**Author details**

J. Filipe Monteiro Pneumology Department, Santa Maria University Hospital, Lisbon, Portugal

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**45**

45-59.

*End-of-Life Ethical Dilemmas*

Publishing 2018. P. 19-30.

[2] L T Cowley, E Young and T A Raffin, 'Care of the Dying: An Ethical and Historical Perspective.', Critical Care Medicine, 20.10 (1992), 1473- 82 <http://www.ncbi.nlm.nih.gov/ pubmed/1395671> (accessed on

[3] Prendergast TJ, Luce JM. Increasing

[4] Lesieur O, Leloup M, Gonzalez F, Mamzer M-F and EPILAT study group.

incidence of Withholding and Withdrawing of Life Support from Critically Ill. Am. J. Respir. Crit Care

Withholding or withdrawal of treatment under French rules: a study performed in 43 intensive care units. Annals of Intensive Care 20155:15 DOI:

[5] Rubenfeld GD, Crawford SW. Principles and Practice of Withdrawing Life-Sustaining Treatment in ICU. In: Managing Death in the Intensive Care Unit. J Randall Curtis and Gordon D Rubenfeld. Oxford University Press

[6] Brown Theodore. n.d. "What Is Moral Authority?" http//www.bigthink. com/articles/what-is-moral-authority/.

[7] Monteiro J. Filipe. The Philosophical Underpinnings of Dysthanasia. In: Dysthanasia: Delaying the Process of Death through Treatment Stubbornness. Cambridge Scholars Publishing 2018. P.

[8] Morin E. Enfrentar a Incerteza. In Reformar o Pensamento. A Cabeça bem Feita (Tr. La Tête bien Faite). Ed. Instituto Piaget. Lisboa, 1999. P. 61.

10.1186/s13613-015-0056

2001. p 127.

Med. 1997:155: 15-20.

**References**

23/10/2017).

*DOI: http://dx.doi.org/10.5772/intechopen.93616*

[1] Monteiro J. Filipe. Deviation from medical Procedures and Ethical Principles. In: Dysthanasia: Delaying the Process of Death through Treatment Stubbornness. Cambridge Scholars

[9] Block E. Le Principe Esperance. Paris: Gallimard Editions. 1982. P. 52-55.

[10] Ordem dos Médicos. Novo Código Deontológico. Revista Ordem dos Médicos. Ano 25; N° 97 Janeiro 2009.

[11] Doctrine of Double Effect. Stanford Encyclopedia of Philosophy. https://plato.stanford.edu/entries/

[12] Consequentialism. Stanford Encyclopedia of Philosophy. https:// plato.stanford.edu/entries/conse

[13] Zohar D, Marshall I. Spiritual Intelligence: The Ultimate Intelligence.

[14] Pope Pius XII. 1957, "Address to an International Congress of Anesthesiologists." http://www. lifeissues.net/writers/doc/doc31

[15] Gracia D. The old and the new in the doctrine of the ordinary and extraordinary means. In: Wildes KW (Eds.). Critical choice and critical care: catholic perspective on allocating resources in intensive care medicine. Kluwer Academic Publishers. Boston

[16] Pope John Paul II. "Evangelium Vitae". https://www.w2.vatican.va/.../

[17] Catechism of Catholic Church. Part 3-Section 2, Chapter 2; Article 5- 2278. www.vatican.va/archive/ccc\_css/ archive/catechism/ccc\_toc.htm

[18] Pope John Paul II. Address to the participants in the International Congress on "Life-sustaining Treatments and Vegetative state: Scientific Advances and Ethical dilemmas. March 2004. https://

john-paul-ii/...hf\_jp-ii\_spe

Bloomsbury Publishing 2000.

resuscitation.html\_

1995. P. 119-125.

P.58.

double-effect/

*End-of-Life Ethical Dilemmas DOI: http://dx.doi.org/10.5772/intechopen.93616*


*Article* **Neurological Impacts of Chronic Methylmercury Exposure in Munduruku Indigenous Adults: Somatosensory, Motor, and Cognitive Abnormalities**

**Rogério Adas Ayres de Oliveira 1 , Bruna Duarte Pinto 1, Bruno Hojo Rebouças 1, Daniel Ciampi de Andrade 1, Ana Claudia Santiago de Vasconcellos 2 and Paulo Cesar Basta 3,\***

**Citation:** Oliveira, R.A.A.d.; Pinto, B.D.; Rebouças, B.H.; Ciampi de Andrade, D.; Vasconcellos, A.C.S.d.; Basta, P.C. Neurological Impacts of Chronic Methylmercury Exposure in Munduruku Indigenous Adults: Somatosensory, Motor, and Cognitive Abnormalities. *Int. J. Environ. Res. Public Health* **2021**, *18*, 10270. https://doi.org/10.3390/ ijerph181910270

Academic Editors: Rejane C. Marques, José Garrofe Dórea and Rafael Junqueira Buralli

Received: 28 June 2021 Accepted: 23 September 2021 Published: 29 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

**Abstract:** There has been increasing evidence about mercury (Hg) contamination in traditional populations from the Amazon Basin due to illegal gold mining. The most concerning health impact is neurotoxicity caused by Hg in its organic form: methylmercury (MeHg). However, the severity and extent of the neurotoxic effects resulting from chronic environmental exposure to MeHg are still unclear. We conducted a clinical-epidemiological study to evaluate the neurological impacts of chronic MeHg exposure in Munduruku indigenous people, focusing on somatosensory, motor, and cognitive abnormalities. All participants were subjected to a systemized neurological exam protocol, including Brief Cognitive Screening Battery (BCSB), verbal fluency test, and Stick Design Test. After the examination, hair samples were collected to determine MeHg levels. Data collection took place between 29 October and 9 November 2019, in three villages (*Sawré Muybu*, *Poxo Muybu*, and *Sawré Aboy*) from *Sawré Muybu* Indigenous Land, Southwest of Pará state. One hundred and ten individuals >12 years old were included, 58 of which were men (52.7%), with an average age of 27.6 years (range from 12 to 72). Participants' median MeHg level was 7.4 μg/g (average: 8.7; S.D: 4.5; range: 2.0–22.8). In *Sawré Aboy* village, the median MeHg level was higher (12.5 μg/g) than in the others, showing a significant statistical exposure gradient (Kruskal–Wallis test with *p*-value < 0.001). Cerebellar ataxia was observed in two participants with MeHg levels of 11.68 and 15.68 μg/g. Individuals with MeHg exposure level ≥10 μg/g presented around two-fold higher chances of cognitive deficits (RP: 2.2; CI 95%: 1.13–4.26) in BCSB, and in the verbal fluency test (RP: 2.0; CI 95%: 1.18–3.35). Furthermore, adolescents of 12 to 19 years presented three-fold higher chances of verbal development deficits, according to the fluency test (RP: 3.2; CI 95%: 1.06–9.42), than individuals of 20 to 24 years. The worsened motor and cognitive functions are suggestive of neurotoxicity due to chronic MeHg exposure. In conclusion, we believe monitoring and follow-up measures are necessary for chronic mercury exposed vulnerable people, and a basic care protocol should be established for contaminated people in the Brazilian Unified Health System.

**Keywords:** mercury exposure; neurological abnormalities; indigenous people; Amazon; environmental impacts; illegal mining

#### **1. Introduction**

Methylmercury (MeHg) is a toxic organo-mercurial compound which acts systemically in the human body [1,2]. The intake dose, exposure duration, and frequency, in addition to the exposure route and age of the individual (or stage of development), are factors that determine the extent of damage caused by MeHg and which organs or systems are worst affected [3].

After the Minamata and Niigata disasters in Japan in the 1950s and 1960s, the international scientific community agreed that methylmercury's main target is the Central Nervous System (CNS) [4]. During that time, it was observed that adult individuals who regularly consumed contaminated fish presented neurological abnormalities such as paresthesia, ataxia, dysarthria, visual field alterations, and hearing impairments. In addition, these events showed that the prenatal period is the most vulnerable stage to the deleterious effects of MeHg. Many women who were exposed during pregnancy gave birth to children with severe congenital malformations and significant cognitive impairments [5–7]. Years later, the clinical manifestation caused by the exposure to high MeHg levels during adult life or pregnancy became known as Minamata Disease [8,9].

The MeHg intoxication episode in Iraq, in the early 1970s, was even more devastating than Japan's tragedy. Because it was an acute exposure episode, there were more than 6000 hospitalizations due to intoxication and hundreds of deaths in a short period of time [10,11]. In that time, the refinement of the spectrometry of atomic absorption technique for analyzing Hg in human tissue made laboratorial measurements more precise. Consequently, it was possible to establish a consistent relationship between the signs and symptoms presented by the exposed individuals and mercury concentration in exposure biomarkers (e.g., hair and blood) [6]. Based on scientific evidence, the World Health Organization established [4] that the neurological effects of MeHg exposure can be found in 5% of adult individuals who ingest 3 to 7 μg daily of MeHg per kilogram of body weight (kg bw). This daily intake dose corresponds to levels of 200 μg/L in blood or 50 μg/g in hair. However, these indexes are constantly revised, and some are updated as a result of new technologies and current research carried out in different parts of the world.

Since the 1990s, research conducted in the Amazon region identified adult individuals with neurological abnormalities probably associated with the consumption of contaminated fish. In those individuals, levels of methylmercury in hair samples varied between 10 and 20 μg/g [12–17]. Similar findings were reported in riparian populations in Bolivia and Suriname [18,19].

