 μm, PTFE, Whatman, Maidstone, UK).Hand wash solutions were checked for residues of chlorothalonil on gloves and hands.The hand and glove wash solutions were extracted using liquid-liquid extraction with dichloromethane, concentrated, and redissolved in acetonitrile for analysis.For the quantitative analysis of chlorothalonil, the separation was conducted using a DB-5MS UI column (30 m × 0.25 mm, 0.25 µm, Agilent Technologies, Santa Clara, CA, USA), and the analysis was performed by GC-MS/MS (SCION TQ, Bruker, Billerica, MA, USA).The detailed instrument conditions are provided in Table S6.Extraction was performed using methanol to analyze flubendiamide residues on operator exposure matrices (inner and outer clothing, gauze, and glass fiber filter).For the analysis of flubendiamide on the hand and glove, each wash solution was analyzed after it was centrifuged 300 rpm for 1 h.The extraction procedure was carried out as described above.For the quantitative analysis of flubendiamide, the separation was carried out using a Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 µm, Agilent Technologies, Santa Clara, CA, USA), and the analysis was performed by LC-MS/MS (Agilent 6420 series, Agilent Technologies, Santa Clara, CA, USA).The detailed instrument conditions are provided in Table S7.

Method validation for quantitative analysis of the pesticide
For the preparation of a 1000 mg kg −1 stock solution, the standard solution of chlorothalonil was diluted with 0.1% formic acid in acetonitrile, and the standard solution of flubendiamide was diluted with methanol.For each body part, a piece of inner and outer clothing (30 × 30 cm), hand and glove washing solution, glass fiber filter, and gauze were extracted as explained in Section "Risk assessment of chlorothalonil and flubendiamide".The extracts were then processed with the solvent standard to produce a matrix-matched standard solution in the range of 0.005-0.5 mg kg −1 for flubendiamide and 0.0025-0.25 mg kg −1 for chlorothalonil, respectively.The method validation is conducted according to previous research 10,17 .The MLOQ was calculated using the instrument limit of quantitation and injection volume, solvent volume of extract, and the standard solution 10 .To verify the instrument's repeatability, various concentrations of standards (MLOQ and 10 MLOQ) were analyzed seven times by LC-MS/MS for flubendiamide and GC-MS/MS for chlorothalonil.The linearity of the calibration curve was confirmed using the various matrix-matched standards.The recovery test was carried out by spiking three levels (MLOQ, 10MLOQ, 100MLOQ) of standard solutions to the control matrices.In-field recovery testing was conducted, with each sample treated to a 100 MLOQ standard under field conditions, exposed to the environment, and then analyzed.All analyses were carried out in triplicate.

Exposure and risk assessment of agricultural workers
The dermal exposure amount (μg) to chlorothalonil and flubendiamide was calculated by considering the residues on clothing based by body part and the amount of solvent used for their extraction.Exposure amounts for gloves and hands were calculated by considering the residue on the wash solution and the amount of solvent used for

Figure 1 .
Figure 1.Exposure distribution pattern (%) of mixing/loading (a) and application (b) chlorothalonil by body part in agricultural workers.

Figure 2 .
Figure 2. Exposure distribution pattern (%) of mixing/loading (a) and application (b) flubendiamide by body part in agricultural workers.

Table 1 .
Dermal and inhalation exposure of chlorothalonil during agricultural work in pepper field.a The 75th percentile of the amount of exposure to agricultural workers.

Table 2 .
Dermal and inhalation exposure of flubendiamide during agricultural work in pepper field.a The 75th percentile of the amount of exposure to agricultural workers.

Table 3 .
Risk assessment of agricultural workers of chlorothalonil and flubendiamide on pepper field.a AOEL (acceptable operator exposure level); b AF (skin absorption rate); c ADE (actual dermal exposure); d AIE (actual inhalation exposure).