Mercury in fish and sediment of Purus River, Acre State, Amazon

1 Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Ministério da Saúde Ananindeua (PA), Brazil. 2 Laboratório de Biologia e Manejo dos Recursos Aquáticos, Universidade Federal do Pará (UFPA) Belém (PA), Brazil. Study carried out at Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Ministério da Saúde – Ananindeua (PA), Brazil. Correspondence: Nathália Santos Serrão de Castro – Instituto Evandro Chagas, Secção de Meio Ambiente – Rodovia BR 316, km 7, s/n – Levilândia – CEP: 67030-000 – Ananidneua (PA), Brazil – Email: n.castro@campus.fct.unl.pt Financial support: The research reported in this manuscript received funding from the Ministry of Health and from the National Council for Scientific and Technological Development (CNPq), Brazil, which supported the undergraduate scientific fellowship of Camila Margalho. Conflict of interests: nothing to declare. Abstract Core subject: To quantify the Hg content of sediment and fish collected along the Purus River (Acre State, Amazon) in order to identify if those samples could be a potential route of Hg exposure to the population of Manoel Urbano (a riverside community). Methods: The total mercury (THg) was quantified using the Cold Vapor Atomic Absorption technique. Results: We collected 06 samples of sediment and 264 samples of fish. The Hg in sediments ranged between 0.038 and 0.065 μg.g–1.The results indicate that sediment is in agreement with “uncontaminated” Amazonian rivers. The carnivorous species presented the highest level of Hg on muscle (mean 0.927 μg/g–1), followed by piscivorous (mean 0.873 μg.g–1), planktophagus (mean 0.566 μg.g–1), omnivorous (mean 0.533 μg.g–1) and detritivorous (mean 0.176 μg/g–1). Fourty four percent (44%) of the total species collected presented mean levels of THg on muscle, a percentage greater than the threshold recommended by WHO. Conclusion: Some species may be a route for Hg exposure. The sediment is within the normality. The authors suggest that other factors, such as culture and society, should be considered for future researches in order to promote the population healths.


▄ INTRODUCTION
Mercury (Hg) is a metal that accumulates along the aquatic food chain, whith carnivorous fish presenting the higher concentrations, and undergoes biomagnification in almost all food chains, which ultimately results in a very high environmental exposure to consumers 1,2 .
The Hg is introduced into the environment thru different sources, such as atmospheric transport and geochemical pools 3 .Its chemical form and physicochemical properties are closed related with its distribution 4,5 .The Hg could be present in the environment as inorganic (Hg 0 , Hg 1+ and Hg 2+ ) and organic forms.The organometallic compounds result from the action of microorganisms that transform the inorganic form into organic forms 6 .
The Amazonian population diet varies according to the season and presents diversity in the consumption of regional fruits 7,8 .Although, this scenario have been changing slowly due to the recent advances in the Brazilian social programs (direct cash transfer programs) which increased the financial support capacity of some populations, diversifying the diet of these individuals and becoming less dependent of the hydrological cycles 9 .In spite of this newly social context, fish continues to be the major source of protein intake for most of the Amazonian population, especially children.Some of the fish species that are commercially available contains Hg levels that extrapolate the safe limit (≤0.50 µg.g -1 ) preconized by WHO [10][11][12][13][14][15] , leading to the development of environmental biomonitoring programs in order to measure the concentration of this metal into different fish species and assess the risk of exposure for this population [16][17][18][19] .
In Amazon, Hg can be released in the aquatic ecosystems by anthropogenic (mainly via gold mining activity) and natural process (such as soils lixiviation) 20,21 .In this context, the mercury levels of Amazonian population, even the ones located at non-canonical exposed area, usually extrapolate the maximum safe limit allowed by WHO [22][23][24][25] .
Brabo et al. 26 conducted a study to quantify Hg in various abiotic and biotic matrices from Acre State and Purus Rivers (both located at the west end of Western Amazonia), including fish samples from Rio Branco (the capital of Acre State), Sena Madureira, Brasiléia and Assis Brazil cities (small riverine villages located at Acre State).The results revealed that water, bottom sediments and suspended solids were in accordance with a non-impacted area and also that Hg lithogenic input is more significant than the external contribution.Regarding the aquatic biota, 1186 commercially samples of fish were captured.Among them, 613 were classified as carnivorous and 513 as non-carnivorous.The Hg content followed the standard profile of bioaccumulation where the concentration was 1.287 μg.g for carnivorous and 0.115 μg.g for non-carnivorous species.
The ratio between fish ingestion and Hg exposure in the population of Acre State is encompassed by some factors, considering the consumer economical status, and, of utmost importance, a great proportion of the population (independent of its social context) shows high levels of Hg on hair 27 .In this context, Martins 28 identified key aspects of feeding habits of non-urban population of Acre that contributed significantly to identify possible and alternatives causes of Hg exposure, such as the cultivation of beans on the small ravines (or beaches) created during the dry season.As a result, sediment and cultivars presented low levels of Hg, but some populations presented high levels of Hg on hair: individuals from Sena Madureira (n=83/ mean =7.2 μg.g Hg) and from Manoel Urbano (n=92/ mean =14 μg.g).
The present study aims to assess if the fish species from the Purus River confer a risk of exposure to Hg for a riparian population living at Manoel Urbano (Acre State).Furthermore, the analysis of the river bottom sediment was performed in order to identify any recent anthropogenic input of Hg.

