# Exploring mercury and selenium dynamics in Amazonian human populations: Insights from urine, blood, and plasma analyses

Gabriel Neves Cezarette, Juliette Esplugas, Jonas Carneiro Cruz, Bruno Alves Rocha, Lucas Cassulatti dos Santos, Maïté Bueno, Zoyne Pedrero, Fernando Barbosa Jr

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Gabriel Neves Cezarette, Juliette Esplugas, Jonas Carneiro Cruz, Bruno Alves Rocha, Lucas Cassulatti dos Santos, et al. Exploring mercury and selenium dynamics in Amazonian human populations: Insights from urine, blood, and plasma analyses. Chemosphere, 2025, 375, pp.144258. 10.1016/j.chemosphere.2025.144258. hal-04985222

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# Exploring mercury and selenium dynamics in Amazonian human populations: Insights from urine, blood, and plasma analyses

Gabriel Neves Cezarette<sup>a, b</sup>, Juliette Esplugas<sup>b</sup>, Jonas Carneiro Cruz<sup>a</sup>, Bruno Alves Rocha<sup>a, c</sup>, Lucas Cassulatti dos Santos<sup>a</sup>, Maïté Bueno<sup>b</sup>, Zoyne Pedrero Zayas<sup>b</sup>, Fernando Barbosa Júnior<sup>a, *</sup>

## HIGHLIGHTS

* Largest human cohort tracing Hg and Se status in Amazonian riverine populations.

* First parallel assessment on Hg and Se levels in human blood, plasma and urine.

* Blood Hg varied significantly while Se remained stable among individuals.

* Frequent fish consumption raises blood Hg but doesn't affect Se levels.

* A positive correlation between Hg and Se was observed in urine.

## GRAPHICAL ABSTRACT

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

Handling editor: Milena Horvat

### Keywords:

- Human biomonitoring
- Riverside population
- Brazilian amazon
- Blood
- Urine
- Plasma

## ABSTRACT

The Amazonian riverside population is one of the most impacted by mercury, interestingly, with distinct selenium values in blood, generally among the highest worldwide. The interaction between Hg and Se remains to be thoroughly investigated and could provide insights into the fate of these elements and the potential for selenium-mediated detoxification. The current study explores the levels of both elements, determined by inductively coupled plasma mass spectrometry (ICP-MS), in blood, plasma, and urine, of 1089 adult individuals from communities of Tapajós and Amazon River. A large inter-individual variability was found for Hg levels (0.2–139; 0.2–27; 0.1–19 µg Hg.L⁻¹ respectively in blood, plasma, and urine), while Se contents show rather homogenous status in blood (arithmetic mean = 183 ± 78 µg Se.L⁻¹) and plasma (AM = 103 ± 34 µg Se.L⁻¹). Hg content in blood was positively correlated with Hg level in plasma, both increasing with the frequency of fish intake. In contrast, Se levels remained stable in blood regardless of how often fish was consumed. However, a positive correlation was observed between Hg and Se excreted in urine. This work represents the largest human cohort in the region, pioneering the evaluation of the levels of both elements simultaneously in blood, plasma, and urine, laying the foundations for understanding the dynamics of Hg and Se in Amazonian riverside populations.

<footnote><sup>a</sup> University of Sao Paulo, School of Pharmaceutical Sciences of Ribeirao Preto, Department of Clinical Analyses, Toxicology and Food Sciences. Analytical and System Toxicology Laboratory, Avenida do Cafe s/nº, Ribeirao Preto, 14040-903, Sao Paulo, Brazil</footnote>

<footnote><sup>b</sup> Universite de Pau et des Pays de l'Adour, E2S UPPA, CNRS, IPREM, Institut des Sciences Analytiques et de Physico-chimie pour l'Environnement et les Matériaux, Pau, France</footnote>

<footnote><sup>c</sup> Institute of Chemistry, Federal University of Alfenas, 37130-001, Alfenas, Minas Gerais, Brazil</footnote>

<footnote>* Corresponding author.
E-mail address: fbarbosa@fcfrp.usp.br (F. Barbosa Júnior).</footnote>
# 1. Introduction

The Amazon region is a focal point for ongoing scientific and global discussions due to its critical role as a sociocultural, economic and, environmental treasure. This region is recognized for the significant impact of mercury on its biome, more specifically in the human population (Watts, 2023; Crespo-Lopez et al., 2021). In the Brazilian Amazon, riverine communities are at risk to high Hg exposure through their unique diet. Riverine habits bring fish as a daily basic food, representing 80% of the dietary protein source (Hacon et al., 2020).

Region dietary Hg daily intake has been estimated to be 1–2 µg kg⁻¹ (Passos et al., 2008), which is much higher than the U.S. Environment Protection Agency reference dose of 0.1 µg kg⁻¹.day⁻¹ or the World Health Organization (WHO) recommendation limit of 0.23 µg kg⁻¹.day⁻¹ (Wong et al., 2022). Even when Hg content in fish does not exceed the maximum levels established by WHO (<0.5 µg g⁻¹ fresh weight), the estimated daily intake is superior to the threshold value recommended by EPA, due to the large fish consumption by river-side communities (Crespo-Lopez et al., 2021).

The Amazon riverside population present a median Hg blood concentration of 15.4 µg.L⁻¹ (Basu et al., 2018) which is higher than values observed in occupationally exposed workers of artisanal and small-scale gold minings, and in some cases, exceeds the WHO chronic exposure limit (5 µg Hg.L⁻¹) by more than 10 times (Santos-Sacramento et al., 2021). Epidemiological studies that report median blood Hg levels in populations reliant on fish consumption, such Faroe Islands (2.5 µg.L⁻¹; Odland et al., 2016), the Seychelles (11.7 µg.L⁻¹; Strain et al., 2015), and the Arctic Inuit (8.6 µg.L⁻¹; Donaldson et al., 2016), highlight that the Amazon population is among the most affected by Hg exposure.

