*Article* **Mercury Exposure in Women of Reproductive Age in Rondônia State, Amazon Region, Brazil**

**Thayssa C. S. Bello 1,\* , Rafael J. Buralli <sup>2</sup> , Mônica P. L. Cunha <sup>3</sup> , José G. Dórea <sup>4</sup> , Fredi A. Diaz-Quijano <sup>5</sup> , Jean R. D. Guimarães <sup>6</sup> and Rejane C. Marques <sup>1</sup>**


**Abstract:** Environmental contamination by mercury (Hg) is a problem of global scale that affects human health. This study's aim was to evaluate Hg exposure among women of reproductive age residing in the Madeira River basin, in the State of Rondônia, Brazilian Amazon. This longitudinal cohort study used linear regression models to assess the effects on Hg levels of breastfeeding duration at 6 months, and of breastfeeding duration and number of new children at 2-year and 5-year. Breastfeeding duration was significantly associated with maternal Hg levels in all regression models (6 months, 2 years and 5 years) and no significant association was observed between the number of children and the change in maternal Hg levels in the 2-year and 5-year models. This longitudinal cohort study evaluated Hg levels and contributing factors among pregnant women from different communities (riverine, rural, mining and urban) in Rondônia, Amazon Region, for 5 years. A well-coordinated and designed national biomonitoring program is urgently needed to better understand the current situation of Hg levels in Brazil and the Amazon.

**Keywords:** Amazon; environmental contamination; mercury; women; breastfeeding

#### **1. Introduction**

Environmental contamination by mercury (Hg) is a problem of global scale that affects human health in high-income countries, such as Sweden, Canada, Japan, the United States of America and Finland [1–4], as well as in lower- and middle-income countries (LMIC), such as Brazil, China, Venezuela, Colombia and Iraq [1,5–7].

In the late 1970s, the Amazon experienced an intense gold rush, and the Madeira River was one of its most active areas, with hundreds of illegal barges excavating bottom sediment to obtain gold by amalgamation with Hg. The recent surge in gold prices in international markets provided a renewed push to the activity [8], and illegal gold mining exploded worldwide. In Brazil, this was dramatically amplified during the Bolsonaro presidency (2019–2022) that dismantled the legal framework underlying environmental surveillance activities and strongly reduced the material support to all institutions involved. The result, among so many others, is that tens of thousands of gold miners invaded indigenous territories, such as the Ianomamis in Roraima and the Mundurukus on the Tapajos River, causing irreversible socio-environmental impacts during these 4 years of total official laxism.

**Citation:** Bello, T.C.S.; Buralli, R.J.; Cunha, M.P.L.; Dórea, J.G.; Diaz-Quijano, F.A.; Guimarães, J.R.D.; Marques, R.C. Mercury Exposure in Women of Reproductive Age in Rondônia State, Amazon Region, Brazil. *Int. J. Environ. Res. Public Health* **2023**, *20*, 5225. https:// doi.org/10.3390/ijerph20065225

Academic Editor: Paul B. Tchounwou

Received: 28 February 2023 Revised: 15 March 2023 Accepted: 15 March 2023 Published: 22 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In addition to the Hg emissions from gold mining, the Madeira River carries an important load of natural Hg from the soil erosion of the Andes and deforested areas [9–11]. In 2017, the Minamata Convention on Mercury—an international treaty to reduce Hg use and emissions—came into force. It is the result of negotiations involving 140 countries under the United Nations Environment Programme (UNEP) [12]. To date, the Convention has 128 signatories, including Brazil [12], though the country signed the convention only a few months before the start of the Bolsonaro period. The convention aims to protect human health and the environment from anthropogenic emissions and releases of Hg and its compounds, especially vulnerable populations, women and children, and through them, the future generations [12,13].

As these groups are the most vulnerable and sensitive to possible long-term effects, there is a general recommendation that pregnant women, children and women of childbearing age should avoid exposure to Hg [14]. By modulating the endocrine system, Hg causes a decrease in luteinizing hormone (LH), estradiol, progesterone and prolactin, and an increase in menstrual disorders [15]. These hormones are essential for the maintenance of the female reproductive cycle, and a disorder can cause adverse reproductive outcomes. Given the adverse effects of Hg compounds, it is imperative to assess exposure so that early predictive effects of toxicity can be detected, especially in vulnerable populations [16].

