Breastfeeding duration in months; <sup>ψ</sup> Number of children increment, compared to last period; \* Models were adjusted for income, years of education, maternal age and place of residence; 2-year and 5-year models also considered the number of children as an independent variable; \*\* *p* < 0.05.

#### **4. Discussion**

This study evaluated pre-Minamata Convention Hg levels among women of reproductive age from the Rondônia State, Brazilian Amazon, and investigated associated factors. Participants are among the population segment most sensitive to Hg toxicity. In the gestational and puerperal period, women can expose their children to Hg through placental transfer and breastfeeding [1,20]. Considering the safe reference levels of 6 <sup>μ</sup>g·g−<sup>1</sup> for women of reproductive age [1], this study points to elevated Hg levels among women in Rondônia, especially among riverine and rural communities (ranging from 1.02 to 146.80 <sup>μ</sup>g·<sup>g</sup> <sup>−</sup>1).

Many factors can influence the body's Hg burden, and some are especially relevant in this study: the global Hg deposition; Hg exposure due to gold mining activities in the Madeira River basin; and exposure to natural Hg present in the Amazonian soils, lixiviated to waterways due to unsustainable soil use practices. All these Hg sources contribute to fish contamination and consequently, the humans who eat them. There is already sufficient evidence that the fish-based diet of the inhabitants of this region is contaminated with methylmercury (MeHg). Barbosa et al. [35] compared populations in the Brazilian Amazon exposed to Hg by different routes (artisanal gold mining vs. fish consumption) and showed that, in general, hair levels were a more reliable descriptor for Hg exposure from fish (mainly MeHg) than for Hg vapor from artisanal gold mining activities.

Brazilian women, especially those from the Amazon, are exposed to high levels of Hg contamination. Hacon et al. [36] evaluated women from Alta Floresta, in the southern Amazon basin, exposed to Hg through work activities (mining) and diet (fish consumption), and found hair Hg levels ranged between 0.05 and 8.2 <sup>μ</sup>g·g<sup>−</sup>1. In urban areas of the Amazon with relatively low fish consumption, a significant correlation was found between Hg levels in mothers and newborns [27,37]. Compared to urban mothers, traditional riverine mothers had significantly higher Hg levels in hair [31,37] and milk [31], which may be explained by higher fish consumption.

Comparing this study results with previous research, similar Hg levels were found with other communities in the Amazon region [30,31,36,38–41] and in South America [42–46], all exceeding internationally accepted safety levels for total Hg in hair [1].

In this study, a difference in Hg levels was found between the analyzed groups, with the riverine and rural communities having the highest levels. The correlation analysis reinforced hair Hg as a reliable biomarker of Hg levels from fish intake as advocated by [2]. These populations with high Hg levels are characterized by remote dwellings, far from major centers, and by having fishing as an important part of their livelihood.

Alves et al. [46] and Baldewsingh et al. [47] found higher levels of Hg among rural than urban communities, as a result of less diverse diet patterns. Drouillet-Pinard et al. [48] and Sakamoto et al. [49] also found that hair Hg levels are mainly associated with fish consumption. Vejrup et al. [50] observed that about 88% of Hg concentration comes from diet, corroborating the MercuNorth study, which found high Hg levels due to elevated fish consumption [51].

The Madeira River basin communities have been strongly impacted by predatory activities related to the region's economic development for at least four decades. In addition to the unbridled search for gold, deforestation, forest burning, opening of roads, and construction of hydroelectric dams triggered intensive migrations to the region. The migration caused socioeconomic and lifestyle changes among the local population [20,52,53], resulting in cultural differences, changes in eating habits, and the search for other nutritional sources, which may partially explain the differences found in the analyzed groups. In general, the differences observed between the studied groups reflected these changes in the Amazonian populations. When analyzing the mining community of Garimpo de Bom Futuro, which receives immigrants from other regions of Brazil, one can see how much the cultural component influences the population consumption habits. In these women, the diet included few weekly meals with fish, while rural and riverine women maintain traditional habits of high fish consumption.

Elevated Hg levels among rural and riverine women appear to be almost entirely due to their diet rich in fish, probably those with higher trophic level and predatory fish, which are known to accumulate significant Hg levels in their tissues [32]. This exposure route among the study women is supported by the collected data, indicating high frequency of fish consumption.

It should be noted that in 2011, a joint committee of the Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO) stated that the health benefits associated with omega-3 fatty acids from fish outweighed the possible adverse neurological effects of ingesting Hg contained in fish [54]. Recommendations for decreased fish consumption in women of childbearing age, pregnant and lactating women are aimed to decrease risks on maternal reproduction and on infant growth and development. On the other hand, fish consumption offers benefits that may provide neuroprotection in children because it is rich in selenium and omega-3 [55,56].

In the Amazon region, fish is an important dietary item that is introduced at a very early age in the diet of the riverine group. Dorea [57] showed that the highly starchy manioc-flour diet is complemented mainly by protein provided by fish consumption. Fish are an exceptional source of nutritional and functional substances. They are a rich source of sulphur amino-acids and bioavailable iodine. These are crucial, on the one hand, to counterbalance cassava compounds that interfere in iodine uptake, and on the other, the iodine paucity in foods derived from the typically iodine-depleted soils of tropical rain forests. In addition to being a good source of selenium and other essential nutrients, Amazonian fish also enhance absorption of zinc and iron from plant foods in the human diet [57].