The selenium blood concentrations in Amazonian riverine populations (median of 222 µg.L⁻¹) are comparable to those found in other fish-eating populations, such as Canadians (median of 199 µg.L⁻¹) and Americans (median of 189 µg.L⁻¹), as reported in cross-sectional studies (Achouba et al., 2016; Tinggi and Perkins, 2022). Local products such as fish and Brazil nuts (Lemire et al., 2010a) from this region are rich in selenium, contributing to the elevated selenium status of this population (Lemire et al., 2010a, 2012; Valentini et al., 2016). Interestingly, Nunavimmiut Inuits exhibit high whole blood selenium levels (199 µg.L⁻¹), but without correlation with plasma levels, in contrast to the linear relationship observed in Amazonian populations in previous studies (Achouba et al., 2016; Lemire et al., 2012).

Studies indicate that a high molar ratio between Se and Hg in human plasma can reduce toxic effects associated with Hg exposure, providing a protective effect of Se against cardiotoxicity and oxidative stress (Barbosa et al., 2009; Barcelos et al., 2012; Carneiro et al., 2014; Grotto et al., 2011). Although being frequently explored, the role of Se as potential antagonist of Hg, particularly of its organic form like MeHg, is not completely elucidated so far (Achouba et al., 2016; Little et al., 2019).

Researches on Hg exposure in human have mainly focused on the monitoring of Hg in hair, urine, blood, and cord blood - biomarkers that provide crucial insights on the type of Hg (organic vs. inorganic) and the timeline of exposure (acute vs. chronic), but don't consider the fate, or potential interactions in the body (Basu et al., 2018). Significant gaps persist in our knowledge about exposures worldwide, largely due to the limited coverage geographic and subpopulations data, and the predominant use of hair as biomarker, with the absence of measurements taken from different biological matrices in specific individuals, which could allow a deeper understanding of the dynamics within the human body (Basu et al., 2018, 2023).

The present work is part of a cross-sectional human biomonitoring investigation which aims to apply the innovative concept of exposome in Brazil, establishing patterns or profiles associated with health outcomes resulting from exposure to metals, metalloids and emerging contaminants, in different Brazilian populations (Cruz et al., 2024; González et al., 2024). Representing the largest human cohort in the Amazon region, this work pioneers the simultaneous evaluation of Hg and Se in blood, plasma, and urine, laying the foundation for understanding their interactions in these riverine individuals.

# 2. Material and methods

## 2.1. Study population

This study is part of a cross-sectional human biomonitoring investigation conducted among participants from 13 communities situated in the Tapajós and Amazon River basins, in the state of Pará, Brazil (Fig. 1) already described in Cruz et al. (2024).

Briefly, based on their geographical locations in rivers course, communities can be split into three subregions: 1) the Lower/Medium Tapajós River Basin (Brasília Legal, Aveiro, Pinhel, and Cametá), 2) the Lower Tapajós River Basin (Boim, Surucuá, Parauá, Vila Amorim, Surucá, Maripá) and 3) the Amazon River Basin (Aninduba, Guajará, Arapixuna).

The majority of the population is composed of "riverine" individuals, who reside along the riverbanks and maintain distinct dietary habits, lifestyles, and patterns of racial mixing, which vary significantly across the different communities. For instance, Aveiro, is a former village, with port terminals for ferries and liner boats, exhibiting more pronounced external influences from urban areas beyond the riverbanks. While most communities are located along the river shore, Arapixuna is an exception, being situated along a stream that connects the Tapajós and Amazon rivers.

In general, the population in these areas demonstrates a high proportion of European ancestry, followed by Amerindian and African ancestries (de Paula Fonseca Arrifano et al., 2018). However, certain communities, such as Pinhel, Surucuá, Vila Amorim, Surucá, and Maripá, retain a stronger indigenous influence, with evident ethnic and cultural traits persisting within the population. Furthermore, the communities differ in population density, which affects local commerce, food availability, and lifestyle.

## 2.2. Sample collection

A sampling campaign was conducted in October–November 2022 during the dry season, period when this population has a greater frequency of fish consumption. The volunteer recruitment was carried out in each village through house-to-house visits and at village meetings, during which the research project was explained, and villagers were invited to participate on a voluntary basis. Trained interviewers administered questionnaires on socio-demographic information, occupational, residential and medical history and dietary information.

A total of 1089 volunteers (676 females; 387 males) agreed to participate in the study, signing the free and informed consent form. The including criteria was volunteers with age between 18 and 80, from both sexes, regardless of health condition, and with fixed permanence in the community. Research protocol was approved by the Ethics Committee of the School of Pharmaceutical Sciences of Ribeirão Preto (CAAE: 77196924.5.0000.5403).

Blood samples (6 mL) were collected as non-fasting samples in trace metal-free evacuated tubes (BD Vacutainer®) using heparin as an anti-coagulant. Approximately 1.5 mL of whole blood was transferred into two separate 2 mL Eppendorf® tubes for whole blood analysis. The remaining blood was centrifuged at 1500 × g for 10 min to obtain plasma. Two milliliters of the resulting plasma were then divided into two 1 mL fractions and transferred into separate 2 mL Eppendorf® tubes. Plasma samples exhibiting hemolysis were excluded from analysis.

The urine collection procedure was performed by the volunteers themselves. During the interview, the volunteer was guided about the
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Study area highlighting the investigated riverine communities and their location in Brazilian territory. From Tapajós river basin: 1. Brasília Legal 2. Aveiro 3. Pinhel 4. Cametá 5. Boim 6. Surucuá 7. Parauá 8. Vila Amorim 9. Suruacá 10. Maripá; Amazon River basin: 11. Aninduba 12. Guajará 13. Arapixuna.</figcaption></figure>

procedures for the collection. Spot urine samples were packed in a sterile container, hermetically sealed, from which 10 mL subsamples were transferred into conical tubes (Corning®). All samples were transferred in tubes previously identified with a numerical ID, thus maintaining the anonymity of the volunteers.

## 2.3. Transport and storage of samples

The transportation and storage of samples adhered to the protocols recommended by Sabé et al. (2000). Samples were processed on-site immediately after collection, centrifuged for plasma separation, and then immediately stored at -20°C aboard the research boat. They were subsequently transported to Santarém city (PA, Brazil) while maintaining the -20 °C temperature. From there, they were shipped with dry ice by air to Ribeirão Preto city (SP, Brazil), and finally storage at the laboratory at University of São Paulo, at -80 °C until analysis.