It is worth noting that the peculiar characteristics of the Amazon's ecosystem and the social dynamics of its traditional populations may be responsible for the different thresholds for the onset of neurological effects. In that region, the majority of the mercury human exposure is due to artisanal small-scale gold mining (ASGM) [20]. ASGM causes the liberation of large quantities of Hg in the ecosystem and, at the same time, mobilizes natural Hg present in the soil. In addition to ASGM, the construction of hydroelectric plants and dams, deforestation, and fires also alter the Hg cycle in the region, increasing the risk of human contamination [20–24]. Moreover, the traditional people living there are considered to be among the largest consumers of fish in the world [25–27].

In the aquatic environment, the Hg released by the ASGM suffers methylation, mediated by microorganisms, transforming it into methylmercury (MeHg). Most of the toxicity attributed to MeHg is due to its biomagnification capacity in aquatic trophic chains and to the ability to overcome blood–brain and placenta barriers. Because fish consumption is the main protein source in many traditional populations in the Amazon, chronic exposure to high levels of mercury can cause various negative consequences and create health problems in local populations [20,28–32]. After intake of contaminated fish, almost all MeHg is quickly absorbed by the gastrointestinal tract. The absorbed MeHg forms a conjugate with the cysteine molecules present in the blood and, afterward, is distributed to virtually all human body tissue. When the MeHg reaches the CNS, it undergoes oxidation and tends to accumulate, leading to potentially irreversible cerebral tissue damage [33].

Experimental evidence shows that MeHg neurotoxicity is marked by an increase in oxidative stress, by the presence of mitochondrial lesions, by the release of excitatory amino acids, and by proteomic expression alterations, which can lead to cellular dysfunction and death [34–36]. Furthermore, the chronic exposure to high levels of MeHg has been associated with memory and learning deficits, motor function abnormalities, hearing loss, and a reduced visual field [3,20,37–42].

In the Amazon region, few clinical studies have correlated MeHg exposure levels with neurological symptoms [12–14,16,17]. Nevertheless, none of these studies included indigenous populations. Therefore, there is insufficient evidence about neurotoxicity concerning indigenous populations chronically exposed to MeHg in the Amazon Basin.

With the lack of evidence on this topic in mind, this study aimed to describe neurological clinical manifestations, focusing on somatosensory, motor, and cognitive abnormalities, in adults indigenous from three Munduruku villages in the Amazon Basin. The ultimate goal was to explore possible associations between neurological abnormalities with chronic methylmercury exposure.

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

#### *2.1. Study Design and Field Work*

We conducted a cross-sectional clinical-epidemiological study including indigenous people older than 12 years old living in three villages: *Sawré Muybu* (SM), *Poxo Muybu* (PM), and *Sawré Aboy* (SA), in the *Sawré Muybu* Indigenous Land (IL), located at Middle-Tapajós River, in the southwest region of Pará state, Brazilian Amazon.

Data collection occurred between 29 October and 9 November 2019, according to a multidisciplinary and inter-institutional study developed to clarify the consequences and assess the impacts of illegal mining in the *Sawré Muybu* IL. For additional information, see Basta et al. [43].

#### *2.2. Evaluated Population*

We performed a census in the three villages mentioned above, and all indigenous individuals aged 12 or older were invited to participate in the study. Therefore, probabilistic sampling methods were not used to select participants. A 15-year-old man from *Sawré Muybu* village, who had cerebral palsy, was excluded from the study.

#### Fish Consumption Estimates

Like the majority of the traditional groups living in the Amazon Basin, Munduruku people consume large quantities of fish. Recently, Vasconcellos et al. [44] carried out an investigation to estimate the fish consumption of the Munduruku indigenous people living in the study area and compared their findings with the acceptable levels proposed by the FAO/WHO [45] and by the U.S.EPA [46].

During one week, the authors collected 88 fish specimens representing 17 species and four trophic levels to analyze the total mercury (THg) concentration in muscle tissue. They also developed an empirical method to estimate fish consumption from catch effort. The average daily fish consumption estimated for adult men was 216.75 g, and for women of childbearing age was 168.58 g. Considering that about 90–95% of total mercury species detected in fish samples is in the form of MeHg [47–49], we assume that all of the Hg detected in the fish samples is compounded by MeHg.

The average MeHg levels in samples of non-piscivorous fish (n = 57) was 0.10 μg/g (SD = 0.09) and the average for piscivorous fish (n = 31) was 0.44 μg/g (SD = 0.34). The highest level of methylmercury in muscle tissue (1.95 μg/g) was observed in the *Serrasalmus rhombeus* (also known as *piranha preta* in Portuguese), a fish not only located at the top of the food chain, but also highly appreciated and regularly consumed by the Munduruku [44].

Moreover, the authors devised four scenarios to estimate the MeHg exposure, considering the fish consumption from catch effort: (i) in the rainy season; (ii) in the dry season; (iii) based on the weighted average of medium mercury levels detected in piscivorous and non-piscivorous species; and (iv) based on the 95th percentile of mercury concentrations in piscivorous and non-piscivorous species (1.42 and 0.29 μg/g, respectively).

According to the authors, scenario (iii) most closely matches the local reality, in which the estimates of MeHg ingestion for Munduruku adult men ranged from 1.84- to 8.28-fold above the safe daily methylmercury intake recommended by FAO/WHO [45] and U.S.EPA [46], respectively. The estimates ranged from 3.17- to 7.29-fold above the FAO/WHO [45] and U.S.EPA [46], respectively, for women of childbearing age.

Summarizing, the studied Munduruku villages are at severe risk of harm due to the ingestion of MeHg-contaminated fish.

#### *2.3. Neurological Examination*

All participants were submitted to a systemized neurological examination protocol, specifically developed for this research, to make the evaluation feasible in the adverse conditions of the fieldwork in the villages. The evaluations were conducted by neurologists (BDP, BHR, and RAAO) familiar with clinical neurological semiology and duly trained to apply the protocol and use the examining instruments.

The explanations about the clinical procedures for the neurological exam were verbally presented to the participants in Brazilian Portuguese. When necessary, for indigenous individuals that had difficulties with Portuguese, an indigenous translator (indigenous health agents, teachers, or chiefs) from the village would assist.

#### 2.3.1. Static Balance and Walking Evaluation

The evaluation of static balance and walking was performed by visual inspection. Participants were asked to remain in an orthostatic position to perform the Romberg test, with eyes open and closed, sequentially. Then, participants were asked to walk towards the examiner.

#### 2.3.2. Cranial Nerve Examination

The following parameters were evaluated: visual campimetry by confrontation, pupil reflex and external ocular motricity, facial motricity and symmetry, facial sensibility, palate elevation, gag reflex and tongue motricity.

#### 2.3.3. Sensory Testing

The following instruments were used to evaluate the respective somatosensory modalities:

The sensitivity was tested in upper and lower limbs at three distinct points for each sensory modality, in the proximal and distal segments. The sensitivity was classified as normal (0) or abnormal (1) depending on the abnormality occurrence in at least one parameter.

For the clinical diagnosis of distal symmetric polyneuropathy, criteria from the American Academy of Neurology were used, which consider the occurrence of distal sensory deficits, in addition to the clinical signs of neuropathy, defined as the presence of abnormalities in at least one somatic sensory domain and/or alterations in the ankle jerk reflex [51].

#### 2.3.4. Motor Function

Muscular strength was evaluated through maneuvers against resistance in all four limbs, on proximal and distal segments. Muscular hypertonia and bradykinesia were evaluated through passive mobilization of the four members, through finger tapping maneuvers, facial expression mimicking, and spontaneous mobilization. Coordination was evaluated through finger to nose, heel to knee, and diadochokinesis (ability to make alternating quick movements) tests.  Toe amyotrophy was investigated through inspection.

#### 2.3.5. Cognitive Evaluation

All individuals were submitted to a cognitive evaluation by the authors (BDP, BHR and RAAO), immediately after the neurological exam, as part of the evaluation protocol.

An indigenous translator from the village was present in the interviews to help with the communication with indigenous individuals who had difficulties with Portuguese.

The Brief Cognitive Screening Battery (BCSB) [53], the verbal fluency test in the animal category [54], and the stick design test [55] were used as instruments of cognitive evaluation. These instruments were selected given their appropriateness for populations with low levels of education and for the easy application in adverse conditions in the field.

#### Brief Cognitive Screening Battery (BCSB)

The BCSB is an instrument developed by the Cognitive and Behavior Neurology Group of the *Hospital das Clínicas* of the Medical School of the *Universidade de São Paulo* [53], which entails naming and remembering 10 drawings of simple and known objects in black and white presented in a single picture. This instrument evaluates, in sequence, the following domains: (a) visual perception and naming, (b) incidental memory, (c) immediate memory, (d) learning, (e) verbal fluency test (described below), (f) 5 min belated memory, and g) recognition.

The following cutoffs were used in the tests to evaluate the domains' normality: immediate memory (≥5), learning (≥7), verbal fluency (≥9), and delayed recall (≥6). Alteration in at least one of these domains was considered a criterion for abnormality for the BCSB, finally classified as: (0) normal or (1) abnormal.

