Heavy Metal Concentration in Neotropical Aquatic Snakes (Helicops pastazae) and Its Potential as a Bioindicator of Water Pollution

The purpose of this study was to test the potential role of the aquatic snake Helicops pastazae as an indicator of water pollution caused by heavy metals. In particular, we tested whether the total heavy metal concentration is related to (1) the position (upstream vs downstream) of the sampling point and its distance from the point where wastewater is discharged; (2) the taxonomic group studied: piscivorous snakes vs characid fish that occupy the same habitats; and (3) the organ or tissue examined: snake liver versus muscle. We used atomic absorption spectrophotometry with electrothermal atomization to quantify cadmium (Cd), chromium (Cr) and lead (Pb) and found significant differences between some of the sampling points, with particularly high metal concentrations detected upstream at point 1. However, we found no clear spatial pattern nor any significant differences in the concentration of any of the metals in fish and snake muscle, suggesting that both species accumulate similar amounts of the sampled elements. With regard to interactions, snake liver had the highest concentrations of Cd, while muscle had the highest concentrations of Pb and Cr, which may indicate tissue affinity differences for certain metals. Altogether, our results indicate that H. pastazae accumulates contaminants differentially, depending on the tissue and location, which highlights their potential as bioindicators of water contamination. Further research is necessary to understand their role as bioindicators based on extensive sampling and environmental contaminant data.

resulting organometallic complexes may inhibit structural changes being made to protein and thereby affect the transport of essential elements and the metabolism of reactive oxygen species or of free radicals related to oxidative stress, among others (Zorrilla 2011).
Snakes may serve as biological indicators of environmental pollution, given their position as primary or secondary predators, their restricted migration and their long life cycles (Heinz et al. 1980;Campbell and Campbell 2001;Burger et al. 2005Burger et al. , 2007Burger et al. , 2017Albrecht et al. 2007;Haskins et al., 2019). Despite the potential value of aquatic snakes as bioindicators, they are not commonly used in studies on environmental pollution (Campbell and Campbell 2001;Campbell et al. 2005;Burger et al. 2005Burger et al. , 2007Heydari and Riyahi 2015;Haskins et al., 2019) given that the information related to their life history and ecological traits is scarce and incomplete in comparison with other reptiles. Existing limitations in terms of their detection and capture and the cultural biases regarding snakes make research related to this group more difficult to conduct and interpret (Haskins et al. 2019).
Recently, aquatic snakes have been considered as potential bioindicators of contaminants showing spatial and temporal trends in bioaccumulation (Haskins et al., 2021a(Haskins et al., , 2021b. Haskins et al. (2019) highlight the potential of different snake species including aquatic snakes as biomarkers of mercury contamination given their natural history traits such as diet, reproductive strategies and habitat that lead them to accumulate high amounts of mercury and potentially other metals and metalloids. The Helicops pastazae (Colubridae) snake, distributed from Colombia to Argentina (Uetz et al. 2019), has been classified in the Least Concern group according to the IUCN. It has semi-aquatic habits, as evinced by the adaptations to their nostrils and eyes in dorsal position and keeled scales (Segall et al. 2016). Snakes in this genus feed on fish, anurans, lizards and other snakes, and although there is a lack of information on the specific diet of H. pastazae, it is thought to feed on fish and to be an opportunistic predator (Almendáriz et al. 2017;Muñoz-Saba et al. 2019).
To assess its potential as a bioindicator, the purpose of the present study was to estimate the concentration of some heavy metals in different tissues of H. pastazae and to compare bioaccumulation of the snakes to those in a fish species of the Characidae family that co-occur with them. The sampling site covered a section of the Bata River (eastern Colombian Andes), which receives residual water from the sewage system of the municipality of Santa María, through the streams of La Argentina, El Toro and Caño Cangrejo, where there is no liquid waste treatment and the networks are in poor condition (Corpochivor 2003). Upstream from this section, the river receives water from the Chivor reservoir, which probably concentrates the contamination by sediments, residues and dissolved nutrients (Corporación Autónoma Regional de Cundinamarca et al. 2006). Given that the Bata River is home to a various species of fauna, that there are no studies on heavy metals in this area, and that snakes can potentially serve as bioindicators, a study was undertaken to quantify total Cd, Pb and Cr concentrations in the liver and muscle of Helicops pastazae snakes, and in fish muscle.
The following hypotheses were tested: (1) The total heavy metal concentration depends on the position (upstream vs downstream) of the sampling point and its distance from the point where wastewater is discharged and is expected to be greater downstream from the discharge point; (2) the total heavy metal concentration depends on the taxonomic group studied and is similar in snakes and fish from the same sites; and (3) it also depends on the organ or tissue examined and is presumably higher in the liver than it is in the muscle.