## 2.4. Mercury and selenium analysis

Total Hg and Se concentrations in samples were determined by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), according to the method described by Palmer et al. (2006), in a NexION 2000 (PerkinElmer®, Massachusetts, USA) quadrupole-based instrument equipped with a triple cone interface, operating with high-purity argon (99,999%, Air Liquide, Sao Paulo, Brazil). Sample introduction system was composed by quartz cyclonic spray chamber and a Meinhard® nebulizer connected by Tygon® tubes to the ICP-MS's peristaltic pump (at 20 rpm).

Samples were prepared according to Batista et al. (2009). Briefly, blood samples were diluted 1:50 (40 µL blood + 1.96 mL of 0.5% v/v HNO₃ and 0.005% v/v of Triton X-100), plasma samples were diluted 1:20 (250 µL plasma + 4.75 mL of 0.5% v/v HNO₃ and 0.005% v/v of Triton X-100), and urine samples were diluted at 1:20 (0.5 mL urine + 9.5 mL of 0.5% v/v HNO₃ containing 0.005% v/v of Triton X-100), before analysis.

All reagents used were of analytical grade and the solutions were prepared using high-purity water with a resistivity of 18.2 MΩ cm⁻¹, obtained from a Milli-Q Plus water purification system (Millipore, Bedford, MA). Nitric acid (Merck, Germany) was double distilled into a quartz sub-boiling before use. All the solutions were prepared in a clean laboratory within a laminar-flow hood class 100.

Quality control assurance was guaranteed by analyzing Standard Reference Materials from the U.S. National Institute of Standards and Technologies (NIST 955d, 1598a and 2670a). Reference samples were analyzed before and after ten ordinary samples. All results were within the standard values.

To assess potential losses of mercury and selenium during storage and transportation, we analyzed a second replicate fraction of 50 samples. These samples were collected in the same evacuated tube, fractionated on-site, and stored in separate Eppendorf tubes for both blood and plasma. The selected samples included those with lower selenium levels. After applying a paired t-test (99% confidence level), no statistically significant differences were observed between the paired sample data.
## 2.5. Data analysis

General descriptive statistics (arithmetic mean, standard deviation, median, and ranges), Kruskal–Wallis test followed by Dunn's post hoc test or, pairwise Mann-Whitney test were used to assess differences between concentrations distribution between groups. Correlation's analysis (Spearman's rho) was performed to explore the relations between element concentrations in the different samples, age, gender and fish consumption frequency. Graphs were plotted and statistical analyses were performed using the GraphPad Prism software, version 8.0.1 (GraphPad Software Inc., San Diego, USA) and Jamovi, version 2.5 (Jamovi, 2024).

# 3. Results and discussion

## 3.1. Hg levels in blood, plasma and urine

A large inter-individual variability of Hg levels was observed in blood, plasma, and non-adjusted urine, with concentrations ranging from 0.2 to 139 µg.L⁻¹ in blood, 0.2–27 µg.L⁻¹ in plasma, and up to 19 µg.L⁻¹ in urine (Fig. 2, Table 1). The average Hg concentrations in the biological matrices were 26 ± 20 µg.L⁻¹ in blood, 4.0 ± 2.7 µg.L⁻¹ in plasma, and 2.0 ± 2.4 µg.L⁻¹ in urine. According to the Mann-Whitney test, Hg levels in all biological matrices were significantly higher (p < 0.0001) in male individuals than in females (Figure S2). No correlation with age was found (Figure S3).

Among the studied communities, variations were particularly noticeable in whole blood Hg concentrations (Fig. 2). While some statistical differences between communities were also found regarding plasma and urine concentrations, the most important variation concerned blood levels, with the community of Cametá and Surucuá exhibiting the highest median values, and Brasília Legal and Suruacá showing the lowest.

Despite the limited number of studies in this region using biological matrices other than hair, a general comparison with previous data suggests

a reduction in Hg content across blood and plasma. Previous studies reported mean Hg concentrations of 49 ± 36 µg.L⁻¹ in blood (from n = 91 individuals) and 7.1 ± 6.6 µg.L⁻¹ in plasma (n = 108) for communities along the Tapajós River (Carneiro et al., 2014; Grotto et al., 2010). These values, corresponding to sampling carried out in 2006 are nearly two times higher than those found in the present study. However, our results show Hg levels that are still high compared to other exposed populations. Recent studies in the Peruvian Amazon reported mean blood Hg levels ranging from 3 to 7 µg.L⁻¹ (n = 554) in 21 communities near small-scale gold mining areas (Weinhouse et al., 2021). Values measured in the current study are in the same order of magnitude as those reported for occupationally exposed gold miners of 28 ± 23 µg.L⁻¹ (n = 106) and 25 ± 25 µg.L⁻¹ (n = 219) from São Chico and Creporizinho, in the region of Itaituba - Brazilian Amazon (Castilhos et al., 2015).

Few studies have investigated Hg levels in plasma in the Brazilian Amazon region, reporting values ranging from 6 to 10 µg.L⁻¹ (Carneiro et al., 2014; De Oliveira et al., 2014; Grotto et al., 2010; Lemire et al., 2010b). Carneiro et al. (2014) reported that Hg in plasma accounted for about 34% of Hg content in whole blood. Our results indicate a similar proportion of 28%, which is still almost six times higher than the safety limit set by the WHO, which is 5% (WHO and UNEP, 2008). However, the interpretation of plasma Hg levels in biomonitoring studies requires careful consideration due to potential sources of error during sample collection and processing. Hg is known to accumulate primarily in erythrocytes, with only a small fraction present in plasma (Clarkson et al., 2007). Consequently, even minor contamination of plasma with red blood cells during blood collection or centrifugation can lead to artificially elevated Hg levels in plasma. In this study, all samples were processed under controlled conditions to minimize the risk of contamination, and any samples showing signs of hemolysis were excluded. To ensure accuracy, plasma should ideally be analyzed for traces of hemoglobin, which represents a limitation in this study.

