Essential and non-essential elements levels in �sh species highly consumed in the middle Miranda River, Brazilian Pantanal

The Miranda River basin spreads over about 40.000 km² and consists of one of the central systems acting in the Pantanal flooding regime in the upper Paraguay River basin. The middle Miranda River has great socioeconomic importance as a source of fishery resources highly exploited by riverine communities, in addition to sustaining very significant fishing tourism in southern Pantanal. The present study investigated the levels of As, Ag, Al, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Pb, Se, and Zn in muscle samples of six fish species ( Pygocentrus nattereri , Serrasalmus marginatus , Mylossoma duriventre , Leporinus friderici , Pimelodus argenteus , and P. pantaneiro ) highly consumed by local riverine and non-resident people in a highly exploited stretch of middle Miranda River. Interspecific significant differences were detected for Ba, Fe, Mn, and Zn levels. However, these seem unrelated to species' feeding habits once pairwise comparison detected significant differences in element levels between species with similar diets. Significant correlations between body size and concentrations were found for Ba in P. nattereri (moderate positive correlation), and for Mn in M. duriventre (weak positive correlation) and P. argenteus (moderate negative correlation). Levels exceeding tolerable daily intake (TDI) were found for Pb maximum concentrations in P. nattereri , M. duriventre , L. friderici , P. argenteus , and P. pantaneiro , and for Cd maximum concentration in P. argenteus . Adopting a Pb monitoring program in biotic and abiotic compartments in Miranda River is strongly recommended.


Introduction
Pantanal biome is one of the most extensive wetlands worldwide, covering, during the rainy period, an area of around 200.000 km² distributed mainly in central-west of Brazil (states of Mato Grosso and Mato Grosso do Sul), in addition to southeastern Bolivia and a small portion of northeastern Paraguay.Pantanal is considered a priority area for worldwide conservation of natural resources by UNESCO (United Nations Educational, Scientific and Cultural Organization).However, despite its importance to the conservation of biodiversity, fisheries, and water resources, Pantanal is currently under serious threat of environmental disturbance by the input of contaminants generated by anthropic activities developed in the surroundings of the wetlands (plateau regions and mountain ranges), which include the use of pesticides in agriculture, mining, industries, and the increasing urbanization (Alho and Vieira 1997;Vieira et al. 2011;Viana et al. 2023), besides petroleum derivatives contamination due to vessels navigation, mainly by boats of fishermen.
Given the Pantanal susceptibility to pollution from human activities, some studies have been developed mainly over the last four decades, aiming to evaluate the concentrations of some potentially toxic elements in native edible fish species in the Brazilian Pantanal.As example, a broad-sampling study in total mercury (Hg) levels in fish from northern Brazilian Pantanal (Mato Grosso State) was conducted by Vieira (1991), that analyzed 475 samples of muscle and liver of Pimelodus maculatus (= currently P. pantaneiro), Pseudoplatystoma fasciatus (= currently P. reticulatum), P. corruscans, Prochilodus lineatus, Acestorhynchus altus (= currently A. pantaneiro), Pygocentrus nattereri, and Hoplias malabaricus from Paraguay, Cuiabá and Bento Gomes rivers.About 30% of the samples showed Hg concentrations higher than the maximum limit established for human consumption by WHO (World Health Organization) (WHO 1990) and the Brazilian legislation (Anvisa 1998).Following, Hylander et al. (2000) evaluated the contents of Hg in P. corruscans, P. fasciatum (= P. reticulatum), P. nattereri and Serrasalmus spilopleura (= S. maculatus) from Cuiabá and Paraguay rivers and found 10% of the samples presenting Hg concentrations exceeding the safety limit for regular fish consumption.Santos-Filho et al. (2016) analyzed the concentrations of cadmium (Cd), chromium (Cr), lead (Pb), and Hg in samples of P.nattereri and Prochilodus lineatus from Cuiabá and Paraguay rivers founding burdens exceeding the maximum concentrations indicated for human consumption for Cr in P. nattereri (95.4% of samples) and P. lineatus (97% of samples), and for Hg in P. nattereri (percentage of individuals not informed).In southern Brazilian Pantanal (Mato Grosso do Sul State), Vieira (1991) analyzed the Hg levels in the Pseudoplatistoma fasciatus (= P. reticulatum), P. corruscans, and P. nattereri in a stretch of Paraguay River at Corumbá municipality, finding samples exceeding the maximum tolerable limits adopted by WHO (1990) and Anvisa (1998).Súarez et al. (2018) determined the levels of chromium (Cr), copper (Cu), iron (Fe), nickel (Ni), and zinc (Zn) in the Apa River basin, finding concentration below the tolerance limits for all elements.
In addition to fish, other studies have investigated the concentrations of toxic metalloids/heavy metals in vertebrate species with a predominantly piscivorous diet.Vieira et al. (2011) determined total Hg and methyl mercury (MeHg) concentrations in samples of tail muscle of yacare caiman (Caiman yacare) individuals from four localities, including the northern (Bento Gomes and Cuiabá rivers) and southern (Nhecolândia and Negro rivers) Pantanal.In this case, relatively low concentrations ranged from 0.02 to 0.36 μg/g w.w.(wet weight) (Vieira et al. 2011).Concentrations of As, Cd, and Pb in caudal crests of C. yacare were later evaluated by Quintela et al. (2019) in the northern (Poconé wetlands) and southern (Miranda River basin) Pantanal.The levels of As and Pb were higher in samples from southern Pantanal, while the opposite pattern was detected for Cd.Fonseca et al. (2005) determined the total Hg levels in hair, kidney, liver, and muscle of two giant otter Pteronura brasiliensis individuals from Negro Riversouthern Pantanal -, obtaining concentrations varying from 0.17 to 4.59 μg/g.Soresini et al. (2021)  Despite the high importance of Miranda River for the subsistence of local riverine communities and the economic development associated with fisheries, no study concerning contaminants in edible fish has been conducted to date.Herein we investigated the concentrations of arsenic (As), silver (Ag), aluminum (Al), barium (Ba), chromium (Cr), copper (Cu), iron (Fe), mercury (Hg) manganese (Mn), lead (Pb), selenium (Se) and zinc (Zn) in six fish species with distinct feeding habits highly consumed in a highly exploited middle Miranda River, especially by the local communities.A significant question to be addressed is the evaluation on human exposure to potentially toxic non-essential (As, Al, Ag, Ba, Cd, Hg, Pb) and essential elements (Cr, Cu, Fe, Mn, Se, Zn), considering that the species herein analyzed constitute protein resources daily exploited by the local communities and non-resident people.