Study Site
Four sampling points were selected: two upstream of the wastewater discharge (known as Caño Cangrejo) and two downstream in the municipality of Santa María (Fig. 1). The sampling was carried out in the months of January and February, during dry season (García-Cobos et al. 2020).
The procedures for euthanasia and manipulation of individuals were approved on October 18, 2018, by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) by analyzing the animal use format COR_C.FUA_18-016. Individuals were collected under the permit: "Permiso Marco de Recolección de Especímenes de Especies Silvestres de la Diversidad Biológica con Fines de Investigación Científica No Comercial," certified under research project PR.6.2018.4967 "Integración de rasgos funcionales y moleculares en estudios evolutivos de comportamiento y fisiología," with mobilization permit P04967S3591_N0004.
Snakes were found and collected under rocks along the riverbank, during the day. Most individuals were adult (length greater than 30 cm), but juveniles were also collected where finding adults became difficult. Individuals were euthanized by intracardiac injection of 3-5 mL of 2% xylocaine anesthetic for adults and a 1-2 mL dose for juveniles. This euthanasia process is approved by the AVMA (American Veterinary Medical Association) and guarantees a non-traumatic death. Subsequently, tissues were extracted and later preserved in a refrigerator with ice until arrival at the laboratory, always within 48 h.
An unidentified fish species, belonging to the Characidae family was abundant in the study area. Specimens were collected during the day on the riverbank and using a net. They 1 3 were euthanized by freezing as approved by the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and some other fish euthanasia guidelines (Barker et al. 2002;Blessing et al. 2010), to be preserved until they reached the laboratory.
We chose to sample three metals of ecotoxicological importance (Henze et al. 2002): Cr, Pb and Cd. The geological baseline of the municipality of Santa María reveals the presence of clays, sandstones, gypsum, aleurolite, among others, which generate mostly iron and aluminum derivatives (Corpochivor 1996).

Analytical Methods
All standard solutions and elements dilutions were prepared in ultrapure water (resistivity of approximately 18 MΩ cm). All solutions were prepared daily by direct dilution in deionized water and high-purity nitric acid. Glassware and polypropylene flasks were submerged in 5% (v v −1 ) nitric acid for 24 h, followed by rinsing with deionized water before analysis. The stock solutions were prepared using 1000 (mg L −1 ) commercial standards of Cd, Pb and Cr (PanReac AppliChem, Spain). All dilutions were acidified by 0.5% (v•v −1 ) nitric acid (65% RE, Pure) purchased from Carlo Erba, Italy. The ammonium dihydrogen phosphate and nitrate magnesium modifiers used in the analysis were NH 4 H 2 PO 4 (100 ± 2 g L −1 ) in H 2 O and Mg(NO 3 ) 2 •6H 2 O (10.0 ± 0.2 g L −1 ) in HNO 3 (ca. 17%). These were commercially acquired from Supelco (Germany).
Analytical measurements were taken in an HR-CS-AAS, CONTRAA 800D (Analytik Jena, Germany) transversally heated graphite furnace. High-purity argon at 2 L•min −1 as operation inert gas and pyrolytically coated graphite tubes with L'Vov platform were used (Analytik Jena Part No. 407-A81.025). Each element was analyzed according to default conditions recommended in the apparatus software as "cookbook" settings. All measurements were taken in triplicate sample injection by 108 positions AS-GF autosampler.