Mercury is usually released in inorganic form and transformed by aquatic bacteria into methylmercury, a potent neurotoxin that biomagnifies along the food chain. As a result, human exposure to mercury is strongly dependent on fish intake frequency. Therefore, lifestyle and diet play an important role in Hg exposure [17]. Kirk et al. [18] found that, due to its neurotoxic potential, Hg exposure through seafood generates a cost of approximately EUR 10 billion per year for the European Union. Consumption of fish with high concentrations of Hg by pregnant women can cause serious problems in the neurological development of the offspring [19]. Dórea and Marques [20] reported that prenatal exposure to this metal may be associated with adverse effects on pregnancy, birth and child development. Lee et al. [21] showed that in late pregnancy, high levels of Hg in maternal blood are associated with an increased risk of newborns with low birth weight.

Xue et al. [22] reported an increased risk of preterm births associated with low to moderate Hg exposure. Women who gave birth before 35 weeks of pregnancy had hair Hg concentrations above the 90th percentile (greater than 0.55 <sup>μ</sup>g·g−1) compared to women who gave birth to term babies (37 to 41 weeks and six days). Dallaire et al. [23] found a negative correlation between cord blood Hg levels and gestational age at birth.

Hg levels in hair are strongly correlated with MeHg intake in an individual's diet [20]. Therefore, the concentration of Hg in hair has been used in many studies as a bioindicator of human exposure [20,24] because it is easy to collect, store and manipulate [25,26]. In addition, its chemical stability facilitates retrospective studies [24,27]. In Brazil, many studies conducted in the Amazon region have used hair as a biomarker of Hg exposure [28–32] among different exposed populations.

Knowing the real influence of Hg exposure on women's health is a matter of great relevance to public health. The development and implementation of appropriate public policies and actions to mitigate the associated risks is fundamental. To the health sector, this context represents a challenge that forces it to constantly review the situation of environmental deterioration and its repercussions on the quality of life.

This study's aims were to evaluate Hg exposure among women of reproductive age residing in the Madeira River basin, in the State of Rondônia, Brazilian Amazon, prior to the ratification of the Minamata Convention, and to investigate possible associated factors. It is hoped that the results can serve as a baseline for evaluating the effectiveness of the implementation of the convention.

#### **2. Materials and Methods**

This longitudinal cohort study with a quantitative and analytical approach evaluated 1433 women of reproductive age who lived in the area covered by the Jamari, Madeira and Mamoré rivers, in the Rondônia State, Brazilian Amazon. All women who were pregnant between 2006 and 2007 were invited to participate in the study. We invited 1668 pregnant mothers; 215 women dropped out within the first semester of the study and 20 during the first year. In the subsequent evaluations, there were no dropouts. All women who agreed to participate were selected according to the following criteria: healthy women during pregnancy, absence of congenital malformations, and residing for at least five years in the study area. Participants were classified as groups of interest derived from rural, urban, riverine and mining areas, according to their place of residence (community). The mining area is located in Bom Futuro, on the banks of the Jamari River, affluent of the Madeira River. It is legal mining, extracting tin from cassiterite, and its activity does not result in any direct Hg emission; it was included in the sampling because it is an important economic activity of the region. Figure 1 shows the study area, which covers more than 733 km along the Madeira River basin and tributaries.

**Figure 1.** Study locations. Source: adapted from Marques et al. [26].

Participants' data were obtained through questionnaires after written authorization. The questionnaire contained open and closed questions in order to obtain participants' information on socio demographic data, eating habits and health status. A questionnaire was applied at the time of inclusion in the study, and a hair sample was collected from each participant. At scheduled home visits (6 months postpartum, 2 years and 5 years), a new questionnaire was applied, women were asked about their daily intake of fish and breastfeeding practices, and hair samples were collected. The study was approved by the Research Ethics Committee of the Federal University of Rondônia (REC/NUSAU/UNIR, file number: 012/CEP/NUSAU).

