Evaluation of digestion efficiency and limit of quantification (LOQ)
One sample of each material was selected randomly (serum - 59; liver - 18; muscle - 1; kidney - 8) to determine the RCC. The following values were obtained: 45.4 ± 0.7% for serum, 26.1 ± 0.1% for liver, 22.53 ± 0.03% for muscle, and 35.7 ± 0.5% for kidney. These values are acceptable when compared to those obtained for other similar materials, such as lean meat (25 - 30%), bovine liver (24 - 32%), and human blood (25 - 29%) (Barin et al. 2019), which indicates that the digestion methods employed were efficient.
Obtaining low RCC values is essential for the proper performance of the mineral quantification step, considering that the introduction of large amounts of non-decomposed organic matter into analytical equipment can cause clogging of the sample introduction system, plasma instability, and intensification of background emission when the ICP OES technique is used (Wiltsche et al. 2015).
Also, the achieved OCC values (2.59 ± 0.08% for serum, 65.9 ± 0.5% for liver, 45.1 ± 0.7% for muscle, and 47 ± 2% for kidney) were compared with data available in the literature for the original carbon content in different samples, such as lean meat (50%), bovine liver (51%), pork kidney (49%), and bovine muscle (41%) (Barin et al. 2019). Except for the liver from the freshwater turtle, which had an OCC value (65.9 ± 0.5%), making it relatively different from the bovine liver (51%), the other results were similar even though they were tissues taken from animals completely different taxonomic groups. The low OCC value obtained for the serum can be explained by the high water content (93%) present in this biological fluid.
The LOQ of the method for each element analyzed are shown in Table 1. The values below the LOQ were expressed with asterisks in Tables 2, 3, 4, and 5.
Multi-elemental composition in serum
In serum, the quantified elements were Al, Cu, Fe, Mn, Pb, and Zn (Table 2), with a significant difference between the areas for Cu with the highest mean concentrations in A2 and A3, Fe in A4, and Pb and Zn in A2 and A4. There was no significant difference in the accumulation of elements between males and females. Among seasons, only Cu showed a higher average in the dry period (Table 2).
Although many elements in the serum have resulted in values lower than the LOQ of the analytical method (Cd, Co, Cr, Mo, and Ni), it was still possible to quantify Al, Cu, Fe, Mn, Pb, and Zn accurately. The results could be compared to the study of Piña et al. (2009), who quantified Al, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, and Zn in whole blood of P. geoffroanus in a river in São Paulo, with similar characteristics of anthropic impacts. The values found for Al (0.09 - 0.11 mg L-1) in that study were much lower than in the present study; while for Cu (1.15 - 2.20 mg L-1), they were higher; for Pb (0.54 - 1.15 mg L-1) and Zn (1.17 - 2.18 mg L-1) were very similar.
Another study carried out with fresh blood of freshwater turtles was developed by Santos et al. (2021) with P. geoffroanus, Mesoclemmys tuberculata, and Kinosternon scorpioides in the Tapacurá River dam in Pernambuco, one of the tributaries of the Capibaribe River which receives the same influences. In this study, the mean values obtained for Al (8.93 mg/L), Cu (1.45 mg/L), and Fe (118.2 mg/L) were higher than those in our work. Considering that the determination of the elements occurred in whole blood, Fe was mainly influenced by hemoglobin, while our quantification occurred in serum.
Blood and serum have much lower mineral concentrations when compared to storage tissues, except for acute absorption with a high level of exposure (Grillitsh and Schiesari 2010), as demonstrated in our study. For elements with high affinity for erythrocytes (e.g., Hg), the use of whole blood in the analysis is more appropriate, as plasma or serum levels are considerably lower, staying close to or below the limits of detection and quantification, resulting in false negatives during an exposure assessment (Day et al. 2007). For biomonitoring purposes, Aitio et al. (2007) and Day et al. (2007) justify that the variability and availability of cellular components in the blood can influence the concentration of certain minerals, which requires correction by hematocrit and hemoglobin.
The concentration of minerals in the blood reflects their recent uptake and redistribution of exposures. However, the turnover of metals in the blood may be lower for elements that have not reached accumulation thresholds (Burger et al. 2006). Consequenty, the extrapolation of the concentrations found in the blood of the other tissues is limited, especially in wild animals (Grillitsh and Schiesari 2010).