Studies on Hg levels in urine are even scarce in this population. Passos et al., 2008, measured Hg levels in density-adjusted urine, re-

<figure><img src="image_5.png" /><figcaption>Fig. 2. Boxplots of concentrations (µg.L⁻¹) of Hg (left panel) and Se (right panel) in whole blood and plasma, according to riverine communities. Different letters indicate a significant difference between groups when tested in the Kruskal-Wallis test followed by the post-hoc Dunn's test (p < 0.001).</figcaption></figure>
<table><caption>**Table 1**
Summary of Hg and Se levels in biological matrices, according to riverine communities on Tapajós and Amazon River Basin.</caption><thead><tr><th rowspan="2">Community</th><th rowspan="2">N</th><th rowspan="2">%</th><th rowspan="2">Biological matrix</th><th colspan="5">Total Hg Level (µg/L)</th><th colspan="5">Total Se Level (µg/L)</th></tr><tr><th>Mean</th><th>SD</th><th>Median</th><th>P5</th><th>P95</th><th>Mean</th><th>SD</th><th>Median</th><th>P5</th><th>P95</th></tr></thead><tbody><tr><td rowspan="3">Brasília Legal</td><td rowspan="3">102</td><td rowspan="3">9.4</td><td>Blood</td><td>15.4</td><td>16.2</td><td>9.8</td><td>0.9</td><td>56.8</td><td>189.6</td><td>96.6</td><td>163.1</td><td>107</td><td>340</td></tr><tr><td>Plasma</td><td>2.5</td><td>2.5</td><td>1.5</td><td>2.1</td><td>10.2</td><td>108.3</td><td>44.0</td><td>98.7</td><td>63</td><td>153</td></tr><tr><td>Urine</td><td>1.5</td><td>1.2</td><td>1.0</td><td>0.4</td><td>8.2</td><td>43.6</td><td>25.6</td><td>38.6</td><td>15.4</td><td>134</td></tr><tr><td rowspan="3">Aveiro</td><td rowspan="3">175</td><td rowspan="3">16.1</td><td>Blood</td><td>31.0</td><td>26.9</td><td>23.8</td><td>4.8</td><td>94.0</td><td>197.0</td><td>84.9</td><td>164.3</td><td>112</td><td>414</td></tr><tr><td>Plasma</td><td>3.8</td><td>3.2</td><td>3.0</td><td>1.1</td><td>7.8</td><td>96.4</td><td>38.9</td><td>86.5</td><td>56</td><td>163</td></tr><tr><td>Urine</td><td>2.2</td><td>2.8</td><td>1.4</td><td>0.4</td><td>6.6</td><td>49.4</td><td>30.4</td><td>45.5</td><td>18.5</td><td>152</td></tr><tr><td rowspan="3">Pinhel</td><td rowspan="3">61</td><td rowspan="3">5.6</td><td>Blood</td><td>25.7</td><td>14.8</td><td>22.4</td><td>6.2</td><td>53.2</td><td>161.4</td><td>60.1</td><td>149.0</td><td>79</td><td>290</td></tr><tr><td>Plasma</td><td>3.2</td><td>1.9</td><td>2.8</td><td>0.9</td><td>11.1</td><td>86.9</td><td>23.1</td><td>84.6</td><td>72</td><td>159</td></tr><tr><td>Urine</td><td>2.0</td><td>1.3</td><td>1.7</td><td>0.4</td><td>3.0</td><td>56.3</td><td>27.8</td><td>56.0</td><td>11.8</td><td>111</td></tr><tr><td rowspan="3">Cametá</td><td rowspan="3">56</td><td rowspan="3">5.1</td><td>Blood</td><td>37.6</td><td>20.8</td><td>33.9</td><td>10.8</td><td>78.5</td><td>189.0</td><td>95.7</td><td>171.5</td><td>69</td><td>363</td></tr><tr><td>Plasma</td><td>4.9</td><td>2.4</td><td>4.5</td><td>1.7</td><td>7.7</td><td>94.4</td><td>37.5</td><td>93.9</td><td>44</td><td>136</td></tr><tr><td>Urine</td><td>3.6</td><td>4.8</td><td>1.9</td><td>0.5</td><td>5.1</td><td>51.7</td><td>26.9</td><td>44.8</td><td>14.3</td><td>105</td></tr><tr><td rowspan="3">Boim</td><td rowspan="3">93</td><td rowspan="3">8.5</td><td>Blood</td><td>22.2</td><td>13.5</td><td>21.5</td><td>4.2</td><td>48.9</td><td>238.4</td><td>103.9</td><td>217.4</td><td>118</td><td>480</td></tr><tr><td>Plasma</td><td>3.4</td><td>1.8</td><td>3.4</td><td>1.3</td><td>11.0</td><td>120.7</td><td>40.5</td><td>115.6</td><td>65</td><td>182</td></tr><tr><td>Urine</td><td>2.0</td><td>1.7</td><td>1.6</td><td>0.5</td><td>9.4</td><td>89.6</td><td>81.4</td><td>72.6</td><td>22.3</td><td>165</td></tr><tr><td rowspan="3">Surucuá</td><td rowspan="3">46</td><td rowspan="3">4.2</td><td>Blood</td><td>46.0</td><td>26.5</td><td>45.5</td><td>5.9</td><td>92.6</td><td>195.0</td><td>52.3</td><td>193.2</td><td>116</td><td>286</td></tr><tr><td>Plasma</td><td>6.5</td><td>3.5</td><td>6.5</td><td>0.9</td><td>7.6</td><td>96.4</td><td>19.9</td><td>97.1</td><td>65</td><td>145</td></tr><tr><td>Urine</td><td>3.4</td><td>3.8</td><td>2.1</td><td>0.3</td><td>2.9</td><td>65.9</td><td>36.2</td><td>55.9</td><td>9.9</td><td>101</td></tr><tr><td rowspan="3">Parauá</td><td rowspan="3">118</td><td