#### *2.1. Determination of Total Hg Concentrations*

Total Hg concentrations were checked in hair samples (five to ten grams) collected from the occipital region of the mothers, close to the scalp. Samples were placed in transparent plastic bags and properly identified, stored at room temperature and taken for analysis to the Laboratorio de Radioisotopos, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.

Hair samples were washed with a 0.01% EDTA solution, rinsed with ultrapure water and dried in an oven at 50 ◦C. Then, they were fractionated as much as possible with stainless steel scissors for better homogenization and increased efficiency of the acid digestion [33]. After being weighed, they were digested with 5 mL of HNO3 and H2SO4 (1:1) and 4 mL of 5% KMnO4 using a digester block at 80 ◦C for 40 min. Total Hg was determined by cold vapor generation atomic absorption spectrophotometry (CV-AAS) on a Perkin-Elmer® FIMS-400 apparatus (Waltham, MA, USA). The results were expressed in <sup>μ</sup>g·g<sup>−</sup>1. Precision and accuracy of Hg determinations were assured by the use of internal standards, triplicate analyses of samples and certified reference materials (IAEA-085 and 086, Vienna, Austria) with recoveries of 92%. All glassware used was washed clean, rinsed with 5% (*w*/*v*) EDTA and double distilled water and left to rest in 5% (*w*/*v*) HNO3 overnight. Before use, it was rinsed again in double-distilled water and dried at 100 ◦C for 12 h.

#### *2.2. Statistical Analysis*

Differences between groups of interest (rural, urban, riverine and mining) were analyzed. The following variables were considered: maternal age; number of children in each period; years of education; family income; number of cohabitants; type of residence (wood, brick, mixed, straw); household situation (owned, rented, from relatives, borrowed); water supply (piped, well, river, piped/well, public tap, piped/river); energy supply (yes/no); gestational period (in weeks); breastfeeding duration (in months); place of delivery (hospital, residence); type of delivery (normal, cesarean); newborn's sex (female, male); fetal maturity (pre-term, term, post-term); number of children (prenatal, 2 years, 5 years); maternal fish consumption (days per week), and levels of total Hg.

The analyses were performed with software R® (RStudio, Version 1.2.5001, 2019, Boston, MA, USA), and Stata 12 (StataCorp, 2011, College Station, TX, USA). Shapiro–Wilk test was used to verify data distribution. Since data did not present a normal distribution, non-parametric procedures were used. Participants' characteristics and differences between groups of interest were presented as median, minimum, maximum and percentiles (P25–P75%). Correlations between Hg levels and exposure variables (number of children, breastfeeding duration and fish consumption) were performed using the Kendall test to assess the degree of association between them.

Based on a DAG model (Figure 2), two versions were established to guide the model adjustments for covariates: (a) Hg levels at 6 months (outcome), considering that the '*number of children*' had minimal variability and, therefore, was naturally adjusted; (b) 2 years and 5 years, in which the variable *'number of children*' was included as an independent variable (births in the period). The DAG's (Figure 2) testable independence implications were evaluated using linear regression models and were not rejected (*p* > 0.05 for all tests), which was interpreted as an indicator of consistency between data and DAG [34].

**Figure 2.** DAG models: (**A**) 6-month period, including only breastfeeding duration as independent variable; (**B**) 2 years and 5 years period, including number of children and breastfeeding duration as independent variables.

Linear regression models of Hg levels in each period (in <sup>μ</sup>g·g−1) on breastfeeding duration at 6 months, and on breastfeeding duration and number of new children at the 2 and 5 years follow-up were performed, adjusting for family income, years of education, maternal age and place of residence (groups of interest). In the 2 years and 5 years models, when the total number of children was equal to or less than that reported at the beginning of the study, an increment value of zero children was allocated assuming reduced breastfeeding possibility. This happened only in 18 (1.26%) and 11 participants (0.77%), in 2 years and 5 years analyses, respectively. A term of interaction between '*number of new children*' (final-initial) and '*breastfeeding duration*' obtained by multiplying those variables, was tested in the multiple model but was not significant. Thus, this interaction was not considered in the final model.

