Potential relationships between exposure to arsenic ( As ) in the environment and 1 endemic disease in southwestern China 2

Donglin Li, Hucai Zhang, Fengqin Chang, Lizeng Duan and Yang Zhang 3 1. Institute for International Rivers and Eco-security, Yunnan University, Kunming, 650504, China 4 2. Institute for Ecological Research and Pollution Control of Plateau Lakes, School of Ecology and 5 Environmental Science, Yunnan University, Kunming 650504, Yunnan, China 6 *Corresponding author: Hucai Zhang 7 Donglin Li , E-mail: donglinli@mail.ynu.edu.cn 8 Hucai Zhang, E-mail: zhanghc@ynu.edu.cn 9 Fengqin Chang, E-mail: changfq@ynu.edu.cn 10 Lizeng Duan, E-mail: duanlizeng2019@ynu.edu.cn 11 Yang Zhang, E-mail: 414064473@qq.com 12 Declarations 13


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Approximately 14 million people in the world are exposed to high-As content living environments, and 68 Arsenic has received widespread attention due to its extreme toxicity and widespread pollution (Ali et

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To probe the possible relationships between As exposure and this endemic disease, the As 85 contents of soil, water and plants within and surrounding Daping village were sampled and analyzed 86 5 comprehensively; based on the analyzed data, the possible sources, migration and enrichment processes 87 of As are discussed in detail. We also assessed the risk of six exposure routes, and the potential 88 relationships between As in the environment and this disease were investigated. This study may  According to a survey, there are 234 people in the village, including at least six patients with 105 unusual diseases (blisters, wounds that are difficult to heal, and ulceration). Patients with severe 106 symptoms were ultimately forced to undergo amputation multiple times to relieve pain, and they all 107 showed symptoms in childhood (Tab. S1). No similar cases were found in adjacent areas.   Mountain streams, irrigation water and villagers' drinking water were collected in plastic bottles; 120 two of each sample (Fig. 1). Plastic bottles were cleaned with 2% nitric acid before sample collection.

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After the tap had run for one minute, drinking water samples were collected. Then, the correct label 122 was placed on the bottle, and the bottle was plugged, placed in a box, and delivered to the laboratory.

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All water samples were collected and stored in sterile polythene cans and filtered through a mixed 124 cellulose ester micro porous filtration membrane with a pore size of 0.45 μm (Chen et al. 1994). To 125 determine the total As (total), 1.25 mL of concentrated hydrochloric acid and 5 mL of thiourea-ascorbic 126 acid mixed solution were added, and the mixture was prereduced at room temperature for 60 min for 127 the analysis of As (total). To determine As (III), 15 mL samples were placed in a 25 mL brown 128 volumetric flask. A total of 5 mL of citric acid aqueous solution (0.5 m/L) was added, the volume was 129 fixed with pure water, and the mixture was shaken well. As (III) and As (total) in the samples were 130 measured by atomic fluorescence spectrophotometry (Jitian AFS-8220).
Where AADSing, AADSde and AADSinh are ingestion, dermal absorption and inhalation intake from 145 soil, respectively; AADWing and AADWde are ingestion and dermal absorption intake from water, 146 respectively; and AADPing is ingestion from plants, with all units in mg As kg −1 BW day −1 . C is the total 147 As concentration in soil, water and plants (mg kg -1 , mg L -1 , and mg kg -1 , respectively), and SIngR, respectively. EF is exposure frequency (day year -1 ); ED is exposure duration (year); BW is body weight 150 (kg); AT is average time (day); ET is exposure frequency (hour day -1 ); SA is surface area (cm 2 ), SAF is 151 the skin adherence factor (mg cm -2 ); ABSS is the dermal absorption factor of soil (unitless); ABSW is the 152 dermal absorption factor of water (unitless); SInhR is the inhalation rate (m 3 day -1 ); PEF is the particle 153 emission factor (m 3 kg -1 ); and FI is the fraction ingested from consumed foodstuffs (unitless). The Where RfDSing is the oral reference dose (ingestion from soil); RfDSde is the reference dose through 168 dermal absorption (dermal absorption from soil); RfDSinh is the dose through inhalation of airborne 169 particles (inhalation absorption from soil); RfCSinh is the inhalation reference concentration given for As;

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RfDWing is the oral reference dose (ingestion from water); RfDWde is the reference dose through dermal    S4, Fig. 2a). The proportion is 74.19% of As contents of soil higher than the Chinese 211 threshold (30 mg/kg). The average content of As (71.03±54.2 mg/kg, ww, n=31) in all soil samples is 212 higher than the average values for the world (6 mg/kg) and China (11.2 mg/kg), with these values being 213 approximately 11.8 times and 6.3 times higher than those for the world and China (Bowen 1979;Wei 214 1990). It clearly shows that soil As pollution is severe (Fig. 2a). The bedrock outcrops around Daping village are black mud shale, biolistic limestone, carbonate 220 rock, basic intrusive rock and intrusive diorite. The village of Daping is located on black mud shale, a 221 large amount of strawberry pyrite was found in the black shale (Fig. 3a, 3d, e and f). In black shale, the 222 As contents in soil are all higher than the Chinese soil threshold (30 mg/kg), except for those of S1, S2, 223 S3, S4, S5 and S7 (Fig. 3a, b). The contents in samples of intrusive basic rocks and carbonate strata are 224 lower than 30 mg/kg, except for that in S6. It is clear that the regional lithology is the main contributor 225 to the As content in soil.  (Fig. 2b, Tab. S4). According to average concentration of As, the different water sources 238 ranked as follows: irrigation water > mountain stream water > residential drinking water. Thus, the 239 concentration of As (total) in the water is lower than the limit of 10 μg/L set by China