#### Verbal Fluency Test in the Animal Category

To evaluate verbal fluency, participants were asked to name as many animals as they could in one minute. The individuals received the following instructions: "*you must say the animal names you remember, as quickly as possible. Any animal will do, four-legged, fish, birds, the more you say, the better*". The cutoff for verbal fluency test normality was ≥9. This test was applied as part of the BCSB, as an interference factor.

#### Stick Design Test

To evaluate logical thinking, participants were asked to reproduce geometric shapes, presented on a sheet of paper one after the other, with matches. The number of sides, the image and stick orientation in each of the four images were criteria considered for the test score, which varies from zero to 12. Scores ≥10 were considered the cutoff for normality and classified as normal (0) or abnormal (1).

#### *2.4. MeHg Exposure Biomarker*

According to the guidance published by the World Health Organization [56], and described by other authors [57,58], hair samples are considered the best biomarker of the MeHg human exposure, because almost all Hg detected in this kind of biological matrix is in the MeHg form. In addition, because the main Hg exposure route observed in the studied population is the consumption of Hg-contaminated fish, and almost all Hg present in the fish muscle is in the MeHg form, hair samples were used as its exposure biomarker [47–49]. Therefore, we assume for this study that all Hg present in hair samples is MeHg.

The hair's samples were collected from the occipital region, with the aid of stainlesssteel dissection scissors, from all participants. The samples were stored in individually identified paper envelopes and sent for total mercury concentration (THg) analysis in the Toxicology Laboratory in the Environmental Section of the *Instituto Evandro Chagas* (IEC).

In the laboratory, before starting the analyses, hair samples were repeatedly washed with Extran detergent (Merck KGaA, Darmstadt, Germany) to remove any exogenous contamination. After drying, the samples were finely homogenized in glass flasks before weighing. This methodology comprises chemical opening, wet digestion, and subsequent

reduction with SnCl2 to quantify total Hg in a Cold Vapor Atomic Absorption Spectrometer (CVAAS).

The protocols for Quality Assurance (QA)/Quality Control (QC) included the following parameters: (i) a method blank; (ii) a 6-point calibration curve (concentration ranging from 0.4 to 4 ng/g); (iii) the Human Hair Certified Reference Material (IAEA-86), whose average recovery rate was 101% (n = 8, recovery ranging from 83.4 to 106.6%) from the International Atomic Energy Agency; and (iv) the relative standard deviation (RSD) of 8.32%. Sample replicates (n = 10), whose RSD was 2.49%, were also randomly selected. The detection and quantification limits (LOD/LOQ) obtained were 0.0083 ng/mg and 0.027 ng/mg, respectively.

For further detail on hair sample analysis, see Basta et al. [43].

#### *2.5. Statistical Analysis*

A descriptive analysis of the participants was undertaken according to sociodemographic variables: sex (female / male), age (12 to 19; 20 to 24, 25 to 29, and ≥30 years old) and village of residence (*Sawré Muybu*, *Poxo Muybu*, and *Sawré Aboy*), in contrast to the evaluated neurological parameters.

To estimate the prevalence of exposure, in the numerator we included participants older than 12 who presented with methylmercury levels ≥10.0 μg/g. In the denominator, the population sampled for the study in the same age group was considered. The prevalence was presented for the three villages under study: *Sawré Muybu*, *Poxo Muybu*, and *Sawré Aboy*.

The main neurological abnormalities detected in the clinical exam were compared between two groups, according to the participants' level of methylmercury exposure: ≥10.0 vs. <10.0 μg/g.

Because mercury levels do not show a normal distribution, the Kruskal–Wallis nonparametric test was used to evaluate differences in methylmercury levels in all comparisons that showed measures of central tendency (medians). The difference in proportion between variables was evaluated using Pearson's chi-squared test.

We used a Poisson regression model with robust variance to estimate the association between alterations observed in the BCSB and the verbal fluency test in the animal category with independent variables: (i) methylmercury exposure level (<10.0 vs. ≥10.0 μg/g); age (12 to 19; 20 to 24, 25 to 29, and ≥30 years old); and (ii) sex (female vs. male). Prevalence Ratio (PR), with the respective confidence interval of 95%, was used as an association measure.

Variables with a *p*-value < 0.20 in the simple analysis were selected and included in the multiple model. Variables that presented significance levels of 5% (*p* < 0.05) remained in the final model. The data was analyzed with the statistical software SPSS version 9.0 (Chicago, IL, USA).

#### **3. Results**

During the field work, 110 indigenous individuals were evaluated, 50 of whom were from the *Sawré Muybu* (SM) village, 37 from the *Poxo Muybu* (PM) village, and 23 from the *Sawré Aboy* (SA) village. Fifty-eight were men (52.7%) and 52 were women (47.3%). The participants age ranged from 12 to 72 years (average: 27.6 years; standard deviation: 13.7). The participants' distribution, according to age group, was heterogenous in the studied villages. There was a predominance of adolescents (12 to 19 years) in PM and SA villages, with 16 (43.2%) and 11 (47.8%) participants, respectively, compared to nine (18.0%) participants in the SM village. In contrast, in *Sawré Muybu* there was the highest concentration of young adults between 25 and 29 years of age (26.0%) (*p* = 0.032) (Table 1).

**Table 1.** Sociodemographic variables, hair-methylmercury level groups, somatosensory and motor functions, and cognitive alterations, according to the village of residence. *Sawré Muybu* Indigenous Land, Pará state, Brazilian Amazon, 2019.

Total 50 37 23 110

#### *3.1. Overall Data by Village*

No participant showed static balance, walking, or cranial nerve function alterations. There were significant somatosensory alterations in the SM and SA villages in contrast to PM, represented here by the distal pinprick perception (*p* = 0.012), distal thermal sensitivity (*p* = 0.004), hallux or thumb vibration sensitivity (pallesthesia) (*p* = 0.042), and feet mechanical detection threshold (*p* = −0.001). Clinical signs of polyneuropathy were detected in 21 (42.0%) participants from *Sawré Muybu* village and 10 (43.5%) participants from *Sawré Aboy* village, revealing a significant superior prevalence compared to *Poxo Muybu* village, where only 4 (10.8%) participants were affected (*p* = 0.003) (Table 1).

Two (1.9%) indigenous people presented with alterations in cerebellar motor alterations ataxy, one from SM and one from SA villages, and three (2.8%) presented with bradykinesia, two of which were from SA and one from SM village, with no significant differences in the distribution between the villages. No signs of hypertonia or rigidity were detected in participants (Table 1).

Five (4.5%) participants were diagnosed with toe amyotrophy, three of which were from PM village and two from SM village (*p*-value = 0.331). There were no recorded cases of amyotrophy in SA.

The absence of ankle jerk reflex was detected in 10 (20%) and six (26.1%) participants from SM and SA village, respectively, and was more prevalent than in PM village, where it was observed in only one (2.7%) individual (*p* = 0.025) (Table 1).

Regarding cognitive manifestations, there were BCSB abnormalities in 39.1% (n = 9) of participants from SA village, whereas in SM and PM the alterations affected 17.6% (n = 9) and 21.6% (n = 8) of participants, respectively (*p* = 0.089) (Table 1).

Impairments in the verbal fluency test were detected in 21.6% (n = 6) of participants from SA village. In contrast, in PM and SM, the verbal fluency affected 10.8% (n = 4) and 6.0% (n = 3) of participants, respectively (*p* = 0.046) (Table 1).

Alterations in the stick design test were observed in only one (2.0%) individual from SM village and in one (4.3%) individual from SA (*p* = 0.466).

#### *3.2. Neurological Abnormalities According to MeHg Exposure Levels*

In general, the median MeHg level in participants was 7.4 μg/g (average: 8.7; standard deviation: 4.5; range from 2.0 to 22.8). In *Poxo Muybu* village, the median MeHg level in participants was 7.3 μg/g (average: 7.3; standard deviation: 2.3; range from 2.3 to 12.9). In *Sawré Muybu* village, the median MeHg level in participants was 6.5 μg/g (average: 7.4; standard deviation: 4.2; range from 2.0 to 22.1). Finally, in *Sawré Aboy* village, the median MeHg level in participants was 12.5 μg/g (average: 13.5; standard deviation: 4.6; range from 4.8 to 22.8), showing a significant statistical exposure gradient (Kruskal–Wallis Test with *p*-value < 0.001).

The MeHg exposure prevalence over 10 μg/g also varied according to the studied villages, 78.3% of which from SA, 22.4% from SM, and 10.8% from PM (*p*-value = 0.001) (Table 1). However, there were no significant differences in MeHg exposure considering age group and sex (Table 2).

#### 3.2.1. Somatic Sensitivity (Somatosensory)

Alterations in distal pinprick perception were found in 14.5 and 12.1% of participants in the exposure groups of <10 and ≥10 μg/g MeHg, respectively (*p*-value = 0.743) (Table 2).

Distal thermal sensitivity alterations were observed in 14.5% of participants of exposure group of <10 μg/g, in comparison to 21.2% in the group of ≥10 μg/g MeHg (*p*-value = 0.384) (Table 2).

Hallux or thumb vibration sensitivity alterations were seen in 6.6% of participants in the exposure group of <10 μg/g, in contrast to 15.2% in the exposure group of ≥10 μg/g MeHg (*p* = 0.154) (Table 2).

**Table 2.** Sociodemographic variables, somatosensory and motor functions, and cognition signs, according to hair-MeHg levels (<10 vs. ≥10 μg/g), *Sawré Muybu* Indigenous Land, Pará state, Brazilian Amazon, 2019.

