Urinary Metal Levels in a Chilean Community 31 Years After the Dumping of Mine Tailings

the local population in this area remains exposed to metals from the mine tailings. Surveillance and remedial actions addressing the Chañaral mine tailings are needed. Patient Consent. Obtained Ethics Approval. The protocols and informed consent documents were approved by the Ethics Committee for Human Research of the School of Medicine of the University of Chile. Competing Interests. The authors declare no competing financial interests.


Introduction
Mining is an important economic activity in many countries. In Latin America, it is a growing industry on which, in part, national incomes depend, as is the case in Chile. Recently, the health risks related to mining have been evaluated due to increased awareness and concern. Environmental risks for the general population are equally important, especially considering vulnerable subgroups such as women and children. There has been increased attention on the evaluation of the physical, chemical, biological, ergonomic and psychosocial health hazards associated with mining. Until now, the focus has been on chemical risks, but heavy metal exposure is especially important due to the abundant worldwide evidence on the Mine tailings contain high concentrations of chemicals and elements that alter the environment. Therefore, they must be transported and stored in dams or reservoirs, where contaminants slowly decant at the bottom, the water is recovered or evaporated, and the material remains as a stratified deposit of fine solid material. 6 Mine tailings are an important source of heavy metal contamination in the environment. Metals such as arsenic, lead, chromium, cadmium, copper, zinc and nickel have been found in mine tailings throughout the world. Communities living around the tailings may be exposed to heavy metals through different exposure routes, such as ingestion of contaminated water and inhalation/ingestion of tailing dust. 4,7 Previous studies have found urinary levels of arsenic exceeding permissible occupational exposures in people residing in close proximity to mine tailings in Mexico, and high concentrations of heavy metals in hair samples of children residing in close proximity to mine tailings in Italy. Such heavy metals exposure may induce poor Research CDC DL ICP-MS Centers for Disease Control and Prevention

Detection limit
Inductively coupled plasma mass spectrometry respiratory health, lung cancer, and neurodevelopmental disorders, as well as various other health disorders. 8,9 In Chile, a total of 603 mine tailings were reported in 2015, and 216 of these (35.8%) were active and associated with mining enterprises in current production. The remaining contained abandoned waste (64.2%), posing a risk for people living near them. From the total number of mine tailings, 81.1% are located in northern Chile, between the Arica and Atacama region, and the largest is located along the coast of Chañaral. 6 Depending on location, mining tailings and associated superficial waters may contain several chemical elements such as silica, aluminum, copper, chromium, nickel, lead, zinc, and mercury. [10][11][12][13][14] The city of Chañaral is located in the north of Chile, 1000 km from Santiago, the capital, and has a population of approximately 13,500 inhabitants ( Figure 1). Copper mining is the city's main economic activity and the two most important mines are Potrerillos and El Salvador, located approximately 100 and 120 km east of Chañaral. The Potrerillos mine operated from 1926 to 1959 and the El Salvador mine opened in 1959 and remains in operation today. From 1938From to 1975 waste materials from these two mines were dumped into the Salado River which carried them into the Chañaral Bay on the Pacific coast. Between 1975 and 1989, waste was instead deposited 15 km north of Chañaral Bay, contaminating the coastline and the ocean. In 1983, Chañaral was classified as a serious case of marine pollution in the Pacific Ocean according to the Organization for Economic Cooperation and Development. 15 In 1989, dumping ceased due to a judicial order, and subsequently waste material has been stored in closed tanks near the mine. 16 During the time the disposal of the waste was permitted, more than 220 megatons of material was accumulated in the bay (mainly porphyry copper type minerals), forming a 10 km artificial beach, covering an area of more than 4 km 2 , with an estimated depth of 10 to 15 meters. This beach is located across the bay from the city, resulting in the coastline moving approximately one km. 16 These mine tailings are mainly composed of active chemical elements including arsenic, nickel, and copper. These elements experience changes that facilitate their migration, solubilization, and progressive transformation. 17 In 2006, Dold measured the metal contents and chemical activity in the mine tailings and identified an oxidation zone at the top of the tailings with liberation of divalent metal cations, such as Copper (2+) , Nickel (2+) , and Zinc (2+) (up to 2265 mg/L, 18.1 mg/L, and 20.3 mg/L, respectively). 17 Based on these findings, it is plausible that residents of Chañaral may be exposed to copper, nickel, and zinc suspended in dust. Furthermore, the presence of H 2 SO 4 (sulfuric acid) or HCl (hydrochloric acid) neo-formations indicate an acidic environment within the tailings, meaning that the sedimentable dusts from the tailing sands have acidic and corrosive properties. However, arsenic