rowspan="3">10.8</td><td>Blood</td><td>28.0</td><td>14.8</td><td>24.9</td><td>8.7</td><td>59.0</td><td>171.6</td><td>59.3</td><td>157.2</td><td>108</td><td>264</td></tr><tr><td>Plasma</td><td>5.5</td><td>2.3</td><td>5.1</td><td>2.0</td><td>12.0</td><td>106.3</td><td>32.1</td><td>104.6</td><td>42.8</td><td>127</td></tr><tr><td>Urine</td><td>2.9</td><td>2.4</td><td>2.2</td><td>0.3</td><td>3.8</td><td>59.7</td><td>32.2</td><td>54.9</td><td>8.9</td><td>91</td></tr><tr><td rowspan="3">Amorim</td><td rowspan="3">35</td><td rowspan="3">3.2</td><td>Blood</td><td>23.8</td><td>10.4</td><td>22.9</td><td>5.4</td><td>44.2</td><td>139.8</td><td>38.6</td><td>135.7</td><td>88</td><td>219</td></tr><tr><td>Plasma</td><td>4.5</td><td>1.9</td><td>4.1</td><td>0.4</td><td>11.5</td><td>103.9</td><td>25.2</td><td>107.1</td><td>69</td><td>204</td></tr><tr><td>Urine</td><td>2.3</td><td>1.5</td><td>1.9</td><td>0.6</td><td>3.2</td><td>58.1</td><td>26.5</td><td>48.9</td><td>11.9</td><td>73</td></tr><tr><td rowspan="3">Suruacá</td><td rowspan="3">58</td><td rowspan="3">5.3</td><td>Blood</td><td>11.5</td><td>6.3</td><td>11.1</td><td>2.5</td><td>25.4</td><td>135.4</td><td>39.6</td><td>134.1</td><td>89</td><td>218</td></tr><tr><td>Plasma</td><td>3.3</td><td>1.4</td><td>3.1</td><td>0.8</td><td>5.0</td><td>109.3</td><td>30.0</td><td>107.6</td><td>79.5</td><td>155</td></tr><tr><td>Urine</td><td>1.3</td><td>1.2</td><td>0.9</td><td>0.2</td><td>2.2</td><td>40.9</td><td>32.8</td><td>32.5</td><td>7.3</td><td>86</td></tr><tr><td rowspan="3">Maripá</td><td rowspan="3">42</td><td rowspan="3">3.9</td><td>Blood</td><td>30.1</td><td>17.0</td><td>27.7</td><td>7.8</td><td>64.4</td><td>194.3</td><td>54.0</td><td>187.8</td><td>114</td><td>298</td></tr><tr><td>Plasma</td><td>4.8</td><td>1.8</td><td>4.6</td><td>2.2</td><td>8.6</td><td>110.5</td><td>24.8</td><td>107.1</td><td>66</td><td>150</td></tr><tr><td>Urine</td><td>1.3</td><td>1.3</td><td>0.8</td><td>0.4</td><td>5.4</td><td>34.0</td><td>27.0</td><td>23.6</td><td>10.4</td><td>113</td></tr><tr><td rowspan="3">Aninduba</td><td rowspan="3">116</td><td rowspan="3">10.7</td><td>Blood</td><td>28.3</td><td>22.2</td><td>21.0</td><td>5.5</td><td>78.7</td><td>134.4</td><td>36.2</td><td>131.3</td><td>88</td><td>203</td></tr><tr><td>Plasma</td><td>5.6</td><td>3.1</td><td>5.0</td><td>0.5</td><td>8.8</td><td>92.0</td><td>24.5</td><td>95.9</td><td>58</td><td>191</td></tr><tr><td>Urine</td><td>1.2</td><td>1.3</td><td>0.7</td><td>0.4</td><td>3.8</td><td>36.2</td><td>26.7</td><td>27.6</td><td>11.5</td><td>100</td></tr><tr><td rowspan="3">Guajará</td><td rowspan="3">60</td><td rowspan="3">5.5</td><td>Blood</td><td>22.8</td><td>18.6</td><td>18.4</td><td>1.4</td><td>58.6</td><td>190.4</td><td>54.8</td><td>189.0</td><td>143</td><td>338</td></tr><tr><td>Plasma</td><td>3.7</td><td>2.3</td><td>3.4</td><td>1.4</td><td>7.1</td><td>109.5</td><td>32.1</td><td>106.4</td><td>68</td><td>177</td></tr><tr><td>Urine</td><td>1.2</td><td>0.8</td><td>0.8</td><td>0.4</td><td>5.2</td><td>55.4</td><td>26.1</td><td>53.6</td><td>7.0</td><td>126</td></tr><tr><td rowspan="3">Arapixuna</td><td rowspan="3">127</td><td rowspan="3">11.7</td><td>Blood</td><td>20.5</td><td>16.7</td><td>16.4</td><td>5.1</td><td>52.0</td><td>189.3</td><td>71.3</td><td>177.5</td><td>145</td><td>261</td></tr><tr><td>Plasma</td><td>2.8</td><td>1.8</td><td>2.4</td><td>0.9</td><td>8.6</td><td>110.0</td><td>22.5</td><td>109.9</td><td>51.1</td><td>156</td></tr><tr><td>Urine</td><td>1.0</td><td>0.8</td><td>0.6</td><td>0.4</td><td>6.6</td><td>37.6</td><td>29.6</td><td>30.1</td><td>15.4</td><td>96.</td></tr><tr><td rowspan="3">Global</td><td rowspan="3">1089</td><td rowspan="3">100</td><td>Blood</td><td>25.8</td><td>20.5</td><td>20.5</td><td>4.2</td><td>65.4</td><td>181.4</td><td>77.8</td><td>164.6</td><td>102</td><td>310</td></tr><tr><td>Plasma</td><td>4.1</td><td>2.7</td><td>3.4</td><td>0.9</td><td>9.5</td><td>103.4</td><td>34.0</td><td>100.9</td><td>58.3</td><td>157</td></tr><tr><td>Urine</td><td>2.0</td><td>2.4</td><td>1.3</td><td>0.4</td><td>5.6</td><td>52.1</td><td>40.9</td><td>45.1</td><td>12.3</td><td>111</td></tr></tbody></table>

porting an average of 7.5 µg.L⁻¹ (range of 0.2–36 µg.L⁻¹, n = 171). Since the Hg content in the current study is not normalized (e.g., not adjusted for creatinine levels or density), the direct comparison with other studies is meted.