This study's participants reported a median breastfeeding time of six months, ranging between 0 and 24 months, and the breastfeeding duration was significantly associated with maternal Hg levels, which may have a negative impact on infants' health. On the other hand, Cunha et al. [58] demonstrate that even in the face of the transfer of Hg by breast milk, breastfeeding has well-defended benefits that offset the adverse effects of Hg. The ability of MeHg to be transferred via breastfeeding is well known. However, it is not possible to define exactly how much MeHg absorbed by the child is excreted through human milk [59]. Bakir et al. [5] and Greenwood et al. [60] found that Hg levels in lactating women were lower than in non-breastfeeding women, indicating the excretion of the metal through milk.

In contrast, Barbosa and Dórea [61] found that placental transfer has a higher rate of Hg transfer than breast milk in an Amazonian population, even when breastfeeding occurred for a long period of time. Barbosa et al. [38] also observed a decrease of up to 20% of maternal hair Hg levels from the first to third trimester of pregnancy, with a return to pre-pregnancy levels in the first trimester after delivery, reinforcing the importance of placental vertical transfer and suggesting that the Hg transfer through breast milk has less relevance.

Furthermore, a negative association (although non-significant) was observed between the number of children and mothers' Hg levels, which may indicate an effect of mercury loss from vertical transmission from mother to fetus during pregnancy. When comparing the participants' prenatal Hg levels and at six months after birth, a decrease was observed in all groups, reinforcing previous findings on placental transfer and breastfeeding. However, after 2 years and 5 years of follow-up, this pattern was not repeated in all groups. In riverine and rural communities, where there is greater fish consumption, Hg levels are much higher compared to urban and tin mining locations, since the protein sources in the diet of these populations is more diversified.

Considering the heterogeneity of the assessed communities, this study's findings on Hg levels of women from the Rondônia State can support the establishment of Hg background levels prior to the Minamata Convention. After five years of follow-up, the mean Hg level was still above the acceptable limit in an important proportion of women, with potential risk of fetal exposure if these women became pregnant. For this reason, monitoring Hg levels in women of reproductive age is of paramount importance, particularly for women who are more susceptible due to high contaminated-fish consumption.

In this sense, the successful implementation of the Minamata Convention depends on adequate scientific research, policy and decision making. In the case of Brazil, since the signature of the Convention, we saw little research and policy, and decision making strongly stimulated Hg use and emission and not the opposite. The Hg risks to human health, especially for women of childbearing age, are multifactorial, and the complexity of social and cultural aspects of the Amazonian communities must be taken into account. However, it is clear that the Bolsonaro period combined all factors that can lead to increased, rather than reduced, Hg exposure in the Amazon, in obvious and frontal opposition with the Minamata Convention's declared objectives.

This study has some limitations that need to be disclosed. Firstly, breastfeeding duration was evaluated in the 2 years follow-up considering the period since the study began, without data on the duration of breastfeeding in subsequent pregnancies. Some participants have breastfed for a long period (up to 24 months), which may have overestimated the effect of breastfeeding on mothers' Hg levels. Still, estimates of the effect of breastfeeding on Hg level changes were consistent across the different follow-up intervals (6 months, 2 years and 5 years). It is also important to highlight that some pregnant and lactating women in the Amazon region have the cultural habit of avoiding eating predatory/top-ofchain fish during the puerperium [62], which may have contributed to the Hg reduction observed. Moreover, the low variability in the number of children born in the study period, especially in the 2 years follow-up may be underestimating the effects of this variable on mothers' Hg levels. In this sense, a follow-up to evaluate this effect for a longer period is recommended. Finally, the existence of residual confounding cannot be ruled out, although the DAG-guided covariate adjustment model was consistent with the data (considering testable implications) and observed associations were consistent across the different analyses.

Lastly, the assessment of children's Hg levels and exactly how much is displaced from mothers during pregnancy and breastfeeding was not the subject of this study, but it is essential knowledge and should be addressed by future research.

#### **5. Conclusions**

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. In general, Hg levels considerably above the limits considered safe by the WHO standards were observed among the participating women, especially those from riverine and rural communities, mainly due to the high consumption of contaminated fish. Moreover, a consistent (significant) association was observed between the reduction in maternal Hg levels and the duration of breastfeeding, in addition to a negative (non-significant) association with the number of children. It is important to highlight

that mothers' Hg level is a balance between her ingestion/absorption and loss, especially through pregnancy and breastfeeding, in this case.

It is noteworthy that the Minamata Convention must be supported and enforced in Brazil, particularly in the Amazonian region. To this end, periodic management and Hg monitoring are essential, as that would help in assessing whether risk management actions are effectively translating into decreased Hg exposure. In addition, 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.

**Author Contributions:** T.C.S.B., R.C.M. and J.G.D. contributed to study design, data collection/analysis and manuscript drafting. R.J.B. contributed to study design, data analysis and manuscript drafting. M.P.L.C. contributed to data collection and manuscript drafting. F.A.D.-Q. contributed to data analysis. J.R.D.G. contributed to manuscript drafting. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by CNPq/MCT grant (project-555516/2006-7; project-575573/2008- 2) and CAPES (Finance Code 001). FADQ was granted a fellowship for research productivity from the Brazilian National Council for Scientific and Technological Development (CNPq), process number: 312656/2019-0.

**Institutional Review Board Statement:** The study was approved by the Research Ethics Committee of the Federal University of Rondônia (REC/NUSAU/UNIR, file number: 012/CEP/NUSAU).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We greatly appreciate the mothers' participation, and the support of the University of Rondônia students and staff. We are also grateful to the Rondônia State Health Secretariat staff, especially the community health professionals.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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