Study area
Sampling was conducted in a stretch of Middle Miranda River, from the mouth of Vermelho River (a tributary of Miranda River) to about 35 km downstream (19°36' 38.74"S, 56°58'49.04"W -19°30'48.51"S, 57°08'30.93"W),municipality of Corumbá, Mato Grosso do Sul State (Figure 1).Surrounding marginal areas comprise mainly flooding plains and pastures, where riparian forests (Seasonal Semideciduous Forest) patchily occur along the sample stretch.Corumbá is also experiencing an increase of urbanization and is currently the most populous city in Brazilian Pantanal (about 96,268 inhabitants;IBGE 2022).Active mining of iron and manganese ores and limestone occurs in the surroundings of the Miranda River in Corumbá (Urucum and Santa Cruz massifs).In upper stretches of MRB, located at the Central Brazilian Plateau in Mato Grosso do Sul, land use is characterized by large areas of agriculture, silviculture, livestock, mining, and several industrial activities, in addition to urbanization.The movements of vessels, mainly boats, are also intense in this area, which presents many fishing ranches in the margins.

Species and sampling collection
We quantified the levels of As, Ag, Al, Ba, Cr, Cu, Fe, Hg, Mn, Na, Pb, Se, and Zn in 101 individuals representative of six fish species: the red piranha Pygocentrus nattereri, the spotted piranha Serrasalmus marginatus, the red-bellied pacu Mylossoma duriventre (Characiformes: Serrasalmidae), the three-spot leporinus Leporinus friderici (Characiformes: Anostomidae), the catfish Pimelodus argenteus and the spotted catfish Pimelodus pantaneiro (Siluriformes, Pimelodidae).The numbers of sampled individuals, sizes, diets, and habits of each species are presented in Table 1.Fish were collected during three field trips conducted from February 2022 to May 2023 through gillnets of varied mesh sizes (3 to 12 cm between knots) and line and hook using artificial bait and baits of fish (small characids and heptapterids) captured in situ.After capturing, individuals were measured (total length in millimeters), packed individually in plastic bags, kept in cold water thermic boxes, and taken to the laboratory.A sample of about 3 x 1 cm of the flank region (epaxial muscle) was extracted from each individual using sterile blade.Tissues were placed in sterile eppendorfs and kept in a freezer (-8 ºC) until the elements determinations.All procedures are in accordance with international protocols for the use of animals in scientific research.Sampling was authorized by the Brazilian environmental agency ICMBio, process nº 62966-4.

Samples preparation and elements determination
Muscle samples were decomposed in a microwave oven, model Multiwave GO, Anton Paar©.For digestion, we used the method for organic samples developed by the oven manufacturer (Digestion of Food and Other Organic Samples for Element Analysis with ICP, Anton Paar©).This method was verified by the manufacturer through the digestion of three certified reference materials (CRM) and subsequent elemental analysis.The digested material was the NIST 1570a (spinach), the NIST 1566b (oyster tissue), and the BCR-414 (plankton).The smallest recovery was 86 ± 15% for Hg in digested CRM NIST 1566b while the largest recovery was 113 ± 3% for As in digested CRM BCR-414.The samples were weighted and transferred to digestion capsules containing 4 mL of HNO3 and 2 mL of HCl (both concentrated reagents).Following, the capsules were closed, placed in a rotator (model 12HVT50), and submitted to the warming program as follows: (1) warming stage of 20 minutes in ramp at 180ºC and 10 minutes of permanence; (2) cooling stage at 70ºC.The digested solutions were collected and stored in 25 mL polypropylene flasks for posterior elemental analysis by optical emission spectrometry with inductively coupled plasma (ICP-OES, model ICPE 9820, Shimadzu©).For the determination of the analytes, all the samples were filtered through a 0.45 µm filter (Chromafil©, Xtra PTFE 45/25).The ICP-OES was calibrated using multi-elemental standards, with concentrations varying from 10 to 200 µg L-1.The limits of detection (LOD) were estimated after the determination of analytes in 11 blank analyte samples (4 mL of HNO 3 and 2 mL of HCl, measured at 25 mL).
The LOD was calculated according to recommendations of IUPAC LOD = 3 σ + blank).The wavelength (nm) and the LOD of each element are shown in Table 2.