Feet mechanical detection threshold (hypoesthesia) alterations were detected in 18.4% of participants in the exposure group of <10 μg/g, in contrast to 33.3% in the exposure group of ≥10 μg/g MeHg (*p* = 0.089) (Table 2).

Finally, the clinical signs of polyneuropathy, indicative of distal symmetric polyneuropathy, were similar in the groups, and were reported in 30.3% of indigenous individuals with the exposure <10 μg/g and in 36.4% of those with exposure ≥10 μg/g MeHg (*p* = 0.531) (Table 2).

#### 3.2.2. Motor Function

Toe amyotrophy was recorded in five individuals (6.6%) in SM village.

The occurrence of bradykinesia was detected in three indigenous individuals: an individual with a methylmercury exposure level of 9.08 μg/g, and in two other participants with exposure levels of 11.68 and 20.18 μg/g (*p* = 0.164) (Table 2).

Cerebellar motor incoordination (ataxia) was observed in two participants with exposure levels of 11.68 and 15.86 μg/g MeHg (*p* = 0.030).

#### 3.2.3. Cognitive Alterations

According to the BCSB, cognitive alterations were observed in 17.1% of indigenous individuals with MeHg exposure levels of <10 μg/g, in contrast with 36.4% of those with an exposure of ≥10 μg/g (*p* = 0.028) (Table 2).

Moreover, the participants with MeHg exposure levels of <10 μg/g showed 7.9% of alteration in the verbal fluency test in the animal category, whereas 21.2% of participants with MeHg exposure levels of ≥10 μg/g presented alterations (*p* = 0.049) (Table 2).

Two individuals (1.8% of the total) presented with alterations in the stick design test, one with a MeHg exposure level of 9.67 μg/g, and the other with 22.75 μg/g (Table 2). It is worth noting that the recorded exposure level in the latter was the highest in the studied population.

3.2.4. Main Findings Considering the Neurological Abnormalities, According to MeHg Exposure Levels

Table 3 summarizes the main results, highlighting neurological clinical variables that presented an association with hair-MeHg levels (≥10 μg/g). Considering the presence of somatosensory signs, abnormalities in the feet mechanical detection threshold affected 33.3% of the participants with MeHg levels above 10 μg/g, in contrast to 18.4% of the participants with MeHg levels below 10 μg/g. Regarding motor function, only two cases of ataxia were detected. However, both were observed in participants with MeHg levels above 10 μg/g.

Regarding the cognition abilities, 36.4% of the participants with MeHg levels above 10 μg/g presented abnormalities in the Brief Cognitive Screening Battery (BCSB). In contrast, 17.1% of the participants with MeHg levels below 10 μg/g showed abnormalities in the BCSB. Finally, concerning the verbal fluency test, 21.2% of the participants with MeHg levels above 10 μg/g presented abnormalities, in contrast to 7.9% of the participants with MeHg levels below 10 μg/g (Table 3).

#### 3.2.5. Poisson's Regression Model Findings, Considering Cognition Abilities of the Participants

Poisson's regression modeling revealed that participants with MeHg exposure levels ≥10 μg/g showed approximately two-fold higher chances of suffering from cognitive deficits in the BCSB (PR: 2.2; CI 95%: 1.13–4.26) and the verbal fluency test (PR: 2.0; CI 95%: 1.18–3.35). Furthermore, the final model showed that 12 to 19 year old adolescents presented with around a three-fold chance of developing deficits in the verbal fluency test (PR: 3.2; CI 95%: 1.06–9.42), compared to 20 to 24 year old young adults, even controlling for the effect of mercury exposure level and sex (Table 4).

**Table 3.** Summary of the main results highlighting clinical variables that presented an association (*p*-value < 0.10) with hair-MeHg levels (≥10 μg/g), *Sawré Muybu* Indigenous Land, Pará state, Brazilian Amazon, 2019.

> **Table 4.** Logistic regression model (crude and adjusted) of prevalence ratios (PR) in the Brief Cognitive Screening Battery (BCSB) and verbal fluency test, *Sawré Muybu* Indigenous Land, Pará state, Brazilian Amazon, 2019.

#### **4. Discussion**

This exploratory clinical-epidemiological study was the first to produce evidence about the neurotoxic effects of consuming contaminated fish by MeHg in adults from the Munduruku indigenous population. The findings were produced with a comparative analysis of systemized neurological evaluation data in contrast to hair-MeHg concentration, used as a biomarker of chronic mercury exposure.

There is strong evidence substantiating MeHg neurotoxicity in experimental models and humans [33–35,42]. The neurological manifestations, classically described after the acute intoxication by MeHg, in Minamata Bay, Japan, in 1953, are characterized by somatosensory dysfunctions, ataxia, incoordination, tremors, and visual and neuropsychiatric symptoms [8,9]. A study conducted by Yorifuji et al. [59] also confirms that the neurological manifestations are due to the methylmercury contamination and are irrefutable evidence that the toxicity produced by this chemical element lasts for decades.

However, in the context of chronic environmental exposure, there is still no solid causal connection between the neurological dysfunction and toxicity due to methylmercury, as highlighted by Puty et al. [60], who conducted a systematic review of this theme. Despite the relevance of this systematic review in consolidating the scientific knowledge, it is essential to emphasize that this study included only six papers, and most of these (i.e., four) do not provide data on mercury concentration in exposure biomarkers. Consequently, we can conclude that almost all selected research in that review presumptively reported human mercury exposure.

Nonetheless, when we focus only on research conducted in the Amazon region, we observe that most of these studies indicate that exposure to MeHg may be responsible for neurological alterations in adult individuals [13–19]. In this region, the mercury levels detected in exposure biomarkers can be up to 10-fold higher than the limits recommended by health agencies [45,46]. The main neurological alterations include cognitive, visual, motor, somatosensory, and behavioral changes [13–19,61–68].

The impact of gold mining on human mercury exposure has been described by some studies that compared exposed populations to control groups (for example, populations living in areas with no previous history of mining activities) [16,69–72]. These studies indicate that hair mercury levels detected in areas impacted by mining activities are considerably higher than those observed in control groups. Even considering the natural mercury available in the Amazon soil, these findings reinforce the hypothesis that the region's primary source of human mercury contamination is mining activities.

In the present study, somatosensory, motor, and cognitive abnormalities were researched in indigenous adults from the Munduruku people who live in three villages, located in the region of Middle-Tapajos River. The participants with higher mercury exposure levels (≥10 μg/g MeHg) presented the highest prevalence of cognitive impairment, according to the BCSB and the verbal fluency test results, and cerebellar incoordination, both indicating CNS dysfunctions. Distal somatosensory deficits and verbal fluency reduction were also reported. These alterations were more frequent in *Sawré Aboy* village, where there was the highest mercury exposure, compared to *Sawré Muybu* and *Poxo Muybu* villages. Again, this finding strengthens the hypothesis that the primary source of mercury exposure in the studied villages is mining activities.

#### *4.1. Somatosensory Abnormalities*

In the group with the highest MeHg exposure (≥10 μg/g), there was also a higher prevalence of abnormalities in the feet mechanical detection threshold, in comparison to the group with lower exposure (33.3% vs. 18.4%, *p* = 0.089). There was a slim predominance of temperature and vibrant hypoesthesia in participants with methylmercury exposure higher than 10 μg/g (Table 2), however, there was no statistical significance. Even considering different methodological approaches, our findings revealed the highest somatosensory abnormalities in comparison to the study conducted by Khoury et al. [63,64], in which there was a decrease in hypoesthesia, tactile sensitivity, and pain, in 11.5% (n = 9) of riparian residents in Barreiras and 13.3% (n = 4) of residents in São Luiz do Tapajós, areas in the Pará State, in the Amazon Basin, equally affected by mining activities. In turn, the somatosensory alterations reported here were higher than those observed in a region known as *Furo do Maracujá* (n = 1; 2.0%), an area considered free of mercury exposure, and therefore classified as a control area by the authors [63,64]. However, due to the small number of participants, the differences highlighted in the study were not statistically significant.

The comparison by village revealed a significantly lower prevalence of somatosensory abnormalities in *Poxo Muybu* village, in contrast to *Sawré Aboy* and *Sawré Muybu* villages (Table 1). The difference in occurrence of alterations was revealed in all sensory modalities investigated, either in the tactile superficial sensory (*p* = 0.001), temperature sensitivity (*p* = 0.004) and pain sensitivity (nociceptive) (*p* = 0.012), and hallux and thumb deep sensitivity (vibration) (*p* = 0.042). In the same manner, *Poxo Muybu* was the village where lower frequencies were reported of ankle jerk reflex alterations (*p* = 0.025) and clinical signs of polyneuropathy (*p* = 0.003).

These findings provide evidence of the toxic effects of methylmercury, because *Poxo Muybu* village recorded the lowest proportion of participants with exposure levels above 10 μg/g MeHg (10.8%), whereas in *Sawré Muybu* and *Sawré Aboy* villages the exposure prevalence was approximately two to eight times higher, reaching 22.4% and 78.3% of participants, respectively.

However, although there is a tendency of major abnormalities in distal thermal sensory, deep vibration, and tactile superficial sensitivity, the magnitude of the somatosensory alterations observed in the comparative analysis by village was not confirmed, according to the methylmercury exposure groups. In part, this contradiction can be explained by the reduced size of our sample. However, it is important to note that the average methylmercury levels in all participants were high (average: 8.7 μg/g; range from 2.0 to 22.8), which makes it difficult for more robust analysis, because we did not have a control group, without mercury exposure, for comparison.