The Inuit population is also highly exposed to Hg due to their reliance on fish and marine mammals as traditional foods (Parajuli et al., 2021). In 15 Arctic subpopulations, totaling 7472 individuals, the median blood Hg concentration was 9 µg.L⁻¹ (Basu et al., 2018). Meanwhile, in 46 subpopulations from the Amazon, representing 18,509 individuals across three countries, median Hg levels were 15 µg.Hg.L⁻¹ in whole blood and 7 µg.Hg.L⁻¹ in urine (Basu et al., 2018). These values were approximately 8 times higher for both urine and blood compared to background populations identified in cross-sectional studies conducted in Germany (Blood<sub>P50</sub>: 0.7 µg.L⁻¹, n = 3973; Urine<sub>P50</sub>: 0.4 µg.L⁻¹, n = 1734), the USA (Blood<sub>P50</sub>: 0.79 µg.L⁻¹, n = 5030; Urine<sub>P50</sub>: 0.24 µg.L⁻¹, n = 1813), Canada (Blood<sub>P50</sub>: 0.65 µg.L⁻¹, n = 1313; Urine<sub>P50</sub>: 0.2 µg.L⁻¹, n = 1048), the Czech Republic (Blood<sub>P50</sub>: 0.65 µg.L⁻¹, n = 302; Urine<sub>P50</sub>: 0.91 µg.L⁻¹, n = 234), and Slovenia (Blood<sub>P50</sub>: 1.2 µg.L⁻¹, n = 1085; Urine<sub>P50</sub>: 0.45 µg.L⁻¹, n = 1020) (Basu et al., 2018). Despite the downtrend observed in the present study when comparing with previously reported values in the Tapajós and Amazon River regions, Hg blood levels of this population still above the Amazon populations median, however, elevated blood

levels do not necessarily point to Hg toxicity. Furthermore, the majority of participants in the current study (~94%) had Hg whole blood levels exceeding the Hg chronic exposure threshold of 5 µg Hg.L⁻¹ set by the WHO (WHO, 2019; WHO and UNEP, 2008).

## 3.2. Se levels in blood, plasma and urine

In contrast to Hg trends, Se contents appeared relatively homogeneous among individuals in all the biological matrices. Age and gender of individuals did not seem to influence Se status (Figures S2 and S3). Although statistically significant differences values between communities were observed, the low magnitude of these differences suggest minimal variable Se intakes or dynamics within the human body. Median concentrations of 165 µg.L⁻¹ in blood (ranging 34–840) and 101 µg.L⁻¹ in plasma (ranging 11–351) indicate a downward trend of Se concentrations compared with median values in previous studies of Lemire et al. (2010b; 2011) of 228 µg.L⁻¹ in blood (ranging 103–1500) and 135 µg.L⁻¹ in plasma (ranging 54–913).

Biomonitoring surveys have documented typical Se levels in blood and plasma among adult worldwide populations (Jain and Choi, 2015; Kim et al., 2017; Komarova et al., 2021; Sobolev et al., 2021; Takeda et al., 2017; Tinggi and Perkins, 2022). In Brazil, the geometric mean Se concentration in blood is 89 µg.L⁻¹ (n = 1125), in the range
68–245 µg.L⁻¹ (Nunes et al., 2010), values that are equivalent to those observed in Australia (GM: 141, 118–224 µg.L⁻¹ – CI 95%), Slovenia (GM: 115 for men, 95 for women, 54–226 µg.L⁻¹ – CI 95%) and, the United States (GM: 146.8, 144.1–247 µg.L⁻¹ – CI 95%) (Tinggi and Perkins, 2022). In comparison, the whole blood Se levels in the current study ranged from 34 to 840 µg.L⁻¹, with an arithmetic mean of 182.3 µg.L⁻¹. These concentrations are higher than those previously reported in other Amazon populations, such as Puerto Nariño in the Colombian Amazon, where a mean of 85 µg.L⁻¹ (60–118 µg.L⁻¹) was recorded (Alcala-Orozco et al., 2020). Furthermore, the studied Brazilian Amazon population appears to have slightly elevated plasma Se levels compared to global averages. A compilation of plasmatic Se data from 28 countries reported a mean of 92 ± 19 µg.L⁻¹ among adults of background populations (Díaz et al., 2023). However, direct comparisons between populations should be made cautiously, as differences in sample size, demographic factors, and methodology could influence the observed ranges.

Studies on populations with a high consumption of marine fish have reported elevated mean Se levels in blood. For example, residents of the Sahtú and Dehcho regions in Northwest Canada exhibit mean Se levels of 170 µg.L⁻¹ (Ratelle et al., 2020). The Inuit population presents even higher average concentrations of 301 µg.L⁻¹ (ranging from 126 to 2362 µg.L⁻¹) in Nunavik, and of 280 µg.L⁻¹ (ranging from 150 to 1500 µg.L⁻¹) in Nunavut (Parajuli et al., 2021). Similarly, in Greenland, mean blood Se values of 488, 225 and 191 µg.L⁻¹ have been reported in districts of Uummannaq, Ittoqqortoormiit and Tasiilaq respectively (Hansen et al., 2004).

These Se levels are likely attributable to the greater Se content found in marine fish compared to freshwater species (Ferraz et al., 2023), particularly considering the specific regions and fish species consumed. Interestingly, coastal communities in Northern Russia (Nenets), despite a high consumption of marine products, exhibit mean blood Se levels of 127 µg.L⁻¹ (ranging from 85 to 192 µg.L⁻¹), which are closer to the values observed in the general global population

(Sobolev et al., 2021). In Inuit populations, the main source of Se is traditional dishes made from seal and whales (Hansen et al., 2004).