#### **3. Results**

Participants' age ranged from 13 to 43 years (median 21 years), and 267 participants (18.6%) were aged less than 18 years. Mean family income was BRL 651.19 per month, which is about USD 130. The national monthly minimum wage was BRL 380 in Brazil when the participants' socioeconomic data were collected for the study (2007). Participants almost doubled their number of children in 5 years. Median number of children was two per woman at the beginning of the study, and four per woman after five years of follow-up (Table 1). Median education was 5 years of study, ranging from 0 to 17 years. Median number of residents per household was six individuals, ranging from 2 to 16 cohabitants. Most participants owned their houses (54.57%), which were mostly made of wood (63.64%), had well water (48.88%) or river water (20.46%). Most participants had electricity (71.24%) (Table 1).


**Table 1.** Socioeconomic and demographic characteristics of studied women (n = 1433), Rondônia State, Brazilian Amazon.

Notes: Min = minimum; Max = maximum; P25–75% = 25th and 75th percentile; y = years; n = number

Participants delivered at 39.09 weeks of pregnancy (median), that is, 90.09% at normal fetal maturity (37 to 42 weeks). Birth environment was hospital for most participants (74.80%), with 58.47% of women having normal delivery (Table 2). Regarding the newborns' sex, 50.52% were girls and 49.48% boys (Table 2). Considering all groups, breastfeeding duration ranged from 0 to 24 months (median 6 months), and was longer among riverine and rural communities (median 6 months), and shorter among the tin mining community (Tables 2 and 3). Riverine and rural groups had the highest number of children at all assessed time periods (Table 3). About 93.64% of participants reported fish consumption, with the highest rates among riverine communities (median 5 days per week) (Table 1), followed by rural (median 3 days per week). Lower fish consumption was observed among mining and urban communities (Table 3).

**Table 2.** Birth and duration of breastfeeding characteristics of studied women (n = 1433), Rondônia State, Brazilian Amazon.


Notes: Min = minimum; Max = maximum; P25–75% = 25th and 75th percentile.

**Table 3.** Number of children, breastfeeding duration, total Hg levels (in <sup>μ</sup>g·g−1), fish consumption frequency, according to place of residence of studied women (n = 1433), Rondônia State, Brazilian Amazon.


**Notes:** Min = minimum; Max = maximum; P25–75% = 25th and 75th percentile; y = years; n = number.

In general, higher Hg levels were found among riverine, followed by rural communities, while lower concentrations were observed among mining communities (Table 3).

In the correlation analysis, fish consumption was significantly related to Hg levels in all groups throughout all assessed periods, except in the tin mining community, where fish consumption is the lowest (Table 4). On the other hand, breastfeeding duration was significantly associated with maternal Hg levels in all regression models (6 months, 2 years and 5 years), after adjusting for family income, years of education, maternal age and place of residence (groups of interest). Each month of breastfeeding was associated with a Hg decrease of 0.053 <sup>μ</sup>g·g−<sup>1</sup> for the 6-month period, 0.037 <sup>μ</sup>g·g−<sup>1</sup> for the 2 y period and 0.053 <sup>μ</sup>g·g−<sup>1</sup> for the 5 years period. No significant association was observed between the number of children and the change of maternal Hg levels in the 2 years and 5 years models (Table 5).


**Table 4.** Correlation between Total Hg levels (in <sup>μ</sup>g·g−1) and number of children, breastfeeding duration and fish consumption in the prenatal period, after 6 months, 2 and 5 years of studied women (n = 1433), according to the place of residence, Rondônia State, Brazilian Amazon.

**Notes:** Tau: Kendall's correlation coefficient; \* *p* < 0.05; \*\* At the six-month assessment, it was assumed that number of children was constant, as all participants had only one child from the study enrolment until six-month follow-up. y = years; m = months

**Table 5.** Linear regression of total Hg levels (in <sup>μ</sup>g·g−1) on breastfeeding duration and number of children among studied women (n = 1433), Rondônia State, Brazilian Amazon, Brazil.