Data analysis
Means, one standard deviation, and ranges of specific concentrations (expressed in μg/g w.w) were calculated for the species that presented at least three individuals showing levels above the limit of detection (>LOD) for a given element.All the data sets that satisfied such conditions were tested for the normality distribution of values through the Shapiro-Wilk test.The data set was composed of 39 specific concentrations, 23 of them showing non-normal distributions of values, while 16 specific concentrations presented normal distribution of values.All elements submitted to statistical tests presented at least one specific data set characterized by non-normal distribution, which is consistent with the application of non-parametric statistics.
The existence of significant differences between interspecific concentrations of Al, Cu, Fe, Mn, and Znelements with data sets composed of three or more specific concentrations >LOD (Al: P. nattereri, S. marginatus, and P. pantaneiro; Cu: P. nattereri, M. duriventre, L. friderici, P. argenteus, and P. pantaneiro; Fe, Mn, and Zn: all the six species)were tested through Kruskal-Wallis tests.For those elements whose interspecific significant differences in concentrations were revealed by Kruskal-Wallis tests, Mann-Whitney pairwise tests were performed aiming to identify the pairs of species that showed significant differences between their concentrations.The existence of significant differences between elements with data sets composed of two specific concentrations showing at least three individual levels >LOD was verified through Mann-Whitney tests.
These analyzes comprised comparisons of Ba and Cr concentrations between M. duriventre and P. nattereri, and Pb concentrations between P. argenteus and P. pantaneiro.Correlations between each element concentration and body size were examined for the species which data set were composed by at least three absolute values >LOD, using Pearson (normal distribution data) and Spearman (non-normal distribution data) tests.For the data sets included in statistical analyses, the individual concentrations below the limit of detection (<LOD) were considered neutral values (zero).The level of significance adopted was p<0.5.
We found no reference regarding the quantity (weight) of human fish consumption per capita for Miranda River.For Northern Brazilian Pantanal (Mato Grosso State), Yokoo et al. (2001)  For this, we multiplied the values by 150, considering that element concentration was given as μg/g w.w.The obtained values were compared to the recommended maximum daily intake of the quantified elementswhen availablefor a 70 kg adult, according to WHO (1992WHO ( , 1997WHO ( , 1998WHO ( , 2011a))

Results
A single individual of P. argenteus presented As concentration >LOD.Individuals from all other species presented Ag levels <LOD.Concentrations of Al >LOD were found in 15 individuals (14.8% of the total sample) from five species (P.nattereri, S. marginatus, M. duriventre, L. friderici, and P. pantaneiro).The highest mean concentrations of Al were found in P. pantaneiro, while the highest individual concentration was present in a P. nattereri sample.Concentrations of Ba >LOD were found in 19 individuals (18.8%) from three species (L.friderici, M. duriventre, and P. nattereri) with the highest mean and individual levels found in M. duriventre.Levels of Cd >LOD were detected in seven individuals (6.9%), five P. argenteus, one P. pantaneiro, and one S. marginatus.The highest Cd level was found in a P. argenteus sample.In relation to Cu, this was present in concentrations >LOD in 54 individuals (53.4%) distributed in all species.The highest mean and individual concentrations of Cu were detected in P. pantaneiro.Levels of Fe >LOD were detected in 95 individuals (94%) from all species, being the highest mean detected in P. nattereri and the highest individual level recorded in M. duriventre.All individuals from all species presented Hg levels <LOD.Concentrations of Mn >LOD were detected in 58 individuals (57.4%) from all species, being the highest mean and individual levels found in M. duriventre.Levels of Pb >LOD were found in four samples of P. argenteus, three samples of P. pantaneiro, and one sample of P. nattereri, M. duriventre, and L. friderici each.The highest mean and individual concentrations were detected in P. argenteus.Levels of Se >LOD were found only in one sample of P. argenteus and one sample of P. pantaneiro, being the highest concentration present in the first.Levels of Zn >LOD were found in all samples except one individual of M. duriventre.The highest Zn mean concentration was present in L. friderici, and the highest individual concentration was detected in a P. nattereri sample.Summary statistics of element concentrations for all species are shown in Table 3.
The Kruskal-Wallis test revealed significant interspecific differences for Fe, Mn, and Zn (Table 4).The Mann-Whitney tests identified significant differences in Fe levels for the following pairs of species: P. nattereri and S. marginatus, P. nattereri and P. argenteus, P. nattereri and P. pantaneiro, S. marginatus and M. duriventre, S. marginatus and P. argenteus, S. marginatus and P. pantaneiro, P. argenteus and P. pantaneiro.
Significant differences in Mn concentrations were found for the following pairs of species: P. nattereri and S. marginatus, P. nattereri and M. duriventre, P. nattereri and P. argenteus, P. nattereri and P. pantaneiro, S. marginatus and M. duriventre, M. duriventre and P. argenteus, M. duriventre and P. pantaneiro, L. friderici and P. argenteus, L. friderici and P. pantaneiro.Significant differences in Zn concentrations were found for all pairs of species except P. nattereri and L. friderici (Table 5).For the elements with data sets composed of two specific concentrations >LOD, significant differences were found only for Ba (M.duriventre and P. nattereri) (Table 4).
Significant correlations between body size and element concentrations were found for Ba in P. nattereri (moderate positive correlation), and for Mn in M. duriventre (weak positive correlation) and P. argenteus (moderate negative correlation).A barely significant correlation was detected between Fe concentrations in L. friderici (strong positive correlation) (Table 6).Estimates of daily intake based on maximum concentrations of Cd in P. argenteus, and Pb levels in P. nattereri, M. duriventre, L. friderici, P. argenteus, and P. pantaneiro exceeded the TDI's adopted for these elements (Table 7).Estimates of daily element intake and TDI values based on the consulted reports for other elements are shown in Table 7.

