4.1 Contamination assessment in C. americana
Our data reveal a worrying scenario of Hg and MeHg contamination in C. americana in the Teles Pires and Juruena Rivers. The observed concentrations were higher than those reported for Ardea alba (2.2 µg/g) in the Brazilian Amazon (Gomes et al. 2009) and Mycteria americana (0.339 µg/g) in the Brazilian Pantanal (Del Lama et al. 2011). This fact may be related to the bioavailability of Hg in the sampled environments and/or to trophic position, although all sampled species are piscivorous. The average values of 5.514 ± 2.351 µg/g and 11.570 ± 5.226 µg/g in the feathers, respectively, for Juruena and Teles Pires River avians show high bioavailability in the sampled environments as well as the vulnerability of the species in sampled environments.
In our analyses, 58% and 90.5% of feather samples in the Juruena and Teles Pires rivers, respectively, had Hg levels above 5 µg/g. Eisler (1987) evaluated the adverse Hg effects by examining feathers and found that concentrations above 5 µg/g negatively affect avian reproductive success. Evers et al., 2008 documented effects related to behaviour during the incubation period of Gavia immer eggs in Canada; there is a negative correlation between Hg concentration and nesting time. Asymmetry in the remnants (flight feathers) is another physiological effect in G. immer with feather Hg concentrations > 40 µg/g (Evers et al. 2008). We suggest here that the latter effect deserves to be further investigated, since the green kingfisher relies on the ability to fly to capture prey.
4.2 Differences between analysed tissues
The relationship of increasing THg and MeHg concentrations observed in the muscle, liver and feathers of the sampled avians was also demonstrated in other Hg studies (Baron et al. 1997; Zamani-Ahmadmahmoodi et al. 2009). In the present study, the THg concentration in the liver, already observed in different organisms (André et al. 1990; Kunito et al. 2004; Seixas et al. 2008), with a mean MeHg percentage of 36.47% in the Teles Pires River and 38.16% in the Juruena River avians, occurs due to detoxifying physiological functions. The liver likely demethylates MeHg (Joiris et al. 1999) when there are high THg concentrations. The liver is a detoxifying organ where the MeHg present is unavailable for remobilisation to other tissues (Evers et al. 2005), since most of the MeHg is demethylated by means of selenium bonds to form the non-toxic Se-Hg complex (Eagles-Smith et al. 2009).
Assuming the conversion of 69.7% of the Hg concentration in wet to dry weight (Yang and Miyazaki 2003), our THg concentration results in the Teles Pires River avian livers (1.499 ± 0.792 µg/g) would assume a mean value of 1.044 µg/g. This value is higher than concentrations found for three kingfisher species: Ceryle rudis (0.26 ± 0.08 µg/g), Alcedo atthis (0.83 ± 0.31 µg/g) and Halcyon smyrnensis (0.62 ± 0.18 µg/g) in Iran. The mean values for the Juruena River avians (0.589 ± 0.196 µg/g) were assumed to be 0.410 µg/g, above that recorded for C. rudis (Zamani-Ahmadmahmoodi et al. 2009).
Feathers, with THg concentrations 7-9.5-times higher than the liver concentrations found in our study, are an important Hg excretion route (Saeki et al. 2000) during the natural process of moulting. Of the THg present in the Juruena and Teles Pires avian feathers, 96.75% and 97%, respectively, was organic. A similar MeHg pattern was recorded by Bond and Diamond (2009). Thus, as Boeck and Gochfeld (1997) suggest, feathers are efficient Hg detoxifying structures for Hg clearance, because during feather growth, this metal, with high affinity to the sulfhydryl groups in keratin, is carried and fixed in this tissue.
Condon and Cristol (2009) analysed Hg dynamics in avians at different developmental stages. They demonstrated the efficiency of feathers in decreasing Hg concentrations in the blood, and this phenomenon was concomitant with feather growth; there was a subsequent increase in blood Hg concentrations after moulting. Honda et al. (1986), when analysing Hg concentrations in the Pelecaniformes Ardea modesta, showed that THg levels in feathers of juvenile and adult specimens contribute close to 50% of the total body Hg load. Feathers from adult birds, downy feathers from young juvenile birds have low variability in THg concentrations, are highly correlated to juvenile body burdens, and are critical for understanding reproductive and ecological implications of Hg (Peterson et al. 2019).