Multi-elemental composition in liver, muscle, and kidney
In the liver, the quantified minerals were Cd, Co, Cu, Fe, Mn, Mo, Ni, Pb, and Zn (Table 3), with a significant difference between the areas for Cd, which had higher averages in A2 and A3; Co in A3>A2=A1>A4; Cu in A3=A2=A1>A4; Fe in A2 and A3; Mn and Mo in A3. There was no difference between males and females. Among stations, only Mn had a higher average in the dry period (Table 3). The liver was the primary storage site for Cu, Fe, and Zn and, to a lesser extent, for Cd, Mn, Ni, and Pb.
In the muscle, the quantified elements were Cu, Fe, Mn, Ni, Pb, and Zn (Table 4), with a significant difference among the areas only for Zn, which had higher averages in A3>A1=A4>A2. There was no difference related to the sex of the animals. Among stations, only Mn had a higher average in the dry period (Table 4). Muscle showed accumulation for Fe, Ni, Pb, and mainly Zn.
In the kidney, except for Pb, which was below the LOQ, all other minerals showed a significant difference among areas (Table 5), with Co, Cr, and Mn with higher averages in A3; Cd, Cu, Mo, and Zn in A4; while Al, Fe, and Ni with higher averages in A3 and A4. There was a significant difference between sexes only for Al, which was higher in females. Among the stations, the elements Al, Co, Cr, Cu, Fe, Mn, Mo, and Ni showed a significantly higher difference in the rainy season; only Zn had a higher average in the dry season (Table 5). The kidney was the major storage tissue for Al, Cd, Co, Cr, Mn, Mo, and Ni.
The major concentrations of minerals were found consistently in the liver and kidneys, as reported by Grillitsch and Schiesari (2010) for marine turtle, in dry samples: Cd (liver 0.22 – 148 mg kg-1; muscle 0.01 - 39.24 mg kg-1; and kidney 2.49 - 653 mg kg-1) and Pb (liver 0.13 - 14 mg kg-1; muscle 0.08 - 19.78 mg kg-1; and kidney 0.09 - 69.89 mg kg-1). Storelli and Marcotrigliano (2003) studied wet marine turtle samples in which the following concentrations were obtained: Cd (liver 0.3 - 26 mg kg-1; muscle 0.01 - 2.21 mg kg-1; and kidney 0.39 - 70.2 mg kg-1) and Pb (liver <LOD - 4.9 mg kg-1; muscle <LOD - 5.53 mg kg-1; and kidney <LOD - 4.9 mg kg-1).
This typical distribution among the tissues demonstrates that the liver and the kidney have the highest concentrations for most elements, as they have an affinity for metallothionein. Muscle concentrations can be particularly high for Pb after prolonged and high-level exposure. At the same time, the integument, due to keratin, accumulates mainly metal ions such as Hg, As, and Pb in the epidermis and nails (Sakai et al. 2000).
In vertebrates, minerals generally have a specific affinity for certain organs (organotropism), which, in turn, tend to serve as accumulation sites (Meyers-Schone and Walton 1994). Minerals bind to protein peptides, such as albumin and metallothioneins, which have a high affinity for low molecular weight metals and are present in all tissues. However, the induction capacity is more significant in tissues involved in the uptake, storage, and excretion of metals, such as the intestine, liver, and kidney (Sakai et al. 2000). The resulting differences in the distribution pattern among tissues and organs becomes more evident when the exposures are prolonged and in high, but not lethal, concentrations (Grillitsh and Schiesari 2010).
Muscles were the primary storage site for Zn, Ni, and Pb, equivalent to the kidney and liver, indicating high-level exposure to these elements, which was also recorded by Sakai et al. (2000). Burger et al. (2006) compared levels of 17 elements in the blood and muscle of three snake species in the United States and found a high degree of variation between these two types of samples. Muscle bioaccumulation occurs as a final storage process since the renal and hepato-biliary excretions were insufficient (Wood 2011).
