Settings and population
Cerro de Pasco is the capital of the province of Pasco, located in the Central Zone of Perú and has a population size of around 80,000 inhabitants. An open pit mine is located in the same capital, and the mining activities generates air, water and soil contamination21,23.
The study population consisted of children between 3 and 16 years of age who had a permanent residence in the study area: Paragsha zone in Cerro de Pasco and Carhuamayo city. The open-air mine hole is situated at approximately 400 m from Paragsha zone, i.e., Paragsha is directly exposed to the activities carried out in the mine. Carhuamayo is located at 35 km from Cerro de Pasco in a southeast direction but shares similar socio-geographic and atmospheric characteristics to those of Paragsha.
Ethics
The participation in the study was voluntary. Parents of participating children provided a signed informed consent before their recruitment. The study was approved by the ethics committee of the University of Santiago de Compostela (Protocol No. 08102019) and was carried out in compliance with the corresponding legal and ethical requirements (Spanish Acts 14/2007 and 3/2018).
Sample
In 2018, hair samples were collected from 94 children. Hair was the type of chosen samples to measure heavy metal concentration because it allows determining if the children have been chronically exposed to these metals, and at the same time it is also a non-invasive and stable method30. Due to the lack of standards that specify the ‘normal’ and the ‘toxic’ levels of heavy metals in hair, and in line with previous studies, we adopted the reference values for children that were established by the Micro Trace Minerals (ER) laboratory (Table 1)23.
Table 1 Average concentrations of heavy metals measured in hair roots and hair tip samples collected in 2018 from children exposed and unexposed to the mine. Full size table
Seventy-eight children were recruited from Paragsha; the region exposed to heavy metals. Twenty-seven of the 78 exposed children were included in a previous study that was undertaken in 201623, hence data from two periods are available for those children. Sixteen other children were recruited from Carhuamayo and served as the group unexposed to heavy metals.
Anamnesis and examination
All recruited children underwent a complete physical examination and anamnesis. In the anamnesis, we inquired about respiratory, digestive, bleeding and neurological antecedents, among others. In addition, data were collected on vaccine administration, medical and hospitalization history and the reasons for medical assistance or interventions, when applicable.
In the clinical examination, we assessed many ophthalmoscopic and otoscopic aspects such as: children’s development and general condition, as well as abdominal, respiratory, dermatological, ophthalmological, cardiovascular, digestive, auditory, urinary and neurological complaints. Regarding the external examination, information was collected on the presence/absence of skin with an earthy color, breath and acid sweat, bleeding, fever at the time of the evaluation, among others. The presence of specific features potentially related to exposure to heavy metals was investigated, such as: Café-au-lait spots (coffee-with-milk-colored spots) in various parts of the children’s body, deafness, inflamed conjunctiva, bluish lines on the edge of the gums, constipation, diarrhea, cramps and white lines on the nails or Mees lines.
At the end of the examination, hair samples were collected from the nape of the neck.
Laboratory analysis
All the collected hair samples were processed, using 1 cm fragments, and analyzed, in a certified laboratory, by Inductively Coupled Plasma Mass Spectrometry, ICP-MS, following previously published procedures31. The concentration of 21 heavy metals was determined: aluminium (Al), antimony (Sb), arsenic (As), boron (B), barium (Ba), beryllium (Be), cadmium (Cd), cobalt (Co), chromium (Cr), iron (Fe), magnesium (Mn), mercury (Hg), molybdenum (Mb), nickel (Ni), lead (Pb), copper (Cu), selenium (Se), tin (Sn), thallium (Tl), vanadium (V) and zinc (Zn).
The analytical procedure consisted of the following steps: (1) cutting the hair samples into parts of about 1 cm, (2) immersion of the hair samples in a mixture of acetone/ethyl ether (1 + 4 v/v) for 30 min, (3) drying in a stove at a maximum of 40 °C, (4) second immersion in 5% ethylenediaminetetraacetic acid (EDTA) solution for 30 min, (5) rinsing with de-ionized water, (6) drying in a stove at a maximum of 40 °C, (7) after the washing phase, each sample was weighed (about 0.1000—0.4000 g) and subsequently mineralized in a microwave device in high-pressure Teflon vessels, with a mixture of nitric acid and hydrogen peroxide in proportion 5 + 1 (v/v). The thermal program consisted of two steps. In the first step, the samples were incubated at 90 °C for 3 min. In the second step, the samples were placed at 120 °C for 5 min. Subsequently, the samples were cooled down for 30 min. A blank control and a reference material were also mineralized with each sequence of samples. The solution was transferred into a volumetric flask and analyzed by ICP-MS technique.
Results were sub-grouped in order to compare the part of the hair that has been exposed to metals the longest (hair tip, at a 20 cm from the hair root) to the part of the hair that has been exposed for the least duration (hair root). Assuming that the rate of hair growth is 0.6–1.4 cm / month, a hair tip located at 20 cm from the root, would be equivalent to an exposure duration of 12 to 28 months before sample collection32.
Statistical analysis
Chronic exposure to heavy metals
To determine the presence of chronic exposure to heavy metals, we calculated the mean difference of heavy metal concentration between hair root samples of the same individuals collected in 2016 and 2018 and tested the statistical significance of this difference using T-student test.
T-student test for paired samples was also undertaken to compare heavy metal concentrations in hair tip samples collected in 2018 with that of hair root of the same individuals (Table S2 of supplementary materials).
Association of heavy metals with clinical symptoms
We investigated the associations of heavy metal concentrations, irrespective of the place of residence, with the manifestation of clinical symptoms (nosebleed, white line on nails and chronic colic) by calculating Pearson’s chi-squared X2 test p-value. We also estimated X2 P-value of linear association to explore if association between heavy metals and clinical symptoms follows a linear trend. For the purpose of these analyses, children were sub-grouped into terciles of the concentration of heavy metals detected in their hair: low, medium, and high.
To verify if the manifestation of nosebleed and the presence of white lines on nails is related to exposure to heavy metals, data from a mixed population composed of exposed and unexposed children to the mine were compared using T-student test for independent samples (see supplementary material online, Table S3).
Association of the place of residence with heavy metal concentration and clinical manifestation
The association of heavy metal concentration with the place of residence (Carhuamayo versus Paragsha) was examined using T-student test. The association of the place of residence with the presence of clinical symptoms was determined by estimating the odds ratio (OR) using logistic regression models.
Analyses were carried out using SPSS (SPSS Inc. Released 2011. SPSS for Windows, Version 20.0. Chicago.
https://www.nature.com/articles/s41598-021-02163-9