Elements occurrence and concentrations: As, Cd, Hg, and Pb
Miranda River basin has a crucial role in the socioeconomic context of southern Pantanal due to the intensive exploitation of its fishery resources (Catella et al. 2020).In this context, the present study comes to bring essential information about the levels of potentially toxic elements contamination in fish species highly consumed by local riverine communities and also by people from other localities, considering that Miranda River is an axis of commercial, subsistence, and tourism fisheries (Catella et al. 2020).Special attention should be given to non-essential elements As, Cd, Hg, and Pb, due to their high toxicological potential (Hencke 2009; Balali- Mood et al. 2021).In general, the occurrence of samples with detectable levels of those elements was low.
Lead was the non-essential element with the highest frequency of concentrations > LOD, being present in such levels in one sample of P. nattereri, M. duriventre and L. friderici each, four samples of P. argenteus and three samples of P. pantaneiro.Arsenic was found in a concentration > LOD in a single individual of P. argenteus, while Hg levels > LOD were not detected in any sample.Cadmium levels > LOD were detected in one sample of A. argenteus and one sample of P. pantaneiro.When analyzing these distributions, it is remarkable that P.
argenteus was the species with the highest frequency of samples showing levels above the detection limits, followed by P. pantaneiro.The catfishes P. argenteus and P. pantaneiro are benthic species (Froese and Pauly 2023), which implies that these are closely associated to sediments.The biota exposure to sediments is highly susceptible to pollutant accumulation once river sediment layers tend to adsorb the insoluble fraction of the elements.Following, elements are incorporated into fish via trophic webs in which the benthic biota are included.The exploited bottom-dwelling fish inhabiting water bodies with elevated burdens of toxic elements, therefore, represent a potential risk to human health (Ali and Khan 2018;Afzaal et al. 2022).Sampaio (2003) measured the levels of Cd, and Pb in sediments of Miranda River, revealing values 1 mg/kg (or 1 μg/g) and 10 mg/kg (or 10 μg/g), respectively.Those burdens were higher than the concentrations herein found in muscular samples of P. argenteus and P. pantaneiroas well as in non-benthic P. nattereri, M. duriventre, and L. friderici -, which apparently indicate that bioconcentration in relation to sediment compartment is not occurring in these species.However, the Pb levels are alarming and may present a future trend for increasing burdens, considering the intensification of the land uses for agriculture, mining, and other human-related activities currently developed in Southern Brazilian Pantanal and adjacent massifs and plateaus.
Among the four highest hazardous non-essential elements analyzed, Pb was the most representative in our sample.The updated report on the security of Pb in food provided by the European Food Safety Authority (EFSA 2020) indicates a TDI of 105 μg for a 70 kg person as a daily intake with -no appreciable risk‖.The estimated daily intake of Pb based on the consumption of 150 g of muscle containing the maximum detected concentrations exceeded the adjusted TDI for this element for P. nattereri, L. friderici, M. duriventre, P. argenteus, and P. pantaneiro (see Tables 3 and 7), by 0.72, 0.78, 1.02, 2.86, and 1.32 folds, respectively.These data alerts to a possible imminent scenario of Pb contamination of fishery resources in Miranda River.Human exposure to Pb can be highly harmful, leading to severe neurological disorders, anemia, immunological activity decrease, liver injuries, renal insufficiency, and reproductive disorders (Wani et al. 2015).Lead was always found in all caudal crest samples of Caiman yacare from MRB, in concentrations ranging from 0.0005 to 0.195 μg/g d.w.Sampaio (2003) found levels of about 0.25 mg/L and 10 mg/kg (or 10 μg/g) in water and sediment of Miranda River, respectively.Water and sediment compartments, therefore, represent potential sources of Pb to the middle MRB aquatic/semiaquatic biota.Given the data so far available for Pb burdens in abiotic and biotic compartments in MRB (Sampaio 2003;Quintela et al. 2019;present study), is strongly recommended the rigorous monitoring of the environmental levels of the element in MRB, as well as in human matrices (e.g., hair and urine).
Arsenic occurs in aquatic systems both by natural and anthropogenic sources.In humans, exposure to As is associated mainly with carcinogenesis in several organs, skin diseases, and neurological disorders (Rahaman et al. 2021).The last report of the World Health Organization for As (WHO 2011a) stated that the provisional TDI tolerable weekly intake (PTWI) of 0.015 mg/kg of body weight -TDI of 0.007 mg/kg of body weight, which corresponds to a 49 μg/g intake for a 70 kg personpreviously adopted (FAO/WHO 1989) was no more secure, considering that this level falls in the range of benchmark dose lower bound (BMDL 0.5 ), an estimate for carcinogenesis with 5% extra risk.Thus, the secure TDI for As is lower than the value previously adopted (FAO/WHO 1989), but there is still no consensus about this level (WHO 2011a).In our sample, a single individual of P. argenteus presented a detectable As level, determined at 0.34 μg/g.The daily consumption of 150 g of fish presenting that level would result in an As daily intake of 51 μg, a burden conspicuously higher than the TDI previously adopted by FAO/WHO (1989), which was demonstrated to be overestimated (WHO 2011a).Even though detectable As levels had shallow occurrence in our matrices, this element deserves attention considering its high negative effect on human health (WHO 2011a), and the fact that our study was based on a sample and may not represent the actual status of As contamination in the local ichthyofauna, given the diversity and volume of the fishery resources exploited in middle Miranda River.