Sample Preparation and Laboratory Procedures
Sample preparation and digestion was performed using a modified EPA method 3052 (EPA 1996), adjusted on the basis of preliminary tests. Each individual was washed with deionized water to remove dirt and external contamination on the skin (Burger 1992). To obtain the sample for the snakes, muscle and liver tissue were extracted from the individuals via 2-cm longitudinal incision made ventrally in the 1 3 middle of the body, using a ceramic scalpel and stainlesssteel tweezers to avoid traces of metals and pollution (FAO/ SIDA 1983). In the case of fish, muscle tissue was removed by making an incision above the lateral line, between the dorsal and caudal fins (Queensland Government 2018). The material was stored in plastic containers previously washed and sterilized with 5% nitric acid and deionized water and was subsequently refrigerated at − 17 °C until digestion and respective analysis (FAO/SIDA 1983). The digestion procedure followed microwave-assisted acid digestion of siliceous and organically based matrices (EPA 1996), which consisted of weighing the solid material and then adding 10 mL of concentrated HNO 3 to it.
We proceeded to weigh each of the samples in the vials to carry out the digestion and to later estimate the concentration of metal per unit mass. Following this, each sample was predigested for 1 h by adding 10 mL of concentrated nitric acid, allowing the vessels to release gases. Subsequently, the sealed sample vials were put in the microwave. The temperature of each sample rose to 180 °C in approximately 5.5 min and remained at this temperature for 9.5 min until complete decomposition, followed by a cooling step. At the end of the microwave program, the samples were left to cool for further 5 min before extraction. When the seals were removed from each vial, the sample was filtered, and 5% nitric acid was added to complete 10 mL in case of volume loss (EPA 1996). All metal concentrations are expressed in µg g-1 of wet tissue weight. The heavy metals were measured using a ContrAA® atomic absorption spectrometer, with electrothermal atomization, taking into account the measurement of standard samples of known concentration and blanks to be able to estimate the specific calibration curve (Burger 1992). For quantification, 25 µL of sample was diluted in 1 mL of deionized water (25:1000), and following measurement, each concentration obtained was multiplied by 40. The limits of detection (LD) were obtained by multiplying the standard deviation obtained from the absorbance measurement of ten targets by three and then dividing this by the slope of the calibration curve for each heavy metal. The results for LD for each element were (in µg g −1 ) 0.00009 for Cr, 0.0000008 for Cd and 0.000042 for Pb.

Statistical Analysis
All statistical analyses and graphics were performed using the R software (R Core Team, 2013), as implemented in RStudio. Multivariate and univariate normality tests were performed for each set of continuous data. When they did not meet the assumptions of normality and homogeneity of variances and could not be transformed to achieve a normal distribution, nonparametric tests were used.
The Spearman nonparametric correlation test was used to assess the correlation between the concentration of the analyzed metals, while, given that the output variable (concentration) has a lower distribution limit than 0, a generalized linear model (GLM) with a Poisson link was built to model the possible relationship between the concentration of metals in the snakes' different tissues and the body mass.
Linear models following a Bayesian approach were built to test whether the concentration of each metal was predictable from the type of organism (snake or fish), the type of snake tissue (liver or muscle) and the sampling point (the position with respect to the wastewater discharge point in the river). A Gamma Hurdle distribution was assumed, which makes it possible to analyze continuous data that includes a large number of zeros (i.e., zero-inflated data).