It is important to note that distal somatosensory alterations and clinical signs of polyneuropathy are indicative of peripheral nervous system (PNS) dysfunctions that can be induced by several clinical conditions in addition to mercury contamination. The presence of chronic alcoholism, diabetes mellitus, and sexually transmittable diseases was actively researched in our study population, via interview, blood glucose level testing, or rapid HIV, syphilis, and viral hepatitis testing, with no positive cases identified (for further detail see Basta et al. [43]). Nutritional deficits, especially B12 deficiency, are also potential causes of polyneuropathy and somatosensory abnormalities. However, these nutritional deficiencies were not researched and may add a degree of bias to our findings. It must also be noted that the use of a hair sample as a biomarker for mercury exposure may not be adequate, especially in the adult population, for detecting the neuropathy cases [33]. It is possible that the clinical signs of neuropathy detected here may have begun prior to our research for reasons not evaluated.

However, it cannot by overlooked that the clinical signs of polyneuropathy, diagnosed in approximately one-third of all participants, were more frequent in *Sawré Muybu* and *Sawré Aboy* villages, where the highest levels of MeHg exposure were detected.

Similar to the results reported in this study, Takaoka et al. [73] compared an indigenous population in Canada who lived in a region affected by chloralkali industrial waste, with two populations in Japan, one exposed to mercury in Minamata Bay and the other in a nonaffected area (control). The authors reported complaints and neurological abnormalities as more prevalent. In addition, somatosensory signs were more evident in the Canadian indigenous group and in the Japanese group exposed to mercury compared to the control group.

In another study conducted in Japan, Takaoka et al. [38] presented a detailed description of somatosensory symptoms that included perioral numbness, hands and feet numbness, cramps, difficulty in hearing, smelling, and tasting, difficulty in buttoning shirts, and difficulty executing fine tasks with fingers. The authors reported that the symptoms were more frequently observed in individuals who ate fish with Hg concentrations higher than levels considered to be safe. They concluded that their findings indicated that the ef-

fects of Minamata disease were observed almost 50 years after Chisso Corporation stopped polluting water with methylmercury, in 1968.

In our study, the presence of distal hypoesthesia and clinical signs of polyneuropathy were respectively observed in 33.3% and 36.4% in the group with the highest mercury exposure (≥10 μg/g). These findings were similar to those reported by Yorifuri et al. [59], who showed somatosensory abnormalities in 25 and 58% of participants with mercury exposure higher than 10 μg/g in the contaminated areas of Minamata and Goshonoura, Japan, respectively.

In the Amazon region, Khoury et al. [63,64] described somatosensory complaints of numbness of the extremities in 11.5 and 13.3% of participants of riparian communities in two areas of the state of Pará, which are under the influence of mining and, therefore, exposed to mercury.

Due to communication difficulties arising because Munduruku people express themselves in their native language and do not speak fluent Portuguese, in this study, we exclusively evaluated clinical signs through a patterned neurological exam. In contrast to other authors [8,63,64,73], we opted not to include any complaints or symptoms reported by participants in the collected data.

#### *4.2. Motor Functions Abnormalities*

Cerebellar signs were only detected in two indigenous individuals, who presented with methylmercury exposure higher than 10 μg/g. Similarly, Takaoka et al. [39] described alterations in the finger to nose test, which indicates cerebellar dysfunction, in 21 and 47% of two groups with high mercury exposure, in Japan. Cerebellar symptomatology was also described in other empirical studies [15,39,59] and in revision papers [8,60,74]. In turn, Khoury et al. [63,64] described altered walking in participants with high mercury exposure levels compared to the control group. However, there was no mention of cerebellar signs.

Bradykinesia or slowness of movement were identified in three indigenous individuals in our study. They did not present rigidity or tremors. Bradykinesia, usually found in rigid-hypokinetic or parkinsonian syndromes, is not part of the classical manifestation of acute mercury intoxication, as described in Japan [8,59].

Nonetheless, experimental evidence associates mercury toxicity with dopaminergic depletion in the CNS [40] and establishes a possible correlation with parkinsonian and neurodegenerative diseases [75], even considering the latency period after mercury exposure [76]. In this sense, we propose two possibilities: (i) bradykinesia is an isolated symptom, or (ii) it may be a part of an incomplete parkinsonian syndrome falling within the spectrum of neurological manifestations of mercury contamination in this indigenous population from the Amazon. To clarify this question, we suggest a longitudinal evaluation in those populations at risk of chronic methylmercury exposure, with a priority for those cases with bradykinesia and other neurological signs and symptoms. In this manner, we believe that it will be possible to broaden the knowledge on this subject, which is surrounded by controversy and uncertainty.

#### *4.3. Cognitive Abnormalities*

Cognitive evaluation in individuals with a low education level is a challenging task, especially in indigenous populations who live in diverse sociocultural and environmental contexts. Language and communication barriers, in addition to the lack of appropriate evaluation instruments, were some of the difficulties experienced by our team. When choosing the evaluation instruments, we selected those that allowed us to overcome communication barriers, had references to the local population's everyday elements, and were easily applicable in the context of the fieldwork in the villages. The BCSB domains used in this study allowed the evaluation of immediate memory, verbal fluency, and belated memory. It is essential to consider years of schooling, a factor significantly related to some cognitive tests [77] but not related to the ability to solve everyday problems, as highlighted in our evaluation.

Despite the limitations, the proportion of higher impairment rates in the BCSB in participants with high methylmercury exposure levels (≥10 μg/g) was more than two-fold (36.4%) those observed (17.1%) in participants with lower exposure levels (<10 μg/g) (*p* = 0.028). Similarly, Yorifuji et al. [59], in an enquiry that analyzed the Minamata population in the early 1970s, revealed significantly more frequent intelligence limitations (RP 5.2, 95% CI 3.7–7.3) and behavior alterations (RP 4.4, 95% CI 2.9–6.7) in the group exposed to methylmercury, in contrast to the control group.

It is worth noting that the neuropsychiatric manifestations, including irritability, depression, social isolation, aggressiveness, or even psychotic cases ("*mad hatter disease*") were described in association with acute mercury intoxication [74]. In a bibliography review, Jackson [74] concluded that methylmercury poisoning causes chronic neurological illness, mainly due to direct neural lesions in the brain, particularly involving the cerebral cortex and granular cells, and reinforces the importance of detailed clinical evaluation to assess the extent of neurological deficits in chronically exposed populations.

In the Amazon scenario, the neuropsychiatric manifestations are more frequently associated with occupational exposure and are reported in miners who burn amalgam to separate mercury from gold and inhale mercury's toxic vapors. Considering environmental exposure through the consumption of contaminated fish, which is manifested chronically and insidiously, there are few studies that report cognitive deficiencies and neuropsychiatric manifestations. Khoury et al. [63,64] analyzed depression complaints and symptoms such as fear, sadness, and insomnia in the riparian population in two areas in the Amazon with different mercury exposure levels. Despite the effort made in the analysis, the authors found that there were no statistical differences between the exposed and control groups.

In our study, the detected memory and verbal fluency impairments indicate cognitive deficits are more frequent in the indigenous people with the highest methylmercury levels (≥10 μg/g). Moreover, cognitive impairment was more frequent in 12- to 19-year-old adolescents. Because chronic methylmercury exposure may cause cognitive, motor, and somatosensory losses, this finding requires caution in interpretation and warrants deeper analysis. The investigated communities, who live in a highly contaminated environment, at present, in addition to the future generations (women of childbearing age and babies), are under permanent threat. Our findings emphasize the need for new studies that deepen cognitive and psychiatric evaluation to generate even more robust evidence about the MeHg toxicity in vulnerable populations, and demand interventions from the authorities and social leadership.

#### *4.4. Limitations*

Bradykinesia, temperature sensitivity, tactile and deep distal alterations, and clinical signs of polyneuropathy were more prevalent in the group with methylmercury exposure higher than 10 μg/g. However, due to the limited size of our sample, the differences were not statistically significant. The broadening of the study to other areas with the inclusion of a greater number of participants may result in more conclusive data.

In this study, we prioritized the evaluation of somatosensory abnormalities aimed at detecting PNS lesions. Somatosensory alterations from CNS lesions were described in anatomo-clinical studies [39] and clinical trials [8]. The two-point discrimination test, graphesthesia, and stereognosis have the potential to evaluate central somatosensory dysfunctions and should also be considered in future studies.

The absence of a control group with low environmental MeHg exposure, which would allow comparisons, was also a limiting factor in our approach. In the preparatory phase of the study, we expected that *Poxo Muybu* village, which is farther from the mining zones, would be a control group for comparison. However, like the other villages, *Poxo Muybu* also presented high average levels of MeHg exposure (average: 7.3 μg/g; range from 2.3 to 12.9), above the safe levels according to international health agencies [45,46]. Therefore, it was not possible to have a control group.

It is also important to consider that the reference levels of mercury exposure vary between international health agencies, and do not necessarily reflect biological security parameters. The safety limits recommended are based on daily or weekly maximum intake parameters of MeHg. The intake criteria have a strong relationship with MeHg levels detected in blood and hair. Therefore, FAO/WHO [45] recommends maximum intake doses of 1.6 μg/kg (body weight)/week for more sensitive groups such as women of childbearing age and pregnant women, and 3.2 μg/kg bw/week for the general population. Thus, the reference intake limit doses considered to be safe, for those still not reported to have poisonous effects in human beings, correspond to mercury levels of 2.3 and 4.5 μg/g in hair samples, respectively [45].