Arsenic content in plants 247
The contents of As in corn seeds are lower than 0.05 (mg/kg, ww.), which are lower than the 248 Chinese ecological security threshold of 0.2 mg/kg (Fig. 2c, Tab. S4). The range of As contents in rice 249 seeds is 0.03 to 0.17 (mg/kg, ww.), and the content of As in some rice seeds was slightly higher than  The carcinogenic risks owing to exposure to As in the living environment are shown in Tab. S7.

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The carcinogenic risks for children and adults exposure to pathways of ingestion of soil and plants, and 269 the dermal absorption are high (CR > 10 -4 ). The inhalation of particles in the air is safe (CR < 10 -6 ), and 270 the other exposure pathways are acceptable or tolerable risk to human health (10 -4 < CR < 10 -6 ). Plant 271 ingestion has some effects on human health, which is different to non-carcinogenic risk (Fig. 5a) (Fig. 2b). The content of As in plants show significant variance between corn and rice (Fig. 2c). The 288 contents of As in corn rice is higher than the Chinese standard for food. Where is the source of As in

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Ore mining or other enterprises that may cause soil As pollution have not been found near Daping 299 village. Chemical fertilizers and pesticides are necessary for contemporary agricultural cultivation, but 300 there is no significant difference (P<0.05) in the As content between farmland and non-farmland in the 301 village of Daping (Fig. 2a); that is, the soil without human interference is also contaminated by As. At 302 19 the same time, there have been no reports of coal with a high As content near the village of Daping, and 303 the area has a subtropical monsoon climate, is warm year round and has a low level of economic 304 development, mainly involving the use of electricity and firewood for daily cooking. These 305 comprehensive factors cause residents to essentially avoid fossil fuels, reducing the possibility of As 306 settling into the soil due to coal combustion ). Therefore, industrial activities, 307 pesticides, chemical fertilizers and air deposition may not be the main sources of As in the soil.  (Fig. 3a). Our analyses revealed a significant correlation between the As content in soil and rock type 318 (Fig. 3). The partial mismatch between As content values and rock types may be due to the influence of 319 the landscape (Fig. 3 and 1c), thus causing the contents in samples (S1 to S5 and S7) to fall below the 320 threshold (30 mg/kg) (Fig. 3b). Sample S6 has higher As content owing to soil with a high As content 321 was transported and covered the soil with a low As content (Fig. 3a and 1c). The content of As in the 322 soil of the village of Daping does not change with the types of soil usage, but it may be related to the 323 20 lithological properties of the bedrock; that is, it may be due to the weathering of pyrite rich in As in 324 black shale (Fig. 3c, d, e), which releases a large amount of As into the soil and causes the difference 325 under the action of physical transport such as gravity and water flow (Fig. 1c, 2a, 3). The subtropical 326 monsoon climate aggravates the weathering process of bedrock that leads to soil formation.

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The oxidative release of As from As-bearing pyrite in the black shale strata of Daping provides a source 338 of As, and changes from dry soil to wet soil promote the transformation of As, which then transfers into 339 the water body with precipitation, forming a complete "source, transportation and storage" process  Plants can absorb and accumulate As directly from soil and water. A previous study showed that 356 the As contents in 13 kinds of rice seeds planted in 72.2 mg/kg soil ranged from 0.10 to 0.38 mg/kg and 357 showed enrichment differences of root > stem > leaf > husk > milled rice (Chen et al. 2009). In the case 358 of the village of Daping, the As content of maize seeds is lower than the Chinese limit of 0.2 mg/kg 359 (HHCRC 2005), but the As content of some rice samples is slightly higher than the Chinese limit of 360 0.15 mg/kg (HHCRC 2005). Taking into account that the As content of cultivated soil is as high as 361 108.12 mg/kg, which is higher than that of the experiment of Chen and colleagues (72.2 mg/kg). Plants 368 Xiao et al. 2019), which is consistent with the fact that children have lower immunity (Fig. 4 and 5).

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The carcinogenic risk and non-carcinogenic risk assessment of six As exposure routes shows that soil 370 and plant ingestion are the two main risk exposure routes (HQ > 1，CR > 10 -4 ), which was consistent 371 with the results that soil and plants was contaminated by As ( Fig. 4 and 5).

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All the patients in the village of Daping developed the disease in childhood (Tab. S1), and the 373 families and patients consumed the same food and drinking water; in other words, they had the same 374 exposure pathways to As, but the prevalence rates were completely different. This may be due to 375 individual differences in immune ability caused by age and sex (Emenike et al. 2019). Previous studies 376 have shown stark differences in metal loading between members of the same household including twins, 377 brother and sister etc. (Mitchell et al. 1996). It is worth noting that children have special behavioral 378 habits (e.g., finger sucking and crawling), not observed in adults. In addition, based on the economic 379 conditions, toys were covered in dirt soil or dust contaminated by As, which were leaded to that