Area of study
The Purus River Basin has a transfrontier position, being located at Amazonas (73%) and Acre State (21%), as well as Peru (5.5%) and Bolivia (0.5%) 29 .The Purus River (the main tributary of the Basin) presents native forest, low anthropogenic impact and wetlands along the oxbow parts 29 .This river has an extension of 3700 Km, emerges in the Contamana Mountain (Peru) and recedes into Solimões River 30,31 .The waters of the Purus River are classified as white, presenting a bicarbonate profile (rich in Ca 2+ e HCO 3-), weakly acid to neutral pH (from 6.5 to 7) and high dissolved materials and salts that are carried from Andes 32 .
Manoel Urbano is located at the central part of the State of Acre along the Purus River.In 2010, the city had a population of 7,981 habitants and a demographic density of 0.75 hab/Km 2 33 .

Sample collection
The present study collected 264 samples of 18 fish species and 06 samples of sediment along 06 points (with a distance of 5 km between each sample point) of the Purus River, in the period November 2013 (Figure 1).Fish were collected using cast nets and gillnets with different mesh sizes (35, 50 and 70 mm between opposite knots).Then, the specimens were identified using taxonomic guides [34][35][36] .Some additional information as total weight (g), total length (cm), trophic level using the Fishbase database 37 , and stomach content were collected.The muscle and liver samples were placed in plastic bags, identified, frozen and transported in coolers to the laboratory.The samples were stored in glass vials, homogenized and stored at -20 °C until the laboratory analysis.
The sediments were collected using Van Veen Grab sampler.Then, the samples were identified, stored in plastic bags, dried at room temperature, fractionated until reach a granulometry of 270 mesh and stored at room temperature until the laboratory analysis.

Analysis of total mercury
The samples were analyzed according to Akagi et al. 38 .Approximately, 0.5 g of homogenized muscle, 0.3 g of homogenized liver and 0.2 g of sediment were weighted and transferred to 50 mL Pirex ® volumetric flasks, added 1 mL of deionized water, 2 mL of HNO 3 -HClO 4 (1+1), and 5 mL of H 2 SO 4 .Samples were then heated on a hot plate to 200-230 °C for 30 minutes.After cooling, the mixture were completed to 50 mL with deionized water, then THg was analyzed by Cold Vapor Atomic Absorption Spectrometry (CVAAS) with the Mercury Analyser, model HG-201, produced by SANSIO.All analyses were performed in duplicate and were accompanied by quality control from certified reference material (DOLT-4 and DORM-3).
Descriptive statistics and correlation calculations were evaluated (significance level of 95% or p<0.05) using the software MINITAB 17 (Minitab Inc.).

Levels of mercury in fish
A total of 264 individuals belonging to 3 orders, 11 families and 18 species were collected.Table 1 shows the number of collected species, the popular and scientific names, trophic levels, feeding habit and mean HgT content on muscle and liver of each specie The THg content on muscle ranged from 0.004 to 5.384 µg.g -1 .The carnivorous species presented the highest level of Hg on muscle (mean 0.927 µg.g -1 ), followed by piscivorous (mean 0.873 µg.g -1 ), planktophagus (mean 0.566 µg.g -1 ), omnivorous (mean 0.533 µg.g -1 ) and detritivorous (mean 0.176 µg.g -1 ).
Our data indicate a good corratio between the trophic level with the THg into muscles (r=0.644;p=0.001), as well as a good corratio between THg into muscles with length (r=0.505;p=0.001) and with weight (r=0.408;p=0.001).The data is in agreement with other studies that evaluated the same parameters 13,39 .