Figure 1 -Chañaral and relative location of the city and mine tailings (2-3), Third Region, Chile (1).
Research of mining waste into the Bahia. The aim was to determine the prevalence of urinary metals such as copper, nickel, mercury, arsenic and lead, and establish cut-off points for future surveillance of exposures in order to improve the quality of life of the inhabitants of Chañaral and enhance their health.

Population and Sample
The total adult population in Chañaral between the ages of 18-65 years old was estimated to be 8,851 inhabitants in 2007 when this study was conducted.
We recruited a sample population of 205 individuals, considering at least 190 to achieve statistical significance and 10% for replacements.
The sample was selected in three stages. First, a list of blocks and residences was developed and random number generation was used to select blocks. Subsequently, the third house clockwise from the north corner of the selected block was chosen. In each residence, an adult that met the selection criteria was selected using a Kish grid. 31 One subject from each of the predefined residences was invited to participate in the study.
Eligibility criteria included: (1) age 18-65 years, (2) a minimum of 8 years of education, (3) absence of mental illness, and (4) residence in the city for at least 3 years. Subjects were excluded if they had a history of occupational exposure to the mining industry.
A questionnaire was used to collect information about demographics (age, sex, education level, place of residence), exposure to metals (employment history, daily activities, proximity to potential sources of exposure), and lifestyle (active and passive smoking, consumption of alcohol, consumption of fish and shellfish). Questions regarding socioeconomic variables were taken from the 2002 Chilean census. 29 Metal exposure questions were developed by the research team and later validated in 20 volunteers who met the study inclusion criteria, but who lived outside of Chañaral in a coastal region that was not exposed to mining waste. Modifications were made to the questionnaire based on respondent feedback.

Collection and Analysis of Metals in Urine
Participants provided a single sample of urine in two separated flasks. Urine samples were collected by households, and researchers were present during the sample collection and provided detailed instructions. Samples were kept at 4°C during the first four hours after the collection. Nitric acid was added to one of the flasks (flask 2) and both flasks were frozen at -20°C in Chañaral. Samples without nitric acid (flask 1) were transported to the laboratory in Santiago. Samples were analyzed with inductively coupled plasma mass spectrometry (ICP-MS) for total arsenic, copper, nickel, and lead. Mercury was measured by the laboratory of the Metropolitan Regional Health Authority with atomic absorption spectroscopy. Samples with arsenic levels higher than 50 μg/L were analyzed at the Public Health Institute to measure inorganic arsenic and its metabolites. There, samples with nitric acid (flask 2) were analyzed by hydride generation atomic absorption spectrophotometry (Perkin-Elmer).
All laboratory activities were performed in accordance with the requirements imposed by ISO 17025 (http:// www.iso.org/iso/catalogue_detail. htm?csnumber=39883). The detection limit (DL) for each metal was calculated independently using 10 urinary samples.