Results
A total of 28 Helicops pastazae individuals (7 males, 14 females and 7 juveniles) and 28 individuals of fish from the Characidae family were collected; 7 individuals of each species at each sampling point (Fig. 2). Female snakes weighed on average 78.71 ± 44 g (mean ± standard deviation for all measures) and measured on average 43.4 ± 11 cm SVL, while the males weighed on average 39.28 ± 21 g and measured on average 33.7 ± 6 cm SVL. Juveniles (measuring a total length of less than 30 cm, which was estimated based on the smallest female that could be sexed given that no juvenile size data are found in the literature) weighed on average 5.42 ± 0.5 g and measured on average 16.3 ± 0.6 cm SVL.
Snake body mass was inversely correlated with Cr concentrations (Z = -2.411, p = 0.0159, n = 83 samples, correlation coefficient = -0.02995) (Fig. 3c), but not with Pb or Cd. The pairwise correlation of the concentration of heavy metals was far from significant (-0.0826 < Spearman correlation coefficient < 0.0995 and P > 0.37 in all cases), which means that individuals with a high concentration of a particular metal do not necessarily have a high concentration of the other metals. Also, correlations between tissues for each element were not significant for Cd (Spearman correlation coefficient = -0.2953 and P = 0.13) or Pb (Spearman correlation coefficient = 0.1617 and P = 0.42), but were significant for Cr (Spearman correlation coefficient = 0.6643 and P = 0.00015), which could indicate the systemic transport of Cr in snakes.
Considering each sampling point, significant differences were only observed for Cr at point 4, where the concentrations were higher in the snakes than they were in fish (Fig. 4). Fish muscle revealed a higher average concentration of the analyzed metals than the concentration found in the snake muscle. We found no significant differences in Cd, Cr or Pb concentrations found in fish and snake muscle (Fig. 5), nor in the concentration of different metals in the same tissue (Table 1). For Cd, significantly higher concentrations were found in snake liver than in snake muscle (Figs. 6,7a) and Pb and Cr concentrations were significantly higher in the muscle (Figs. 6b, c). These patterns remain when comparing the average concentrations of metals for each of the river points analyzed (Table 2).
When comparing the difference in terms of the sampling points upstream and downstream from the water discharge point, higher Cr and Cd concentrations were found in fish and snake muscle at point 3 than at point 1 and higher Pb concentrations at point 2 than at points 1 and 4 (Fig. 5). In contrast, when analyzing snake tissue, point 1 led to higher concentrations for all metals in comparison with at least one of the other points (Fig. 7). Thus, no clear spatial pattern was found.

Discussion
Female, male and juvenile frequency varied at each sampling point (Fig. 2). An attempt was made to collect only adult individuals, but some juveniles had to be captured given that in some points not enough adults were found. Although the juvenile condition could imply less bioaccumulation of metals, this pattern did not appear in the analyses. There was no evidence of umbilical cord and some of the juveniles were shedding their skin. This growth indicator reveals that food intake has taken place and considering that diet is a main factor involved in heavy-metal exposure (Lemaire et al. 2018;Hopkins et al. 2001), it could be concluded that metal bioaccumulation takes place even with just a few ingested animals. Maternal transfer from gravid females to their offspring is another means by which juvenile snakes can be exposed to metals (Chin et al. 2013), and this may affect the metal concentrations found in this age group.
Another point to consider is that many water snake species exhibit ontogenetic dietary shifts as they age, meaning that as they grow, they could change their dietary preferences (Mushinsky et al. 1982). The dietary information related to Helicops pastazae is limited, and no information is found on differences in their diet based on size, but if there is an ontogenetic dietary shift, differential metal bioaccumulation patterns could be found depending on the age of the individual. Heavy metal bioaccumulation could also be influenced by sex-specific factors such as maternal transfer from gravid females to offspring. Both aspects therefore merit further investigation.