Analysis of HG into sediment
The analytical results of Hg in bottom sediments (n=6) ranged between 0.038 and 0.065 µg.g -1 and mean values observed was 0.050 µg.g -1 (Table 2).
The results are higher when the data are compared with the results obtained by Brabo et al. 26 who found an mean value of 0.023 µg.g -1 for the Purus River (closer to Sena Madureira city) and 0.026 µg.g -1 for the bottom sediment of the Iaco River (one of the main tributary of the Purus River) while the results are similar with the ones observed by Mascarenhas et al. 40 who reported a mean Hg content of 0.054 µg.g -1 for the bottom sediment of Acre River.The values found in this study are below the background set for "uncontaminated" Amazonian rivers <0.200 µg.g -1 .
Table 1.The popular and scientific names of the collected species, the feeding habit, the number of samples and the mean HgT (µg.g -1 ) are shown

▄ DISCUSSION
There is no direct evidence of gold mining activity in the Acre State.Although, its geographical location may be a determinant factor for the comprehension of Hg flow in this ecosystem, given that Peru, Bolivia and Amazonia have been historically affected by gold extraction.In spite of this scenario, most of the studies identified that the abiotic samples in this region were within the normality and were in agreement with a non impacted area [26][27][28] , and our data are in agreement with this idea.
It is premature to evaluate if C. macropterus, H. scomberoides, R. vulpinus, C. coecutiens, S. lima, P. pirinampu, P. squamosissimus and H. edentatus represents any real risk to human health.Although, according to our observations, all of them are commercialized and consumed by the population of Manoel Urbano (except for C. coecutiens).
There are few studies regarding the economic fishing potential of the Purus River.de Almeida et al. 41 characterized the fishing activity in Manoel Urbano and other small villages, unraveling the fishing sector into Manoel Urbano, revealing peculiarities about the fishing activity, such as: the preponderance of lake fishing, the use of motorized canoes rather than boats, gillnetting is the main methods used for fish capture, the absence of a central market for fish commercialization (improving the ratio between fishermen and consumers) and the decreasing of fishing activity during the dry season.This work provided a list of some species that are commercialized in the region, but used the popular their names, thus decreasing our capacity to successfully compare our results.Although, Filhote/Piraíba covers 29% of the tons landed in the main ports of the region; Branquinha (a detritivorous fish also reported in our work) cover 11%.
Most of the species cited in this work migrate (such as C. macropterus, H. scomberoides, R. vulpinious, S. Lima, P. pirinampu, P. amazonica, P. nigricans, S. fasciatus and M. duriventre), and some of them (H.scomberoides, P. Pirinampu and P. nigricans) had being identified into different rivers in Amazonia, such as Solimões and Madeira-Tapajós interfluves 42,43 .C. macropterus and P. pirinampu had been identified also in Colombia and Venezuela where it has commercial potential 44,45 .Other studies also identified the specie R. vulpinus as potential to accumulate Hg into muscle 16,46 .Special emphasis should be attributed to P. squamosissimus and H. edentatus which are significant for the economy of the fisheries activity in Amazon and presented higher values of Hg into muscle 47 .
The socio-economical behavior of the population of Acre (reported by Santos et al. 27 and Martins 28 ) put in evidence that the Hg flow dynamic goes beyond the geopolitical barriers, and may be dependent of anthropologic features, such as cattle activities and ancient diet heritage that corroborate with the idea that those populations have alternative origins of protein intake (rather than fish).Thus, the relationship between food, culture and society should be considered in future studies in order to understand the high values of Hg observed on hair of this population.

▄ CONCLUSION
Considering that species with higher Hg content on muscle were the ones that presented higher trophic position, the ingestion of some fish species may be a route of exposure for the population of Manoel Urbano.
Purus is an international river and merit special attention given that the route of Hg exposure may be associated with gold mining activities in other countries, such as Peru.
The population of Manoel Urbano is composed by a miscellaneous of factors, where the triad food, culture and society should be addressed for future studies in order to better design the route of Hg exposure and, consequently, promote health in this population.

Figure 1 .
Figure 1.Map showing the geographic location of Manoel Urbano and the sampling points along the Purus River