The As levels in Miranda River were determined by Richter et al. (2019), who found values between 0.6 and 0.8 μg/L and concluded that there is no As-contamination in this and other rivers of Pantanal.Phytoplankton is a primary catcher of elemental As in freshwater environments, but the biomagnification of such element in freshwater habitats is not steady (Rahman et al. 2012).The low incidence of detectable levels in our sample could be related to a possible non-occurrence of biomagnification phenomenon in the studied system, which resulted in low transfers of As forms through trophic chains to the fish species herein analyzed.On the other hand, As was found in all yacare caiman (Caiman yacare) caudal crests from MRB, in concentrations varying from 0.005 to 2.84 μg/g d.w.(Quintela et al. 2019).Fishes, especially from families Characidae and Cichlidae, are the main prey of adult C. yacare in Brazilian Pantanal (Santos et al. 1996).The differences between the status of contamination in C. yacare (Quintela et al. 2019) and the fish species herein analyzed could be a reflection of the following factors: (1) fish for the present study were collected in the main channel of Miranda River while C. yacare individuals sampled by Quintela et al. (2019) were captured mainly in pounds of the adjacent flood plains, which represent distinct limnic compartments, where interactions between As forms and factors such as sediment types, hydrological regime, and even the resident biota (microbiota, producers, and low trophic levels; see Rahman et al. 2012) may result in distinct patterns of As levels in the matrices of the studies target species; (2) it is possible that the occurrence of differences in As concentrations between the representatives of the fish families herein analyzed and the families most explored by C. yacare; (3) juveniles and sub-adults of C. yacare broadly explore insects and gastropods highly associated to sediments (Santos et al. 2016), which may represent potential sources for As incorporation into caiman matrices.
Cadmium was also unrepresentative in the analyzed fish species, being present in seven samples (6.9% of the total sample).However, the estimated daily intake based on Cd maximum concentration in P. argenteus exceeded the TDI currently adopted by WHO (1992) by 0.52 fold.Cadmium contents in water and sediments of Miranda River determined by Sampaio (2003) were about 0.008 mg/L and 1 mg/kg (or 1 μg/g), respectively.
Cadmium was also detected in caudal crests of C. yacare from MRB floodplains in levels ranging from 0.001to-0.53μg/g d.w.(Quintela et al. 2019).These data (Sampaio 2003;Quintela et al. 2019; present study) indicate that Cd is present in variable concentrations in biotic and abiotic compartments of middle MRB, which demands attention to the monitoring of levels and patterns of accumulation of this element in the local environments.A particular caution should be given to benthic fish, considering that P. argenteus and P. pantaneiro comprised six of the seven samples with detectable concentrations.Although a single sample of S. marginatus presented a detectable level, this also deserves attention, considering the very intensive use of piranhas in the local culinary.
High levels of Cd in humans are associated with several types of cancer, liver, and renal diseases, and osteoporosis (Genchi et al. 2020).
Interestingly, a study conducted on P. brasiliensis diet in Miranda and Negro river basins (Leuchtenberger et al. 2020) pointed out ‗Serrasalmus sp'.as the second most consumed fish, after only the trahira Hoplias sp.
(Characiformes: Erythrinidae).‗Pimelodus sp.' was also well-represented in the giant otter's diet.The authors also found a variety of other fish genera in P. brasiliensis prey spectrum, in addition to reptiles, insects, mollusks, and crustaceans, the last two consumed in high frequencies (Leuchtenberger et al. 2020).Thus, it is possible that Hg sources which resulted in moderate Hg levels in P. brasiliensis matrices (Fonseca et al. 2005;Soresini et al. 2021) may not correspond to the fish species herein analyzed, but to other prey items explored by the carnivoran.Nevertheless, Soresini et al. (2021) raise the possibility of widespread Hg contamination in fish and their predators in Pantanal due to anthropogenic sources such as mining activity and the use of pesticides in agricultural practices.Ongoing sampling and analyses on Hg from other fish species from middle MRB will bring new data for the patterns of Hg distribution in the local ichthyofauna.