### 3.3. Correlations of Hg and Se levels across biological matrices

Relation between Hg levels in plasma and blood (Fig. 3 and S.4) revealed a positive strong correlation between blood and plasmatic Hg (r = 0.83, p < 0.0001). A mean plasma/blood Hg concentration ratio of 0.2 ± 0.1 was determined, corroborating with previous findings in this region of Lemire (0.15) and Grotto (0.13) (Grotto et al., 2010; Lemire et al., 2010b), and with a slight contrast with those reported by Carneiro et al. (2014) and De Oliveira et al., 2014, whom identified higher ratios approximately of 0.33.

Blood and plasmatic Se concentrations exhibited a moderate positive correlation (r = 0.53, p < 0.0001), consistent with previous findings of Lemire et al. (2010b; 2011). Achouba et al. (2016) and Hansen et al. (2004) investigated Se status in Inuit populations and observed a non-linear relationship between Se levels in whole blood and plasma in contrast to our findings. This discrepancy may be attributed to the intake of different Se species with dissimilar distribution in blood components by those populations. Selenoneine has been found to be the primary Se compound present in the foods consumed by Inuits, and the main Se species in the blood cellular fraction of these individuals (Achouba et al., 2016; Hansen et al., 2004; Little et al., 2019).

Despite following all protocols for preserving selenium integrity in blood samples during collection, transport, and analysis, there remains a possibility that Se levels may have been underestimated due to losses at some stage of the process, for reason beyond our awareness, since we observed minimum Se plasmatic levels very low for a small set of individuals (1.65%), in almost all studied communities.

To assess the potential impact of these low Se values on our findings, a sensitivity analysis was conducted by excluding the subset of individuals with plasmatic Se levels under 40 µg.L⁻¹. Additionally, a robustness check was performed using Pearson correlation with a Bootstrap resampling approach and Iteratively Reweighted Least Squares (IRLS)

<figure><img src="image_6.png" /><figcaption>Fig. 3. Comparisons of Hg and Se concentrations (µg.L⁻¹) in blood and plasma (upper line), Hg and Se in urine (lower line, left panel), and Se:Hg molar ratio in urine and blood (lower line, right panel). Solid line represents the linear regression between variables. Numeric values indicate Spearman correlation coefficients.</figcaption></figure>
method. The results (Table S1) remained consistent to the previously observed correlation. In example, Se blood to plasma correlation yielding a value of r = 0.53, remained similar even after the exclusion of those individuals (r = 0.52). Moreover, the application of robust methods confirmed the reliability of our findings. Both Pearson correlation and its Bootstrap counterpart yielded consistent results (r = 0.63), while the IRLS found a slightly lower correlation (r = 0.46). Although the presence of low Se values may have some influence and should be carefully analyzed in further studies, in this case, they did not substantially alter the overall correlation's structure and strength on the chosen method.

Thus, assessment of Hg and Se levels in plasma and blood with their correlations observed between those matrices (Fig. 3 and S.4), suggests that, despite variability in concentration medians, the characteristic distribution of Hg and Se in the body has remained unchanged in this region, over the past 20 years (Lemire et al., 2010a, 2011).

While blood-to-plasma ratios are crucial parameters for understanding the bioavailability and short-term exposure of Hg and Se in the human body, blood and plasma to urine ratios are interesting to assess their metabolism and excretion after long-term exposure (Kuras et al., 2018b). In this study, relationships with urinary Se and Hg levels were evaluated in two ways: by examining the concentrations of both elements, given that they are subject to the same dilution effect in unadjusted urine samples; and by analyzing the molar ratio of the elements across different biological matrices.

Hg and Se levels were found exclusively correlated in urine (Fig. 3 and S.4, r = 0.60, p < 0.0001). Chen et al. (2006) indicated a similar association for exposed miners from Ghinzou province, China, with a strong correlation of Se and Hg concentrations (r = 0.625, p < 0.001, n = 37). It should be highlighted that since urine is largely considered a biomarker of Hg exposure, and is principally excreted as inorganic Hg, some authors suggested that this relationship may involve a possible Se-induced demethylation mechanism (Khan and Wang, 2009; Kuras et al., 2018a).

The median Se:Hg molar ratio in blood was 21, ranging from 3.5 to 1251. Deleterious effects on motor function were observed at a median blood molar ratio of 15.8 (1.7–137, n = 407) in the same population, as reported by Lemire et al. (2011). Higher and similar molar ratio val-

ues were observed in plasma and urine, with medians of 71 (9.2–1274) and 78 (5.4–1372) respectively.

Significant correlations were observed between blood and urine molar ratios (Fig. 3, lower line, r = 0.493, p < 0.001), as well as between plasma and urine (r = 0.465, p < 0.001). Our results suggest that exist a correlation between Se and Hg in blood and plasma, with those in urinary excretion, Overall, correlations obtained with ratios were stronger than those obtained with individual concentrations in plasma and blood. Further studies are necessary to precisely understand the association between molar ratios and health outcomes (Alcala-Orozco et al., 2020).

### 3.4. Effects of fish consumption frequency on Hg and Se status

The augment of fish consumption frequency resulted in increased Hg levels in blood as recently reported by Cruz et al. (2024), and this is also applied in plasma (Fig. 4, left panel). In contrast, Se levels did not show any relationship with fish consumption frequency (Fig. 4, right panel). This appears to be a singularity of this population. In the Inuit populations of northern and northeastern Canada the high frequency of marine fish consumption leads to similar trends for blood Hg, where levels found (0.2–164 µg.L⁻¹) were comparable to those determined in our study, but additionally, blood Se concentrations exhibited the same trend as Hg (achieving an higher average of 301 µg Se.L⁻¹ in blood), what could not be observed here (Parajuli et al., 2021; Ratelle et al., 2020).