The environmental characterization of the studied areas can help understand the observed differences in concentrations. The highest mean concentrations were observed in areas A2 and A4, where the sources of pollution for the river are, respectively: the sugarcane industry (A2) and its extensive plantation with the everyday use of vinasse or filter cake to fertilize the soil (Anacleto et al. 2017; de Godoi et al. 2019); and the textile industry (A4), in which the dying phase is the critical point, with the use of dyes that contain metallic complexes such as Cd, Co, Cr, Cu, Pb, Zn, among others (Rovira and Domingo 2019).
Area A3, characterized by urbanization and various commercial activities with diffuse effluents, also had high levels of minerals. Lower concentrations were found in the mouth of the river, area A1, with an estuary with mangrove vegetation that acts as an ecosystem filter. In addition, this area has a tidal variation that dilutes the river water with that from the sea. These estuarine conditions have already been pointed out in some studies, such as Teuchies et al. (2013), Fortune and Mauraud (2015), Zang et al. (2015), and Nguyen et al. (2020).
As for accumulation, there was no significant difference between males and females, which was also reported by Piña et al. (2009), Grillitsh and Schiesari (2010), and Santos (2021). Only Al was higher in females in the kidney; however, our study found no explanation to justify this result.
Regarding seasonality, only Cu in the serum and Mn in the liver and muscle were higher in the dry period, while Al, Co, Cr, Cu, Fe, and Mn were higher in the kidney in the rainy period. According to Meche et al. (2010), who evaluated compounds in free-living fish in São Paulo, the rain causes a current that removes the compounds accumulated in the sediment, increasing the exposure of the animals, resulting in mineral concentration in the kidneys, as they serve as filters.
Maximum elemental limits established in the legislation
Concerning the national norms that establish limits of concentration for mineral elements, Resolution n.º 357/2005 of the National Environmental Council [Conselho Nacional do Meio Ambiente] (CONAMA) describes the types of bodies of water and environmental guidelines for their classification (CONAMA 2005), while Resolution No. 430/2011 of this same institution provides the conditions and standards for the discharge of effluents (CONAMA 2011). Bearing that artisanal fishing occurs in three studied areas (A1, A2, and A3), comparing the obtained results with food standards is essential. In this sense, the Collegiate Board Resolution No. 487/2021 and the Normative Instruction No. 88/2021 of the Brazilian Health Regulatory Agency [Agência Nacional de Vigilância Sanitária] (ANVISA) provide the maximum limits of inorganic contaminants in food (ANVISA 2021a, 2021b), can be used to evaluate fish used for human consumption (Table 6).
CONAMA (2005) classifies fresh waters according to use and quality standards, from best to worst: special class, class 1, class 2, class 3, or class 4. The water in the Capibaribe river is class 2 (Pernambuco 2010), being used for public supply after conventional treatment, as well as for fishing, irrigation, leisure, and animal feed. However, the concentrations of elements (Al, Cu, Fe, Pb, and Zn) obtained in the serum of the animals examined exceeded even the limits of water class 3, with lower quality than class 2 (Capibaribe River). Thus, the actions of basic sanitation and control of effluents in the river require a more outstanding commitment from the Pernambuco state and the 42 municipalities included in the Capibaribe River basin to minimize environmental impacts.
ANVISA (2013) determines maximum limits for As, Cd, Pb, Hg, and Sn in food. The results obtained in our study were compared with the limits established for these minerals in fish. Cadmium and Pb were above the maximum limit in the four studied areas and in all types of samples where they were identified. These findings represent a risk to consumers of this animal, as occurs in A3, as well as to the fish population and for other types of food growing or being produced by the riverside. Cadmium causes oxidative stress, liver degeneration, and necrosis (Huo et al. 2017), as well as nephrotoxicity, and Pb acts mainly on the nervous system and gastrointestinal tract (Jaishankar et al. 2014).
The maximum levels of these metals quantified in the liver, kidney, and muscle of P. geoffroanus considerably exceed the limits for animal health and food security products from the Capibaribe River. This result suggests the likelihood of long-term exposure to low or less than acute toxic metal levels in the environment (Grillitsch and Schiesari 2010).
Thus, studies to determine the levels of mineral elements and to evaluate their adverse effects in free-ranging freshwater turtles are scarce, and those already carried out have used only blood (Burger et al. 2010; Piña et al. 2009; Santos 2021). The present study is the pioneer in using different biological samples (serum, muscle, liver, and kidney) for quantifying minerals in P.geoffroanus from a river subjected to different anthropogenic impacts.