Elements occurrence and concentrations: Ag, Al, Ba, Cr, Cu, Fe, Mn, Se, Zn
The elements Ag, Al, Ba, Cr, Cu, Fe, Mn, Se, and Zn have lower toxicity to humans when compared to As, Cd, Hg, and Pb (ATSDR 2022a).Among these, Cu, Fe, Mn, Se, and Zn are considered essential elements in animals due to their crucial role in metabolic paths.However, although essential for the organism's functionality, those elements can cause adverse effects when present in elevated levels.Until recently, Cr was also considered essential due to its probable role in glucose absorption.This property, therefore, is still controversial, and currently Cr is not included in the list of essential elements (Zoroddu et al. 2019).Aluminium, Ag, and Ba are metals with apparent no biological function (Zoroddu et al. 2019), and are associated to harmful health effects at elevated burdens (Drake and Hazelwood 2005;ATSDR 2007;Klotz et al. 2017).
Daily intake estimates on means and maximum concentrations revealed concentrations far below the TDI adopted for Ag, Al, Ba, Cu, Fe, Mn, Se, and Zn (Table 7).Thus, the consumption of muscle of the fish herein analyzed from the middle Miranda River is safe for these elements to date.The exposure to Cr, however, deserves attention.There is still no consensus about the TDI established for this element (EFSA 2014).The US Institute of Medicine Panel on Micronutrients ( 2001), therefore, has established an Adequate Intake (AI) for the element, which corresponds to 35 μg/day and 25 μg/day for young and adult men and women, respectively.The estimates of Cr daily intake herein presented were calculated based on the species mean concentrations and show no value above the AI concentrations.However, intake estimates based on Cr found in P. nattereri, M. duriventre, and P. pantaneiro, the resultant daily estimates intake -111, 354, and 108 μg/day, respectivelyfar exceeds the established AI.Considering that there is no consensus about a TDI for Cr, it is not discharged the potential risk of exposure to Cr high burdens through the consumption of P. nattereri, M. duriventre, and P. pantaneiro from the middle Miranda River.
Chromium is the 21 st most abundant element in Earth's crust (Barnhart 1997).It occurs naturally in rocks, soils, water, and sediments (WHO 1988), and the main anthropogenic sources comprise mining and metallurgical processes, pigments, wood preservatives, and leather tannings (Barnhart 1997).In aquatic environments, Cr is very persistent in sediments (Jaishankar et al. 2014), which can lead to contamination and mortality of benthic fauna (Berry et al. 2004).Surface water may also retain soluble Cr forms (EFSA 2014).
Therefore, the wide distribution of Cr in aquatic compartments represents a condition favorable for the element bioaccumulation in benthic P. maculatus, as well as for benthopelagic M. duriventre and pelagic P. nattereri.Sampaio (2003) found Cr levels of 0.014 mg/L and 10 μg/g in Miranda River water and sediment, respectively.
The Cr levels determined in P. nattereri, M. duriventre, and P. pantaneiro are below the burdens detected in river sediment, indicating that bioconcentration phenomena are still not occurring in these species.However, the regular monitoring of Cr levels in exploited fish from the middle Miranda River is suggested, considering the adverse effects of Cr in high concentrations.In humans, Cr toxicity is associated with carcinogenesis (DesMarias and Costa 2019), skin and mucous ulcerations, gastro-enteritis, hepatic disorders, and renal deficiency (Baruthio 1992).