Although our results may show promise in revealing higher cognitive deficits in participants with the highest methylmercury exposure levels, the BCSB has not yet been validated for use in traditional Amazonian populations. The use of simplified and validated instruments for evaluation of cognition in populations chronically exposed to mercury and ethnically differentiated can produce new evidence to consolidate the knowledge on this topic. Tools such as the mini-mental state examination [78], previously used in Amazonian populations [77], appear to be equally promising, and mainly associated with the BCSB.

#### **5. Conclusions**

The present study produced evidence concerning the association between motor and cognitive impairments in the CNS with chronic MeHg exposure among the Munduruku indigenous adults. The majority of people who suffered from neurological abnormalities presented MeHg exposure levels above 10 μg/g.

Somatosensory abnormalities reported in the villages where the highest methylmercury exposures were registered also indicate toxicity signs in the PNS. However, the differences reported between the two exposure groups were not statistically significant. Due to the limited sample size, we believe it is necessary to conduct broader studies to deepen the knowledge about the neurotoxic effects of methylmercury in the traditional populations living in the Amazon Basin, and particularly indigenous populations.

In conclusion, despite its limitations, this study indicates that neurotoxicity due to chronic environmental methylmercury exposure in the Munduruku indigenous population provides evidence of the long-lasting persistent environmental impacts in the Amazon [20–24]. The context revealed here highlights the risk looming over the present and future generations, who represent a substantial social, ethnic, and culturally diverse community, and should be considered and treated as the country's most significant wealth.

Finally, we believe monitoring and follow-up measures are necessary for the vulnerable populations chronically exposed to methylmercury who live in the Amazon basin. Moreover, we consider it essential to establish a basic care protocol for contaminated people in the Brazilian Unified Health System (SUS) with the support of specialists.

**Author Contributions:** Proposal design, study design and methodology, R.A.A.d.O., D.C.d.A. and P.C.B. Fieldwork data collection, R.A.A.d.O., B.D.P. and B.H.R.; writing—original draft preparation, R.A.A.d.O. and P.C.B.; writing—review and editing, R.A.A.d.O., D.C.d.A., B.D.P., B.H.R., A.C.S.d.V. and P.C.B.; supervision, obtaining resources and project management, P.C.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Vice-Presidency of Environment, Care and Health Promotion (VPAAPS) of Fundação Oswaldo Cruz (Fiocruz) through Decentralized Execution of Resources Document No. 175/2018, Process: 25000.209221/2018-18, signed between the Fiocruz and the Special Secretariat for Indigenous Health, both under the Ministry of Health. The non-governmental organization WWF-Brazil offered financial support to disseminate the results of the research.

**Institutional Review Board Statement:** The study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Research Ethics Committee of the National School of Public Health at Fiocruz (REC/ENSP) and the Brazilian National Research Ethics Commission of the National Health Council (CONEP/CNS), CAAE: 65671517.1.0000.5240, with Opinion No. 2.262.686 favorable to its performance. In compliance with Convention No. 169 of the International Labor

Organization (ILO), the study began with a pre-study consultation, carried out in August 2019, during a visit to the villages, in which the author PCB, and local indigenous leaders participated. At the time, the study objectives were presented and discussed (https://www.youtube.com/watch?v= oFEYEGxNmns&t=704s, accessed on 20 September 2019). After answering questions and approval of the proposal by the communities, we received support from the coordination of the Special Indigenous Sanitary District of the Tapajós River through the multidisciplinary indigenous health team to carry out the study. In addition, the interviews and data collection started only after the participants had their questions answered and formal consent was given via the Informed Consent Form (ICF) by the children's guardians.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Written informed consent was obtained from the patients to publish this paper.

**Data Availability Statement:** Data sharing not applicable.

**Acknowledgments:** On behalf of chiefs Juarez Saw, Jairo Saw, and Valdemar Poxo, and the leader Alessandra Korap, we thank the Munduruku people for the trust placed in our team and the support in carrying out the research. On behalf of Rio Tapajós Indigenous Special Sanitary District, we thank the coordination Cleidiane C.R. Santos and the nurses Alan Marcelo Simon and Lygia Catarina de Oliveira. We also thank to Marcelo de Oliveira Lima, Iracina Maura de Jesus and João Paulo Goes Pereira who performed the mercury laboratory analysis. We also thank Marcelo Oliveira-da-Costa from World-Wide Fund for Nature (WWF-Brazil) for technical and financial support for this scientific communication.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


**4. Discussion**

The reports of ICCT and Sphera present average methane slip values of 5.5 g/kWh and 3.8 g/kWh for LPDF engines, respectively [14,15]. However, the methane emissions from actual LNG vessels have not yet been widely studied. Anderson et al. [8] conducted onboard studies on an LNG ship resulting in total hydrocarbon emissions of 1.1–6.7 g/kWh (depending on engine load). Ushakov et al. [12] report average methane emission of 5.26 g/kWh based on on-board measurements. Peng et al. [6] also did on-board studies and reported methane levels of 3.7–25.5 g/kWh, the lowest level corresponding to highest engine load and the highest methane level corresponding to engine load of 25%. They also publish the actual methane concentration of 1000 ppm at 90% engine load and higher concentrations at lower engine loads. Lehtoranta et al. [5] reported methane levels from one medium speed engine (dual fuel, natural gas) at two load modes of 85% and 40% resulting in 5.6 g/kWh and 13.8 g/kWh, respectively. These levels correspond to methane concentrations of 1800 ppm (at 85% load) and 3750 ppm (at 40% load) (Table 2). Wei and Geng [30] review also reports total hydrocarbon concentrations above 2000 ppm from natural gas/diesel dual fuel combustion. Comparing these published results, there is a large variation in the methane levels reported, depending, most probably, on different engines (e.g., engine model, size, age) used as well as on engine loadings. Most of the values reported are in g/kWh while there are few publications reporting in concentrations (ppm) also. By comparing these values, it can be concluded that all the methane slips reported are in the order of 1000 ppm or higher. Furthermore, comparing this to the present study, the methane level (1000 ppm and 1500 ppm) in the present study was in the same order of magnitude. However, we used a smaller engine to investigate the performance of MOC but with a target to have similar exhaust gas composition to medium speed DF engines operating with NG as the main fuel. Therefore, we consider the results of the present study to be interpreted as indications of the possibilities to use MOC in LNG vessels.

In the present study, MOC was found to decrease the methane emission by 70–80% at the exhaust temperature of 550 ◦C. This efficiency, however, decreased significantly within time, even with only 0.5 ppm SO2 in the exhaust. Regeneration was done once a day and was found to recover the efficiency. This regeneration was done in nearly stoichiometric conditions, the very low O2 enabling SO2 release from the MOC while also the temperature increase observed during the regeneration phase might influence the SO2 release. In addition, the H2 might play a role here too, since H2 was found in the exhaust in the regeneration phase and has also been used for methane catalyst regeneration purposes in earlier studies [26,27].

Overall, it seems that the temperature is in significant role, both in methane oxidation efficiency as well as in regeneration. One regeneration performed in the present study resulted in higher exhaust temperature downstream of the catalyst and released higher amount of SO2 compared to other regenerations.

In addition to MOC, a SOx trap was studied in connection to MOC, for the first time (to the authors' knowledge). The SOx trap was shown to protect the MOC against sulfur poisoning to some extent, since tripling the exhaust SO2 level (from 0.5 ppm to 1.5 ppm) was found to have, in practice, no effect on MOC performance when trap was used upstream the MOC.

Other hydrocarbon emissions (ethane, ethylene) and carbon monoxide are easier to oxidize and were found to be nearly totally diminished by the MOC over the whole ageing test. The NG in the present study was high in methane content, while the fuel gas composition from different suppliers can have lower methane content (see e.g., [12]) and higher share of ethane and heavier hydrocarbons favoring better performance of MOC.

Formaldehyde, HCHO, is defined as carcinogenic substance and contribute also to other severe health effects, such as asthma, see, e.g., [31,32]. Therefore it is important to also consider the HCHO increase by LNG use (e.g., [6]), especially in coastal areas. In the present study, formaldehyde measurements were employed and showed that the

MOC very effectively (by 95–99%) reduced the formaldehyde emissions and practically no decrease was found in the formaldehyde oxidation efficiency during the catalyst ageing studies. This indicates that formaldehyde emissions from NG engines can be diminished with the MOC employment.

The results of the present study give indication of the possible use of MOC in LNG ships to control methane slip emissions. However, the regeneration process in real sized lean-burn marine engine is an issue that needs to be solved. Optimization of the regeneration interval and duration depending on the actual target of application is needed as well. Further catalyst development, regarding the efficiency and sizing, is to be done to have the best possible catalyst suitable to be installed at high-temperature conditions in the exhaust line.

Currently, LNG is a viable marine fuel deployed to substantially reduce pollutant emissions (NOx, SOx and particles) from ships. The use of LNG has therefore a notable effect on air quality and human health. In addition, LNG can provide reduction in GHG emissions if methane slip is controlled. This methane slip challenge needs to be solved to maximize LNG's potential to contribute to climate neutrality. The present study shows that MOC can play a role here. As it is an after-treatment system, it has potential both in new vessels as well as to retrofit to existing vessels. However, further studies are needed to solve the optimized solution for MOC and performance on different engine loadings as well in transient loading relevant in vessel operation.