Comparison Between Sampling Points
Two statistical models were developed to assess whether there were differences between sampling points: one considering the organism and another considering the type of tissue. In the first model, significant differences were obtained for Cr and Cd concentration, with these being higher at point 3 than point 1 (Fig. 5). In the second model, lower concentrations were observed near the water discharge zone, particularly at point 3 for Pb and Cr (Fig. 7). This may indicate that both snake liver and muscle can reveal different concentrations along the river. As a general trend, metal concentrations are higher in point 1 when analyzing snake tissue.
The absence of a spatial gradient in metal concentration-expected to be lower upstream from the wastewater discharge point and higher downstream-may be due to the fact that the Bata River is located downstream from the Chivor reservoir, which receives sewage from the basin and has dammed waters that are highly contaminated by sediments, residues and dissolved nutrients (Corporación Autónoma Regional de Cundinamarca et al. 2006). This would cause the upstream points to show higher metal concentrations than some of the other points, which was the general tendency shown by the second Bayesian linear model (Fig. 7). Similarly, according to the municipality's residents, the Chivor dam discharges very high flows into the Bata River, approximately once a year and for several days, and also during periods of long inflows to the reservoir. The sudden The increase in river flow could drag some organisms downstream, possibly disrupting the expected pattern due to spatial migration. However, based on the current data, it is not possible to establish the potential source of higher metal concentrations in the river, given that no predictable results were found at spatial level. Not many studies have been conducted on Helicops pastazae, and nothing is known about its movement or migration patterns. In Nerodia sipedon, a Nearctic aquatic snake, different movement patterns were found depending on the time of the year, presenting changes in its range of activity without occupying an established or permanent home range (Macartney et al. 1988). However, other studies indicate that aquatic snakes have limited home ranges (Campbell and Campbell 2001;Hopkins et al. 1999;Heinz et al. 1980). Given that the sampling points are not far from each other, the snakes could migrate from one point to another, diluting the expected spatial patterns. However, based on the current data and the lack of information in the literature, it is not possible to establish the influence of snake migration on the results found at a spatial level.

Interspecific Comparisons
We have no information on the predator-prey relationships between the sampled snake and fish. Although the former could prey on the latter, both could be high-level predators in the aquatic community. This first scenario would suggest that both species accumulate similar amounts of heavy metals. On the other hand, given that the biotic community of the Bata River does not comprise large vertebrates (Muñoz-Saba et al. 2019), the snakes studied may very well be the top predators in this ecosystem, as they are considered opportunistic predators (Almendáriz et al. 2017) and possibly feed on different species of fish (Muñoz-Saba et al. 2019). Under the second scenario these predatory snakes were expected to reveal a higher concentration of heavy metals with respect to fish, due to bioaccumulation. However, there were no significant differences in metal concentrations in fish and snake muscle (Fig. 5), supporting the first scenario. Under the second scenario, our data would not support bioaccumulation along the food chain.
Under the second scenario, obtaining statistically equal concentrations between fish and snakes may indicate that they have some mechanism by which to excrete heavy metals. Various studies infer that snakes can excrete metals through different mechanisms, through feces, skin shedding and egg production (Campbell and Campbell 2001;Jones & Holladay 2006;Cusaac et al. 2016). High concentrations of chromium, lead, manganese and mercury have been found in the skin of the Nerodia sipedon aquatic snake compared to all of its other body tissues, suggesting that they can sequester these metals in their skin and excrete them through frequent shedding and, therefore, decrease the pollutant load  1 3 (Burger 1992;Campbell et al. 2005). Similarly, when comparing the levels of heavy metals between males and females of some snake species, it was concluded that there may be a transfer of metals from the female to the eggshell Burger et al. 2017), which may be possible considering that the study species is oviparous (García-Cobos & Gómez-Sánchez 2019). We cannot rule out the existence of different chemical and kinetic dynamics between species. In fact, a study conducted on a Thamnophis sauritus snake and anuran larvae found that the snakes had significantly lower levels of Pb and Cd than the larvae, indicating that these metals were not biomagnifying to superior trophic levels (Albrecht et al. 2007). It has also been established that Pb and Cd concentrations do not increase along the trophic chain in surface water ecosystems, and these metals have even been found in lower concentrations in the tissues of predatory fish than in lower levels of the food chain (Jezierska and Witeska 2006;Kenšová et al. 2010). Although some studies indicate a generalist diet for the Helicops genus, there are no studies that determine the diet of H. pastazae, except for one by Almendáriz et al. (2017), which indicates that it may feed on fish of the Hypostomus pyrineusi species. Not having definitive information about the diet of this species of aquatic snake in the study area limits our ability to present conclusive data on interspecific comparisons.