Interspecific differences in element concentrations and relationships between concentrations and size
In the present study, marked interspecific differences were found for Ba, Fe, Mn, and Zn concentrations.
Many studies attribute interspecific differences in element concentrations in freshwater fish to divergences in species diets and use of habitats (Petkovšek et al. 2012;Rajeshkumar and Li 2018;Súarez et al. 2018).Our results partially agree with those propositions, considering that the distribution of the elements' highest means and maximum concentrations varied considerably among the species, characterized by distinct diets and habitats in the riverine environment.However, it is remarkable that the species with the highest trophic levels, represented by the piranhas P. nattereri and S. marginatus, did not show the highest concentrationsmean and maximum levelsfor a great part of the elements.For Ba and Mn, both the highest mean and the maximum concentration were found in M. duriventre, an herbivore species and possibly the lowest trophic level in the set of the analyzed species (Resende et al. 1998;Froese and Pauly 2023).In contrast, the highest Fe mean concentration was found in P. nattereri, a predominantly piscivorous species (Ferreira et al. 2014) while Fe maximum concentration was detected in a M. duriventre individual.Concerning Zn, the highest mean level was found in L. friderici, also an herbivore species (Balassa et al. 2004;Froese and Pauly 2023), while a P. nattereri individual presented the highest concentration.Thus, it is possible that factors other than feeding habitsand trophic level positionsact in the bioaccumulation of Ba, Fe, Mn, and Zn in the fish species herein analyzed, such as specific levels of natural binding proteins and other parameters of metabolic routes (Ali and Khan 2018;Paschoalini and Bazzoli 2021).This assumption is further supported by the results of pairwise comparisons, which detected significant differences in concentrations between species of similar diets, such as P. nattereri vs. S. marginatus (Fe, Mn, and Zn), and P. argenteus vs. P. pantaneiro (Fe, Zn) (see Table 4).It is also known that many elements are not susceptible to biomagnification along the aquatic trophic levels.There are suppositions for the occurrence of biomagnification of Ba in aquatic environments, but more studies are needed to draw further conclusions (ATSDR 2007(ATSDR , 2022b)).There is no evidence of Fe, Mn, and Zn biomagnification in freshwater trophic levels (Barceloux 1999;Oberholster et al. 2012;Cardwell et al. 2013).
It is suggested that benthic species are more susceptible to bioaccumulate metals and other elements than species that inhabit the upper zones of the water column, considering that the first are in direct contact with the sediment (Yi et al. 2011;Paschoalini and Bazzoli 2021).In our study, this trend was observed for As, Al, Cu, Pb and Se, which bioaccumulation levels were more representative in benthic catfish P. argenteus and P. pantaneiro.Pimelodus argenteus was the single species showing As level above the limit of detection and also had the highest Pb maximum concentration and the highest number of Cd individual concentrations above the limit of detection.Pimelodus pantaneiro, in turn, had the highest mean concentrations of Al and the highest mean and maximum levels of Cu.Selenium was also detected only in the two catfish species.In Tarumã microbasin, a tributary of the upper Paraná basin in the central-western plateau surrounding the southern Pantanal, Viana et al. (2018) found the highest burdens of Cu in the benthic armored catfish Hypostomus ancistroides (Loricariidae) while the also benthic catfish Rhamdia quelen (Heptapteridae) had the highest levels of Cd and Pb.Among several fish species with diversified habits In Piracicaba River, southeastern Brazil, muscle samples of benthic Hypostomus punctatus presented the highest concentrations of As, Cd, and Se, while the highest Pb levels were detected in benthic Pimelodus maculatus (Meche et al. 2010).In Sinos River, southern Brazil, liver samples of the detritivore bottom-dweller Cyphocharax voga (Curimatidae) presented higher concentrations of As, Al, Cd, Cu, and Pb, when compared to the pelagic carnivore Oligosarcus sp.
(Characidae) (Weber et al. 2013).The bioaccumulation pattern of heavy metals and other elements in fish assemblages is, therefore, a reflex of the elements distribution in the environment.Considering that element burdens in sediment are generally higher than in the water column, benthic species are more susceptible to incorporating the elements directly through gills and skin and via digestive tract, through the consumption of sediment-associated food/prey (Ali and Khan 2018).In Miranda River, Sampaio (2003) found the concentrations of Cd, Cu, and Pb in sediments exceeding the levels in water at about 93, 1,5000, and 31 times, respectively.
Little evidence for relationships between element concentrations and individual sizes was found in the selected species.The types of association between fish size/age and levels of metals, metalloids, and other elements are shifting in literature, and there is no clear pattern for those relationships (Ali and Khan, 2018).Our study found a moderately significant positive relationship between P. nattereri body size and Ba levels.In contrast, Donald (2017) found negative relationships between Ba muscle concentrations and the size of 11 fish species from Lake Diefenbaker, Canada, but only for five species these relationships were significant.Pourang et al. (2005) also detected significant weak relative relationships between Ba muscle levels and size in five sturgeon species from Caspian Sea.According to Donald (2017), more studies addressing a broad variety of fish species are needed to elucidate the aspects of Ba bioaccumulation and individual development.We also detected disparate significant relationships between Mn concentrations and body size in M. duriventre and P. argenteus, with weak positive for the first and moderate negative for the later.Łuczyńska and Tońska (2006) found negative correlations between muscle Mn levels and size of perch Perca fluviatilis and pike Esox luscious in lakes of northeastern Poland.The yellow catfish Tachysurus fulvidraco (= T. sinensis) from Xiang River, southern China, also presented a moderate negative correlation between Mn levels in muscle and the body size (Jia et al. 2017).Negative correlations were also observed for Clarias gariepinus and Labeo umbratus from the Upper Olifants River and Klein Olifants River, South Africa (Coetzee et al. 2002).Thus, there are indicators that Mn levels in fish muscular tissue tend to decrease with age and corporal growth.An intraspecific condition of an element concentration decrease in determined organs as a function of the increase in fish body size implies that tissue renewal occurs at a greater rate than the storage of the element in such organ (Cross et al. 1973).In the case of P. nattereri, it is possible that the intake and fixation of Mn in muscular tissues are swifter than the muscular cell renewal, leading to increased bioaccumulation.Manganese may be bioconcentrated in freshwater lower trophic levels and producers (ATSDR 2012), and P. nattereri explores these levels through the consumption of insects, small fish, and plants (Ferreira et al. 2014).