Statistical Analysis
Urinary metal concentrations are expressed in μg per liter of urine (μg/L). Levels below the DL had an imputed released from sulphur is immobilized and remains at the bottom of the tailings and in the sediments, where it is dissolved in the sea. 17 Although waste material is no longer deposited in Chañaral Bay, its accumulation and transformation in the environment may continue to represent a risk to human health and surrounding ecosystems. 18-28 Arid conditions in the region and lack of rain precipitation limiting the removal of salt from soil may exacerbate exposure. 16 In addition, the population in Chañaral is characterized by high social vulnerability, with the highest unemployment rates in the region (11.5% vs 3.7% in the nearby city of Caldera) and the lowest annual per capita spending on health in the region (US $42 in Chañaral vs US $146 in Diego de Almagro, 61 km north), a condition that could worsen the potential adverse health effects of exposure to mine tailings in this population. 29,30 The mine tailings in Chañaral represents a unique scenario due to the lack of mitigation projects and poor environmental control. This makes it difficult to make a comparison with other cities with or without nearby mine tailings. These characteristics, followed by the scarcity of policies addressing the problem and the remoteness of universities and high quality toxicology laboratories, constitute a scenario that complicate the study of the tailings and their potential health risks to Chañaral's population. Chañaral represents a typical case of a contaminated site that calls for an integral approach with available and well-known methodologies. Although this is not the intention of the present study, we will consider a few specific elements from those methodologies.
Our study was conducted 31 years after the prohibition of the dumping Research our study. Reference values for copper, lead and nickel were defined as 13 µg/L, 2.6 µg/L and 4.1 µg/L, respectively. [33][34][35][36][37] We defined the reference value for total arsenic using national expert opinion (50 µg/L), but for inorganic arsenic, an occupational reference from the United States was used (35 µg/L). 37 Multivariate models were used to evaluate the risk factors for high metal levels in urine. Levels higher than the reference value were defined as high metal exposure. Multivariate models were adjusted for covariates.

Results
A total of 215 residents of Chañaral were invited to participate in the study and 205 agreed to participate (95.3%). The mean age was 43.6 years (± 11.2). More than half of the participants were women (67.3%). Of the total participants, 23% had a middle school level of education (8 years or less of education), and 7% had more than 12 years of education. The mean distance between homes and mine tailings was 1560 meters (range 100-6000 meters). Among study participants, 24.4% reported contact with chemical agents at work, including 5.4% who smelted lead to make fishing weights. More than half (54.9%) reported regularly eating fish and shellfish ( Table 1).
Almost all participants (204) provided adequate urine samples (adequate volume and lack of contamination). Median metal concentrations by sex, age group, distance (meters) to tailings, residence in the north, consumption of fish and shellfish, and occupational exposure to metals were compared using the Kruskal-Wallis test.
As an indicator of metal exposure, the 95th percentile level of urinary metals for the residents of Chañaral were compared with other populations described in the literature by reviewing studies from the period 2005-2010 that measured urinary metals in adults. These studies included adults from the general population (not occupationally exposed to metals) of similar ages, as in

Table 1 -Participant Demographics (No significant differences by sex using Kruskall-Wallis test)
Research in people younger than 44 years old, living in the northern area of the city and working in small-scale fishing. In the multivariate analysis, residence in the northern area of the city was a risk factor for having a urinary copper level higher than the reference value of 13 µg/L. 35 Preparing fishing weights was identified as a risk factor for an elevated mercury level ( Table 3).
Preparing lead fishing weights was also associated with higher concentrations of nickel and lead, but the association did not reach significance. We found that people living fewer than 1000 meters from the tailings had a two times higher risk of showing a urinary nickel level above the reference value (4.1 µg/L) (odds ratio = 2.5; 95%, confidence interval = 1.08 -5.82) (data not shown).
In the multivariate analysis, fish and shellfish consumption remained a statistically significant predictor for having a urinary arsenic level >50 µg/L (odds ratio = 2.3; 95%, confidence interval = 1.05 -4.87) (data not shown).