Comparison Between Tissues
No significant differences were found between the concentration of different metals in the same tissue (Table 1). However, different H. pastazae tissues did bioaccumulate metals differentially (Fig. 7). Cd was significantly more concentrated in the liver than in muscle, and Pb and Cr had significantly higher concentrations in muscle. This corroborates that tissues differ in their affinity for certain metals and that } not all tissues are useful for evaluating traces of a particular metal. For example, Cd appears to be accumulated mainly in the kidney and liver of fish (Jezierska and Witeska 2006;Panchanathan and Vattapparumbil 2006), and fish muscle tissue tends to have the lowest levels of metals (Jezierska and Witeska 2006). The higher concentration of metals in the liver may be due to the fact that it is a metal storage and detoxification organ. However, this does not explain the high concentrations of Pb and Cr found in muscle. Some studies show Cd concentrations to be higher in fish liver, while Pb concentrations tend to be homogeneous in the sampled tissues (Zorrilla 2011). For snakes (Pituophis melanoleucus), it has been found that metals have a greater affinity for the skin. This is especially true for Pb, which was found in higher concentrations in the skin than in other body tissues (Burger 1992). In alligators (Alligator mississippiensis) in contrast, the liver had the highest concentrations of cadmium, arsenic, manganese, mercury and selenium, while the muscle had the highest concentrations of Pb and Cr (Burger et al. 2000). The highest concentrations of Cd in liver were found in aquatic snakes (Nerodia fasciata) fed with contaminated and uncontaminated prey (Hopkins et al. 1999), as in Nerodia spp., where Cr and Pb were higher in the skin (Burger et al. 2007), which is consistent with other studies involving Nerodia sipedon . A study on marine snakes (Lapemis curtus) showed no differences in concentration between muscle and liver for Pb and Cd. In this study, in accordance with other studies on aquatic snakes (Burger et al. 2007;Hopkins et al. 1999;Campbell et al. 2005), higher concentrations of Cd were found in the liver than in the muscle. Similarly, higher concentrations of Pb and Cr were found in the muscle than the liver, which is consistent with other studies on aquatic snakes and other reptiles (Burger et al. 2000(Burger et al. , 2007Campbell et al. 2005). The diversity of results  in the literature shows that it is difficult to predict the tissue in which each type of metal will accumulate. Several studies have found that the liver accumulates high concentrations of metals, regardless of the route of incorporation, and is considered a good indicator of water contamination, since the concentrations that accumulate in this organ are proportional to those found in the environment, especially for copper and cadmium (Jezierska and Witeska 2006). The differential concentration of metals in each tissue could be explained given the differences in metal affinity for sulfhydryl, amino, phosphate, carboxyl and hydroxyl groups. These are functional groups whose concentration can vary between tissues generating different chemical reactions for the formation of an organometallic compound (Zorrilla 2011). Pb has great erythrocyte-binding capacity and can easily be substituted with divalent cations (Zuluaga et al. 2015); thus, it may, as most heavy metals, have a high affinity for tissues with high blood flow (van der Brink 2004). High affinity has been found between Cd and the kidneys, liver and bones (Zuluaga et al. 2015). Given these differences in levels of affinity, it can be inferred that tissues vary in their value as bioindicators of different metals.