Final considerations and recommendations
The present study brings new data on a broad set of non-essential and essential elements in edible fish from the highly exploited middle Miranda River.Most previous studies concerning the fish ecotoxicology in Pantanal focused on determining the Hg levels in the northern basins (Mato Grosso State).There, Hg was found in variable concentrations, some exceeding the limits established by Brazilian and international protocols, as the case of P. nattereri and Pseudoplatystoma spp. in Paraguay River (Vieira 2001;Santos-Filho et al. 2016), P. nattereri, Serrasalmus spilopleura (= S. maculatus), and Pseudoplatystoma spp.and P. maculatus (= P. pantaneiro) from Cuiabá River (Vieira 1991;Hylander et al. 2000), and Acestrorhynchus altus (= A. pantaneiro), Hoplias malabaricus, S. nattereri, and P. fasciatus (= P. reticulatum) from Bento Gomes River (Vieira, 1991).
The state of Mato Grosso has a long-term history of gold mining, and Hg is heavily used for gold amalgamation in the many mines distributed in the state.Surprisingly, Hg was not detected in any fish sample from the middle Miranda River analyzed in our study.However, more fish species need to be tested, considering their historical use in the region and the Hg burdens already detected in piscivorous giant otter P. brasiliensis from MRB (Soresini et al. 2021).However, our study calls attention to the possible status of heavy Pb contamination in the fish assemblage from the middle Miranda River.Although not widespread in our sample, the high levels of Pb detected mainly in benthic P. argenteus and P. pantaneiro are worrying, which demands new investigations in other benthic species of socioeconomic importance, such as the large-sized bottom-dwelling cat fishes Pseudoplatystoma corruscans, P. reticulatum, Sorubim lima, Zungaro jahu, Pinirampus pirinampus, to mention ones, as well as analysis on abiotic compartments such as sediment as water.
Lead is present in the formulation of glyphosate-based and other herbicides and fungicides.In compounds such as Glyfos ® , Matin ® , and Starane ® -all of them freely traded in Brazil -, Pb was found in concentrations far above the WHO (2011b) guideline values (Defarge et al. 2018).Considering the new safety values suggested by EFSA (2020), these exceeding burdens are of greater magnitude.The plateau surrounding Pantanal has been extensively modified through the deforestation of the Cerrado Biome (savannah phytofisiognomies) and the establishment of large agricultural and silviculture areas.In this scenario, herbicides and other pesticides applied in plateau crops may disperse over great distances into Pantanal rivers and flood plains from the upper stretch of hydrological basins (Alho and Vieira 1997).Another aspect of the current 'Pantanal in transformation' is the enlargement of mining activity in areas surrounding the middle and lower Miranda River, such as the Urucum massif.Although there is no available data on the geochemical background of Urucum and other massifs around MRB, mining activity could be acting on the mobilization of Pb into water bodies.Natural content of Pb in rock matrices of the region could not be discharged, considering that Pb was not detected in P. nattereri from the Paraguay and Bento Gomes rivers located in a northern Pantanal landscape highly impacted by anthropic activities (Santos-Filho et al. 2016).Another aspect to be raised is the long-term inclusion of tetraethyl lead in gasoline in Brazil, which persisted until 1989 (Costa 2001).It is known that Pb is highly persistent in river sediments (Dunlap et al. 2001).The studied stretch of Miranda River has a long-term intense traffic of motorized vessels, which may represent a potential resource of Pb input into the river compartments.
The Brazilian National Health Surveillance Agency -ANVISAhas established the maximum tolerance limits for Pb level in fish and other fishery products, which is specified as 2.0 mg/kg (or 2 μg/g) (Anvisa 1998).
According to this threshold, the consumption of 150 g of a fishery product containing this concentration would result in a Pb burden of 300 μg, which exceeds the EFSA (2020) TDI of 105 μgestablished based on rigorous protocols on human exposureby 1.8 times.Thus, it is strongly recommended the review of Brazilian legislation about the maximum levels of Pb in commercialized fish, aiming to adopt a more secure standard on contaminants in fishery products.It is also recommended that a Pb monitoring program be developed in biotic and abiotic compartments in all Brazilian Pantanal.3).
measured the concentrations of total Hg in hair of P. brasiliensis from Miranda and Vermelho rivers (southern Pantanal), finding concentrations conspicuously higher, ranging from 2.01 to 12.06 μg/g d.w.(dry weight).The Miranda River basin (hereafter MRB) is an important affluent of the left margin of the upper Paraguay River basin.It constitutes one of the main systems acting in flooding the Southern Pantanal plains.The MRB drains an area of about 43.000 km² in the state of Mato Grosso do Sul and is divided into two sub-basins: Miranda and Aquidauana.Both sub-basins have tributaries located both in the Plateau areas that surrounds Pantanal wetlands (Bodoquena and Maracaju mountain ranges) and in the Pantanal sedimentary basins itself (Pott et al. 2014).The middle portion of Miranda basin has great importance to the subsistence, professional, and tourist/sportive fisheries conducted in Pantanal, which represents one of the main socioeconomic activities developed in the state of Mato Grosso do Sul.Only in 2018, 114 tons of fishery resources were obtained from rivers Miranda and Aquidauana.That year, Miranda and Aquidauana rivers stood out as the second and third basins in the exploited fishery volume, respectively, after only the Paraguay River.That volume corresponds to 37.4% of all the fishery resources obtained in 2018 from all river basins in Mato Grosso do Sul (Catella et al. 2020).The fishery resources from MRB, however, have invaluable socioeconomic importance to Southern Pantanal.Due to fishery and the well-preserved ecosystems, MRB currently represents an ecotourism hub in high development in the Pantanal region, benefiting directly and indirectly the local communities (IMASUL 2016).
estimated a per capita consumption of 153.4 g in Baixada Cuiabana, while Ceccatto et al. (2015) estimated a per capita consumption from 140 to 200 g in Poconé-Cáceres region.Thus, we estimated the daily intake of the elements herein analyzed based on a per capita fish consumption of 150 g, for two parameters: (1) mean values (data sets composed of specific concentrations with at least three individual levels >LOD); (2) maximum concentration values (data sets composed of specific concentrations with at least two individual levels >LOD or single specific concentration values >LOD).
, US Institute of Medicine Panel on Micronutrients (2001), NSCPS (2004), Hadrup and Lam (2017), EFSA (2014, 2020), and U.S. National Institute of Health (2021) reports.When the reference value was given as provisional tolerable weekly intake (PTWI), we converted it to a tolerable daily intake (TDI) value adjusted for a 70 kg person.The TDI's for Cd (WHO 1992), Cu (WHO 1998), Fe (US Institute of Medicine Panel on Micronutrients 2001), Mn (US Institute of Medicine Panel on Micronutrients 2001), and Se (U.S. National Institute of Health 2021) were established without body weight references (specified as TDI for ‗adults') and therefore were considered for estimates as informed.
Significant differences are marked in bold.

Fig. 1 .
Fig. 1.Location of the study area in South America (A), Mato Grosso do Sul State (B), and the studied stretch of Miranda River (C; highlighted in red)

Table 1 .
Species analyzed in the present study, and respective number sampled individuals (N) sizes (mean ± one standard deviation (range)), diets, and habits

Table 2 .
Wavelengths and limits of detection (LOD) recovered for the analyzed elements

Table 3 .
Summary statistics of elements concentrations (μg/g wet weight) given as mean ± one standard deviation (range)

Table 4 .
Results of tests for significant interspecific differences in element concentrations

Table 7 .
Tolerable daily intake (TDI) of elements (μg) for a 70 kg person according to international protocols and estimates of daily intake (μg) of elements through the consumption of 150 g of muscle of the analyzed fish