In the long term, the transition from fossil NG to renewable NG is needed. This means also that further studies on, e.g., biogas (LBG) is needed to be able to develop suitable systems for biogas emissions since, e.g., possible biogas impurities may play a major role in the catalyst performance.

**Author Contributions:** Conceptualization, K.L., H.V., K.K. and T.M.; methodology, K.L., P.K., H.V.; investigation, K.L., P.K., H.V., K.K. and T.M.; original draft preparation, K.L.; writing—review and editing, K.L., P.K., H.V., K.K. and T.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was part of INTENS-research project, funded by Business Finland and several Finnish companies.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

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


*2.2. Disk Dynamics and Structure*

Determination of the kinematics within dense circumstellar environments requires the use of reliable tracers. High-resolution near-infrared spectroscopic observations have revealed that the band heads of the CO emission from B[e]SGs typically display a characteristic shape, consisting of a blue-shifted shoulder and a red-shifted maximum. For the generation of such a band head profile, the individual CO rotation-vibration lines, superimposing within the region of the band head, must display double-peaked profiles (see Figure 2). Such line profiles can originate either from a circumstellar ring of gas expanding with constant velocity (constant outflow), or from rotational motion of a ring of gas around the central object. To discriminate between the two scenarios, complementary tracers are needed.

**Figure 2.** Sketch of the generation of the typical CO band head profile. (**a**) Spectrum around the (2-0) band head of the CO first-overtone bands for a hot gas with velocity dispersion of a few km s−1. (**b**) Profile of a single line from a rotating gas ring with a velocity, projected to the line of sight, of 66 km s−1 as seen with a spectral resolution of 6 km s−1. (**c**) Total synthetic CO band head spectrum resulting from the convolution of the band transitions in (**a**) with the profile of the ring in (**b**). (**d**) CO band head observations of the Galactic B[e]SG CPD-57 2874 [58].

The SiO band emission seen in four Galactic B[e]SGs displays a similar shape of the band heads. Detailed modeling revealed that in each object the SiO bands required a slightly lower value of the velocity [45] than the CO bands. The SiO molecule is less stable than CO, meaning that it can form and persist only at lower temperatures. This fact naturally places the region where SiO molecules are expected to form, and hence the SiO band emitting region, at slower orbital velocities and greater distances from the central object.

As CO is the most stable molecule, its formation and emission region marks the inner edge of the molecular disk. Closer to the star, tracers for the kinematics need to be found from line emission of the atomic gas. Here, the lines from forbidden transitions are most suitable, because their emission is optically thin, so that their profile shapes contain the full velocity information of their formation region [56,57]. Of particular interest are hereby the [O I] lines, because they are one of the defining characteristics of the B[e] phenomenon and hence observed in all B[e]SGs. The ionization potential of O I is about the same as the one for H I, which means that within the [O I] line forming regions, hydrogen should be basically neutral as well, restricting the formation region of the [O I] line emission to the neutral regions within the circumstellar disk. While recombination in the equatorial region close to the star might be achieved, e.g., with the model of a latitude dependent wind [70–72], the requirement of a hydrogen neutral environment severely limits the number of free electrons that will be available to collisionally excite the levels within O I from which the forbidden transitions emerge. Consequently, the [O I] lines arise in regions with high total density, but low electron density.

The profiles of the [O I] lines often display double-peaks, in line with their formation in the disk. Typically, the [O I] *λ*5577 line, which arises from a higher level than the *λλ*6300,6364 lines, is broader, indicating spatially distinct formation regions of the emissions, with the [O I] *λ*5577 line being formed at higher velocities and higher densities and hence closer to the star than the other two lines.

With the identification of the lines of [Ca II] *λλ*7291,7324 in the spectra of numerous B[e]SGs, a further highly valuable tracer for the disk kinematics has been found. These lines typically display double-peaked profiles as well, with velocities comparable to or even greater than the one traced by the [O I] *λ*5577 line [53,57,58]. This implies that they form in the same region, or at least very close to each other, which is in agreement with their comparably high critical density. Since the [O I] *λ*5577 line is not always detectable, the [Ca II] lines thus provide a suitable, complementary benchmark for the dynamics within the disks of B[e]SGs.

In summary, the optical and near-infrared spectra provide emission features from several species, which are suitable to pin down the kinematics within the disks of B[e]SGs at various distances from the star, and Table 2 includes the information on the detection of the individual tracers in the MC sample. Based on the physical constraints outlined above, the logical order of the appearance of the divers tracers from inside out would be: [Ca II], [O I], CO bands and SiO bands. The velocity information carried by these species thereby implies a decrease with increasing distance from the star. While an equatorial, outwards decelerating outflow might be able to explain some of the observed line profiles [56], the velocity patterns seem to be in better agreement with (quasi-)Keplerian rotation. In this respect, it is interesting to note that Keplerian rotation has been made directly discernible by means of spectro-interferometric observations. The rotational motion of the CO gas has been derived based on the differential phase spectrum [38,52], and the rotational motion of the ionized gas based on the spatially resolved Br*γ* emission [49].

While, in general, the rotational motion of the material within the circumstellar disks of B[e]SGs seems now to be well established, possibly in connection with a (very) slow outflow component [57], recent investigations of the spatial distribution of the circumstellar gas revealed that it is more likely accumulated in multiple rings, partial rings, and possible spiral arm-like structures rather than in a smooth disk [43,44,58]. These rings might result from multiple mass ejection phases caused by (pulsational) instabilities acting in the outer layers of these luminous objects (e.g., [73–75]), or from binary interaction in close systems, as seems to be the case for some of the Galactic objects [58], in which the rings are circumbinary rather than circumstellar. Other disk-forming mechanisms that have been proposed in the literature over the years include equatorial mass-loss from a critically rotating star [76,77], the rotationally induced bi-stability mechanism [78], the slow-wind solution [79], and the combination of the latter two [80]. For an overview including a detailed description of the various models and their limitations, see [81].

The circumstellar material of many MC objects appears durable. This is evidenced by their emission features and their infrared photometry that both display no considerable variability over several decades, in combination with chemically processed dust displaying emission from crystalline silicates [82]. It is tempting to imagine that in such an environment even minor bodies might have formed from the long-lived disk material, creating gaps within the disk in radial direction and hence leading to the formation of the presumed ring structures [43]. These minor bodies or possible planets can also stabilize the neighboring rings, in analogy to the shepherd moons in planetary systems. However, thus far, there is insufficient observational evidence that might support the validity of such a scenario.

### *2.3. Current Evolutionary State of B[e]SGs*

The formation mechanism of the observed gaseous and dusty rings or disk-like structures around B[e]SGs is certainly one of the most important yet unsolved issues. Equally important questions arise: What is the evolutionary phase of B[e]SGs? What is their evolutionary connection to other evolved massive stars? Does such a connection exist at all? While the question on the relation between B[e]SGs and other evolved objects is beyond the scope of this review, we briefly elucidate the current knowledge about the evolutionary status of B[e]SGs. Considering the MC sample, it is obvious from Figure 1 that all objects have evolved off the main sequence. Whether this occurred only recently, or whether B[e]SGs might be on a blue loop or blueward evolution after having passed through the turning point on the cool edge of their track, is still an open issue, in particular, since we lack clear methods for age determinations of these emission-line objects, which only very rarely are detected in clusters2.

In this regard, the detection of clear signs of the 13C isotope in the form of 13CO band emission from a number of MC B[e]SGs ([41,86], see Table 2) was one major step forward. The appearance of these bands has been predicted based on theoretical model computations for a variation of the carbon isotope ratio 12C/13C [87]. As surface abundance calculations have shown, this ratio will drop during the evolution of massive stars from an initial, interstellar value of ∼90 down to values <5, depending on the initial mass of the star and its initial rotation speed. The surface material enriched in 13C is transported via winds to the environments, where it will cool and condense into 13CO molecules, whose emission can be observed in the K-band, together with the emission from the main isotope, 12CO (see Figure 3). Hence, the detected amount of 13CO is a measure for the stellar surface enrichment in 13C at the time the material, which is currently traced in the molecular emission, has been released from the stellar surface.

**Figure 3.** Synthetic spectra of the combined emission from 12CO and 13CO for different values of the 12C/13C ratio. The computations have been performed for the following physical parameters: a 12CO column density of 2 × 1021 cm−2, a gas temperature of 3000 K, a line-of-sight rotational velocity of 66 km s−1, and a spectral resolution of 50 km s−1.

From the MC sample of 15 objects, seven have been found to display CO band emission, and all of them display clear indication of enrichment in 13C (see Table 2). Interestingly, all these objects with CO emission cluster in the same region of the HR diagram, as indicated by the dotted black square in Figure 1, i.e., in the luminosity range log *L*/*L* = 5.0–5.8. None of the three most luminous stars (S 22, S 111, and S 127) or of the four low-luminosity objects (S 23, S 59, S 93, and S 137) displays clear signs of CO emission. One outlier in the luminosity domain occupied by the CO emitting B[e]SGs is the star S 89, which also has no detectable CO band emission [41].