Positive correlations between blood Hg and Se levels have been consistently documented in fish-eating populations, including those from the Amazon region (Hacon et al., 2020). Amazonian fish serve as the most important protein source for local riverine populations, and fishing remains one of the most significant and traditional economic activity throughout the region (Basta et al., 2021). In the current study, 7% of participants reported consuming fish once a week or less, with only 5 participants (0.45%) reporting that they never consume fish. A total of 13% indicated a fish consumption of 2 days per week, while 24% and 20% of participants consumed fish 3–4 days and 5–6 days per week respectively. The majority of participants (36%) reported consuming fish

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Mercury and selenium levels (µg.L⁻¹) in blood and plasma according to weekly fish consumption (days per week). Different letters indicate a significant difference between groups when tested in the Kruskal-Wallis test followed by the post-hoc Dunn's test (p < 0.001).</figcaption></figure>
daily, at least once a day, underlining the importance of fish as an essential element in their diet (Cruz et al., 2024).

Comparing blood Hg levels (Fig. 1) with reported fish consumption by community (Fig. S5), we found that communities with frequent fish intake, such as Maripá (where 88% individuals are consuming fish daily or at least five times a week), do not always have the highest Hg concentrations (27.7 µg.L⁻¹). Surucuá, as example, had the highest median concentration (45.5 µg.L⁻¹) with 61% individuals reporting frequent consumption. Conversely, Brasília Legal, which presented the lowest median (9.8 µg.L⁻¹) and number of frequent consumers (25%), shared low Hg levels with other communities like Suruacá (11.1 µg.L⁻¹) and Arapixuna (16.4 µg.L⁻¹), where 41% and 65% consumes fish frequently, respectively. These finding suggest that, beyond consumption frequency, the fish species consumed may also influence Hg and Se status should be considered in future studies.

Given that fish is the primary source of methylmercury exposure in humans, understanding its metabolic fate within the body is crucial. Several studies explored the hypothesis that methylmercury undergoes demethylation in humans. For instance, a significant relationship between weekly fish meal frequency and urinary Hg levels among fish consumers has been reported in Italian populations (Apostoli et al., 2002; Carta et al., 2003). Increased urinary Hg fractions were also found in children with high fish consumption (Levy et al., 2004), and a 15-fold increase in urinary levels among adults who frequently consume freshwater fish compared to infrequent consumers (Johnsson et al., 2005).

A recent short-term intervention study by Kuras et al. (2019) evaluated the effect of two weeks of daily marine fish consumption on Hg and Se levels in 67 adults individuals. Significant increases in blood Hg and plasma Se concentrations were observed after the first week of consumption, alongside a rise in urinary Se and Se-antioxidant proteins. Unlike in earlier studies, fish consumption did not affect Hg urinary levels. Interestingly, during the second week of fish consumption, Hg concentration was at its peak in blood, while Se plasma remained stable compared to the first week of consumption, which may indicate that the frequency of fish consumption can hardly impact the Hg status, but not the Se, as also observed in our study.

In a Japanese population with high fish or seafood consumption, correlations were found between whole blood Hg and plasma Se, as well as between consuming fish more than once per week and blood Hg (Ser et al., 2017). In similarity to our study, no correlation was obtained between the frequency of consumption and blood or plasma Se. In this study, authors suggests that Hg exposure may interfere with Se metabolism and excretion, indicating a possible threshold of methylmercury above which Se metabolism and/or selenoproteins function might be altered.

Regarding the lack of correlation between fish consumption frequency and Se levels, and the presented literature data, we propose two hypotheses. First, although fish are an important source of Se for the Amazonian riverside population (Lemire et al., 2010a; Lino et al., 2018), another significant source of Se, like Brazil nuts (Lemire et al., 2010a) potentially impacts Se status of studied population. Additionally, a regulatory mechanism, might maintain constant Se levels regardless of the fish consumption frequency. Considering Se potential antagonistic effect against Hg toxicity, further research is essential to investigate these mechanisms at the molecular level. Elucidating how Se interacts with Hg could provide valuable insights into the protective roles of selenoproteins and inform strategies to mitigate Hg exposure and its adverse health effects in vulnerable populations.

## 4. Conclusions

This study provides the first comprehensive evaluation of Hg and Se levels across blood, plasma, and urine in Amazonian riverine populations, describing intraindividual levels, ratios as well as possible inter-

actions of these elements. Positive correlations were found for Hg concentrations between biological matrices and with fish consumption frequency. In contrast, Se levels exhibited remarkable homogeneity across different communities and showed no correlations with the frequency of fish consumption. To improve the knowledge on this field, further research should consider the assessment of Hg and Se status in this population employing Se and Hg speciation and omics strategies, such as proteomics and metabolomics. It may elucidate the interaction between Hg and Se species, and distinguish the role of biomolecules that are effectively involved in the genesis of Hg toxic effects, from those that bind Hg but do not affect the metabolic functions.

### CRediT authorship contribution statement

**Gabriel Neves Cezarette:** Writing – original draft, Methodology, Formal analysis, Conceptualization. **Juliette Esplugas:** Investigation, Formal analysis. **Jonas Carneiro Cruz:** Investigation, Formal analysis. **Bruno Alves Rocha:** Investigation, Formal analysis. **Lucas Cassulatti dos Santos:** Investigation, Formal analysis. **Maité Bueno:** Writing – review & editing, Supervision, Investigation, Conceptualization. **Zoyne Pedrero Zayas:** Writing – review & editing, Supervision, Investigation, Funding acquisition, Conceptualization. **Fernando Barbosa:** Writing – review & editing, Supervision, Investigation, Funding acquisition, Conceptualization.

### Acknowledgments and funding

This study was made possible through the participation of all villagers and support of nurses, community health assistants and the health departments of Santarem and Aveiro. We would like to also acknowledge Maria Aparecida Buzeto, Eleni José Linjardi and João José Franco for their technical support. We extend our gratitude to FAPESP for financial support and research fellowships (FAPESP - process numbers 2018/24069-3, 20/07488-2, 2022/03476-5, 22/07979-1, 22/08135-1, 2023/12363-2 and 2023/03654-3), and to the Brazilian National Council for Scientific and Technological Development (CNPq - 406442/2022-3). The authors thank the Agence Nationale de la Recherche (SeMerAMAZON Project No. ANR-22-CE34-0020). This project also received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 101007962.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2025.144258.

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