Discussion
This is the first study to determine levels of multiple metals in urine samples of adults living in Chañaral in order to assess their current exposure.
Urinary metal concentrations indicate that Chañaral residents are exposed to metals, possibly from the mine tailings in Chañaral Bay, even though mine waste discharge ceased 31 years prior to the present study. The study confirms the findings of Dold, who reported in 2006 that mine tailings undergo various chemical transformations facilitating the migration and solubilization of metals, making them more bioavailable. 17 The chemical composition of these mine tailings is a mineral surface of eriochalcite (Copper(II) chloride·water) and halite (NaCl), strongly enriched with copper at levels Research between 1000 to 24100 mg/kg. Other metals in the mine tailings included nickel (5-370 mg/kg) and arsenic (30-281 mg/kg). These results suggest that residents living in Chañaral have been exposed to copper and nickel suspended in dust carried by wind from the mine tailings to the city. In our study, the copper and nickel levels found in study subjects were consistent with findings reported by Dold. 17 Metal levels in other matrices such as soil, food, or dust in suspension were not evaluated in this pilot study. For exploratory information only, drinking water samples were collected from the residences of volunteer participants (n=10). The highest levels found for nickel (5.6 µg/L), arsenic (7.6 µg/L), copper (10.4 µg/L), and lead (1.3 µg/L) were within the national drinking water standards. 38 Regarding food exposure, key informants indicated that locally-harvested seafood is not consumed in Chañaral due to obvious discoloration by copper. We were not able to obtain data on metals in foods more frequently consumed in the area.
We did not collect blood samples to measure lead, which is the preferred method of assessing recent lead exposure. We use urinary lead level as a qualitative indicator of chronic exposure, as has been done previously in the United States. 32 This is the first publication of general population data on urinary lead levels in Chile. Notably, the highest urinary lead level (58 µg/L) was found among people who stated that they smelted lead for small-scale fishing, an activity unrelated to the exposure to mine tailings. Moreover, when these subjects were excluded from the analyses, the population mean level of lead was comparable to other international studies. 32 Although all metals were elevated in some urine samples ( Table 2), the prevalence of copper exposure, with 67.5% of subjects exceeding the reference level, deserves further attention. These copper levels have not been previously reported in the international literature on environmentally-exposed populations, and further investigation is necessary to develop better biomarkers for monitoring at the population level. [39][40][41] In addition, this is the first study in Chile to publish data on mercury levels in a general and non-occupational population from a coastal location. The 95th percentile for mercury (6.3 µg/L)

Research
Residence in the northern area of Chañaral, which is strongly affected by air masses that bring dust from the mine tailings, is the best proxy for exposure. In fact, subjects living in proximity to the mine tailings had higher urinary copper levels than those not living nearby, suggesting that the main source of exposure in the area could be the mine tailings.
This study used ICP-MS, a polyelemental technique, to simultaneously measure various chemical elements at a low cost. This method is ideal for monitoring metal exposure in a population. 43 Additionally, this method of screening allows researchers to select complementary methodologies on a case-by-case basis, according to the context of the exposure being evaluated. On this occasion, the study focused on speciation of arsenic in subjects with high total arsenic values, significantly reducing costs.
The use of the 95th percentile as an indicator to monitor a population is strongly recommended by a number of international authors. 32,33,43 This technique allows results to be compared across exposed populations and to measure changes in exposure levels due to environmental interventions. Values reported in this study can be used in the future to assess the impact of subsequent interventions in the area by comparing post-intervention metal levels in a representative sample of the adult population.
The adverse health effects related to chronic exposures to copper, nickel and arsenic are more varied and multisystemic than the effects evaluated in this population. Preliminary findings showed the prevalence of cough, asthma, chronic obstructive respiratory syndrome and dyspnea to be higher than the national prevalence (18.4%, 27.0%, 46.6% and 37.7% vs 8.1%, 10.1%, 25.7%, 18.6%, respectively). Other findings reported differences in lipid profiles, with higher levels of cholesterol > 200 mg/L, triglycerides > 200 mg/L and high-density lipoprotein cholesterol than the national level (64.4%, 51.5% and 69.5% vs 43.3%, 35.2% and 47.3%, respectively) (data not shown). This suggests that the probability of disease and alterations in organs and systems in the population in Chañaral and others sites affected by mining wastes is high and additional studies of this issue are needed. However, the health risk assessment methodologies employed in the present study provide an opportunity to obtain more and better information from sites affected by mining.

Conclusion
The urinary levels of copper, nickel, and arsenic found in residents of the city of Chañaral confirm significant exposure to these metals 31 years after the cessation of the dumping of mine tailings in the area.
Effective interventions to stop emissions from tailings are needed to prevent further exposure to Chañaral's population. This study provides a reference point to assess the effect of future interventions in the area that effect a change in exposure profile. Questions regarding health effects associated with the exposure identified in the present study, in particular the interaction of these various metals and their health effects, require future indepth studies. Finally, we are grateful to the Chañaral community for their participation.