The absence of measurable CO band emission might have different reasons. Either the intensity of the emission is too low to be detectable against the strong near-IR continuum3, or the density of

2 Currently, only four B[e]SGs are reported to be cluster members: the two LMC objects LHA 120-S 111 in the compact cluster NGC 1994 [26] and LHA 120-S 35 in SL482 [44], and the two Galactic sources MWC 137 in SH 2-266 [83] and Wd1-9 in Westerlund 1 [84,85].

3 This spectral region suffers from strong telluric contamination, which is not always easy to remove, so that especially weak CO emission features might be hidden within telluric remnants.

the molecular gas might be very high, resulting in optically thick emission, which no longer has a characteristic band head structure. Another possibility would be that the CO emission from these stars might have variable CO band emission and they have thus far always been observed in phases of no emission. In this context, it is interesting to refer to the SMC object LHA 115-S 65, in which CO band emission suddenly occurred, while observations taken about nine months earlier did not detect any molecular features [40]. Alternatively, since we now know that the material is most probably concentrated in rings, the conditions within the circumstellar environment in terms of density and temperature might not be favorable for the excitation of the first-overtone bands. For those stars, observations in the spectral region of the fundamental bands might therefore be a possibility to search for cooler CO gas.

The measured 12C/13C isotope ratios of the MC B[e]SGs are all very similar, spreading from 9 to 20. This might point towards a similar formation history of the circumstellar material, i.e., a similar phase in the evolution (considering they are single stars or at least unaffected by a possible companion) when the enriched material was ejected. Considering that stars are typically born with some intrinsic rotation velocity, rotational mixing in combination with enhanced mass-loss may drive the enrichment of the stellar surface with 13C already in early stages of the evolution of massive stars. This can be seen in Figure 4, where the evolutionary tracks of a 32 M star with solar metallicity [88] and for a variety of initial rotation velocities are shown. The covered rotation speeds spread from *v*/*v*crit = 0 to *v*/*v*crit = 0.4, which correspond to values of Ω/Ωcrit from 0 to 0.568. The interpolation of the tracks has been performed with the SCYCLIST tool4 provided by the Geneva group.

**Figure 4.** Evolution of the 12C/13C isotope ratio along the solar metallicity tracks of a star with initial mass of 32 M and initial rotation speeds *v*/*v*crit ranging from 0 to 0.4 (corresponding to Ω/Ωcrit = 0.0; 0.1; 0.2; 0.3; 0.4; 0.5; 0.568). The individual tracks have been obtained from the interpolation tool SCYCLIST provided by the Geneva group. For clarity of the plot, we truncated the evolutionary tracks within the red supergiant regions. Included are the positions of the MC B[e]SG sample from Table 2 with known 12C/13C ratio, following the same color coding as for the tracks. The Galactic objects are excluded due to their highly uncertain luminosities. Depending on the initial rotation speed of the star, the observed ratio can be reached either in the pre-RSG (moderate rotator) or post-RSG (slow rotator) phase.

The color coding along the tracks refers to the values of the 12C/13C isotope ratio on the stellar surface. Figure 4 also includes the positions of the seven MC B[e]SGs with known values of the

4 https://www.unige.ch/sciences/astro/evolution/en/database/syclist/.

12CO/13CO isotope ratios. Their colors correspond to the same color coding as the evolutionary tracks. Obviously, the observed ratios for the sample stars might be reached either along or after the main-sequence evolution for stars rotating initially with rates Ω/Ωcrit ≥ 0.3. However, they might also be reached during or after the red supergiant stage for stars rotating initially with rates smaller than Ω/Ωcrit ≤ 0.3. As we do not know the initial rotation speeds of the progenitor stars of these B[e]SGs, the measured values of the 12CO/13CO isotope ratio alone cannot solve the issue with the current evolutionary state of the objects.

If B[e]SGs represent a specific phase in the evolution of massive stars, then these objects should also exist in other environments with high content of massive stars. Searching for representatives of B[e]SGs in other galaxies and studying their properties and number statistics at various metallicities might help to unveil their disk/ring formation mechanism, to pin down their evolutionary phase (preversus post-RSG), and to set constraints on the evolution of massive stars in general.

### **3. Identification of B[e] Supergiants in the Local Universe**

Identifying and classifying B[e]SGs is a tedious job, whether in the Milky Way, where we face large amounts of foreground extinction and the issue with the often unknown distances, or in other galaxies, where we have strong contamination with foreground sources, often crowded regions, and the faintness of the objects. While the first B[e]SGs have been found rather accidentally, nowadays dedicated surveys can make use of the established classification criteria. As mentioned in Section 2, there are basically four characteristics a star should fulfill to be classified as B[e]SGs [18,25]. It should display:

Despite these defining criteria, the identification of extragalactic B[e]SGs is not as straightforward, because suitable candidates need first to be found based on other means. These candidates can then be further investigated to search for these, mostly spectroscopic, characteristics.

A suitable approach is to search for B[e]SG candidates among the luminous, blue objects identified in imaging surveys that have been performed for several galaxies over the past ∼30 years. For instance, the early photographic and photometric surveys of M31 [89,90] and M33 [91] revealed (amongst many other objects) the most luminous hot stars and the brightest blue supergiants, which could then be studied spectroscopically to obtain indications for their possible nature (e.g., [92]). A milestone for the identification of evolved massive stars was certainly the Local Group Galaxies Survey (LGGS) project [93,94], which resulted in the discovery of numerous putative emission-line stars in M31, M33, and seven more dwarf galaxies. Spectroscopic follow-ups were used to sort out H II regions, and to match the remaining objects with the various known categories of evolved massive stars. A major result of this survey was the identification of numerous objects that were dubbed as luminous blue variable (LBV) candidates [6,95], based on the appearance of their blue emission-line or P Cygni-type spectra that resemble confirmed LBVs in quiescence. Since the blue optical spectra of LBVs in their

5 We would like to stress that the high-luminosity B[e]SGs barely display photospheric absorption lines, whereas the low-luminosity B[e]SGs typically do.

quiescence state display a number of common characteristics with B[e]SGs [95–97], it was expected that this sample contains a number of B[e]SGs.

Surprisingly, when analyzed in more detail [97–99], this bunch of newly identified LBV candidates in M31 and M33 turned out to be a mixed bag containing not only B[e]SGs candidates, but also so-called Fe II emission stars (with neither [O I] nor [Fe II] emission and lacking warm dust), and warm hypergiants (with lots of dust, possible [O I] and/or [Fe II] but of spectral type A–F), with only a few objects left to be considered as LBV candidates (with no [O I] emission and lacking hot dust).

As both LBVs and B[e]SGs are luminous blue supergiants, they share the same optical colors. Hence, one step to distinguish these two groups of objects is to inspect their location in infrared color–color diagrams (e.g., [16,20,41,97,99]). The hot ( ∼1000 K) circumstellar dust of B[e]SGs results in significantly increased near-IR emission. On the other hand, LBVs can be associated to cold, dusty environments such as the circumstellar shells recently discovered around many LBVs and LBV candidates with the Spitzer Space Telescope at 24 μm ( e.g., [100,101]). The separation of B[e]SGs from quiescent LBVs, based on their diverse IR properties, is demonstrated in Figure 5 for the known samples of MC objects, limiting to the confirmed and generally accepted LBVs in the LMC [102] and including one confirmed object from the SMC (R40, [103]). Shown are two different color–color diagrams (J–H versus H–K and W1–W2 versus W2–W4). The IR colors of the objects are listed in Table 3. They result from the JHK-band magnitudes obtained from the 2MASS point source catalog6 [104], and from the mid-IR magnitudes (W1, W2, W4) collected with the Wide-field Infrared Survey Explorer7 (WISE [105]). From the many possibilities of near- and mid-IR color–color diagrams, these two show the clearest separation between the two groups of objects.

**Figure 5.** Demonstration of the separation of the B[e]SGs from the quiescent LBVs within the near-IR (J–H versus H–K diagram (**left**)) and the WISE diagram (W1–W2 versus W2–W4 (**right**)). Shown are the positions of the classical MC B[e]SG sample and of the MC LBV sample. IR colors of the objects are provided in Table 3. The solid line represents the positions of regular supergiants with empirical colors taken from [106] for solar metallicity stars.

Another clear distinctive feature between B[e]SGs and LBVs is the S Dor cycle of the latter, while B[e]SGs are typically not undergoing this type of variability. However, to identify such S Dor excursions of the stars within the HR diagram is a time-consuming (though important) task, because it requires regular monitoring of the whole sample.

6 http://cdsarc.u-strasbg.fr/viz-bin/cat/II/246.

7 http://cdsarc.u-strasbg.fr/viz-bin/cat/II/311.

In addition, dedicated spectroscopic observations are required to search for the characteristic forbidden emission lines of [O I] and possible [Ca II] in the red portion of the optical spectra, and to search for possible molecular emission of CO in their near-IR spectra, because LBVs typically do not show these sets of forbidden lines and CO band emission8 [21,35,41].

**Table 3.** IR colors of Magellanic Cloud B[e]SGs and LBVs.

Note: IR photometry for all objects is taken from the 2MASS point source catalog (J, H, and K [104]), except for the stars LHA 120-S 111 [21] and LHA 120-S 89 [108], and from the WISE All-Sky Data Release (W1, W2, and W4 [105]). a Despite of the lack of WISE colors, the presence of warm dust is proven by its IR excess seen in the Spitzer data, and its IR spectrum that looks like a twin of the one of LHA 120-S 73 (see [82]).