The sampled limnological variables, which exhibited relatively low pH, favour the presence of organic matter commonly found in the Amazonian rivers (Souza and Barbosa 2000) and Hg2+ methylation (Bisinoti et al. 2007), a phenomenon that would make it available for entry into the food chain. These results suggest the need for biomonitoring in other environmental matrices, given the possibility that the sampled environment is a potential hotspot for Hg bioaccumulation and biomagnification in the Amazonian environment.
Hg concentration dynamics in the avian muscle are still not well understood. In migratory avians, Hg concentrations increase with natural muscle atrophy (Seewagen et al. 2016; 2020) due to long periods of fasting. The THg concentrations we observed in the muscle were lower than in the other tissues. Since there is energetic withdrawal of the Hg-containing food source for muscle growth, the concentration of this metal is diluted (March et al., 1982; Lewis and Furness, 1991). In cases where high dietary Hg absorption occurs, MeHg can be sequestered in muscle tissue, where it is available for later remobilisation (Evers et al. 2008). Proportionally, the MeHg percentage of THg in the muscle (70.5% for the Teles Pires River and 71.37% for Juruena River avians) is larger than for the liver and other tissues with detoxifying functions, such as the kidney (Scheuhammer et al. 1998). Considering the proportionality between the mass of other tissues, muscle shows higher Hg accumulation values, as previously discussed by Zamani-Ahmadmahmoodi et al. (2009). Therefore, Hg stock structures can enter the food chain.
Although there are no safe Hg concentration thresholds for avians, the values we found are above the recommended 0.5 µg/g for fish intended for human consumption (WHO 1990). Fish-eating avians are top trophic level organisms, and thus they are more vulnerable to Hg contamination, due to biomagnification of this metal (Pérez-López et al. 2006; Provencher et al. 2014).
The mean values found in the muscle of the Teles Pires River avians (0.672 ± 0.280 µg/g ) are above the values found for other organisms of the Brazilian Amazon, such as in the muscles of carnivorous/piscivorous fish Hoplias malabaricus (< 0.57 µg/g) and Cichla spp. (< 0.52 µg/g) (Bastos et al. 2007, 2008; Soares et al., 2016) and the omnivore/carnivorous Serrasalmus rhombeus (< 0.59 µg/g) in the Madeira and Negro Rivers (Soares et al. 2016).
4.4 General considerations
The high degree of fidelity to the foraging and breeding sites of the species of Alcedinidadae (Davis and Graham 1991), related to the historicity of anthropogenic practices in the sampled area, favour Hg entry into the aquatic trophic chain. This phenomenon explains the highest Hg concentrations in the Teles Pires River avians.
Individual physiological differences contribute to the intraspecific variation in Hg concentration, especially excretion capacity (Bearhop et al. 2000). This factor may contribute to non-covariance between the tissues and the weight and length variables, as found in our study. Additionally, the negative relationship between biomass and Hg concentrations may occur in cases where there are temporal variations in feed (Marshall et al. 2016) or the chemical form of Hg in food material, already observed in fish (Barbosa et al. 2003), because Hg biomagnification occurs with MeHg bioaccumulation.
Historically, the Amazon region features intense deforestation activities for livestock rearing, agriculture and mining activity. The greater activity in the surroundings of the Teles Pires River compared to the Juruena river is notorious. The average 50% lower Hg concentration in the Juruena River avians reinforces the importance of maintaining protected areas, since it is in the vicinity of Juruena National Park and indigenous lands. One of the Hg stocks in Amazonian aquatic ecosystems is derived from natural soil erosion (Roulet et al. 2001), a process that is enhanced with anthropogenic remobilisation. Thus, with the natural occurrence of Hg in soils (Roulet and Lucotte, 1995), higher methylation potential in the areas sampled in the Teles Pires River (Lázaro, et al. in preparation) may contribute to higher concentrations found in this site.
There is a crucial need to expand the knowledge about Hg in Brazil and Latin America, as well as the identification of contaminated sites, as recommended in the Minamata Convention (UNEP 2013), of which Brazil is a signatory. Our results contribute to the evaluation of Hg effects in the Amazon, since it is bioavailable for accumulation in most aquatic organisms (Bastos et al. 2008; Gomes et al. 2009; Schneider et al. 2009; Souza-Araujo et al. 2015) and required attention for biodiversity conservation strategies.