Snakes as Bioindicators
Compared to similar studies, the present study reflects large quantities of metals, especially of Pb and Cr, found in different tissues of aquatic snakes. No differences were found between the concentrations of metals found in fish and snakes, which shows that aquatic snakes can potentially be a good indicator for water pollution. For a species to be a useful bioindicator, certain conditions must be taken into account: (1) there is a relationship between tissue contamination levels and dietary exposure (Hopkins et al. 2001);(2) there is a relationship between the levels of contamination in the tissues and the levels in the ecosystem, that is, that the contaminants found in the ecosystem are concentrated at detectable levels in the tissues; and (3) the species should reflect the levels of contamination in a specific area (Heinz et al., 1980).
Regarding the first condition, as the snake's diet is unknown, we cannot establish a relationship between tissue contamination levels and the level of exposure from diet. Despite this, finding detectable levels of metals in snake tissue can help us conclude that they absorb contaminants from their diet or via maternal transfer. The second requirement is met, as detectable heavy metal levels were observed in the snake tissue, which reflect the contamination present in the ecosystem in which they live. Beyond this, the differences in concentration in each tissue and organism may depend on toxicodynamics (Burger et al. 2007), on the different rates of metal incorporation and excretion, along with environmental factors, among others. In terms of the third condition, this study did not reveal the expected spatial results with respect to each sampling point in the Bata River. This may be due to snake migration or wastewater discharges, but aquatic snakes did reflect heavy metal pollution differentially at the different sampling points along the river.
Additionally, although specific migration patterns for H. pastazae are unknown, aquatic snakes are believed to be relatively sedentary, which may make them good indicators of local contamination (Heinz et al. 1980). The abundance of this species of snake in the Bata River and its long life cycle makes it a potentially good indicator of contamination during long temporal scales (Burger 1992), and it can be monitored at different points of the river without threatening its population. Besides this, aquatic snakes are primary or secondary predators in the trophic chain and may be susceptible to bioaccumulation of environmental pollutants, making them useful for evaluating compounds that can be transferred by trophic mechanisms (Campbell and Campbell 2001). Taking into account all of the above, Helicops pastazae's potential as bioindicators of heavy metal accumulation is highlighted, but a more extensive sampling and research related to environmental contaminant data is encouraged in the Bata River in order to stablish the relationship between the levels of contaminants in the ecosystem and in the snake tissue.

Conclusion
This study was conducted in order to provide information about the concentration of heavy metals in the liver and muscle of an aquatic snake (Helicops pastazae) and the muscle of fish of the Characidae family, as a means to assess the contamination present in the Bata River and adding further support to the importance of aquatic snakes for monitoring heavy metal contamination. Differential concentrations of metals were found in the liver and muscle tissue of H. pastazae, with the three metals showing significant differences in their concentration in tissues: Cd was higher in the liver and Pb and Cr in muscle, indicating that tissues have different bioindicator potential for monitoring different metals. Given the patterns of bioaccumulation and detection of heavy metal levels in the tissues of aquatic snakes, it is suggested that Helicops pastazae can potentially be a useful bioindicator of heavy metal accumulation at different temporal and spatial scales. Snakes are organisms that are usually not taken into account when conducting environmental pollution studies, so it is not possible to fully analyze the risks generated by a potential pollutant in an ecosystem. It is important to consider that the analysis of a greater number of bioindicators in an ecosystem provides information of different trophic levels and habitats that allow a complete evaluation of the pollution in a specific ecosystem. Accordingly, ecotoxicological studies in snakes are encouraged, given the importance of monitoring metals in different tissues and organisms in the trophic chain at local level and of comparing the results with those found for different locations. It is worth highlighting the importance of exploring non-lethal methods for long-term biomonitoring without putting the population of studied organisms at risk, as these have been useful in various studies. Research on the snake's diet and life history traits could help elucidate heavy metal spatial trends and biomagnification in this system.
Author's contribution MJHM wrote the research proposal, conducted the data analysis, did the field work, interpreted the results and wrote the initial manuscript. MRS and AA reviewed and approved the research proposal, results and conclusion, and they commented and contributed to drafting the manuscript. All authors read and approved the final manuscript.
Funding The departments of biological sciences, civil and environmental engineering, and chemistry at Universidad de los Andes provided the financial support for this research.

Availability of Data and Material
The datasets created during and/or analyzed during the current study are stored as Electronic Supplementary Material and available from the corresponding author on reasonable request.

Code Availability
The codes generated and used during the current study are available from the corresponding author on reasonable request.

Conflict of interest
The authors declare no conflicts of interest.

Ethical approval
The procedures for euthanasia and manipulation of individuals were approved on October 2018 by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) by analyzing the animal use format COR_C.FUA_18-016. Individuals were collected under the permit: "Permiso Marco de Recolección de Especímenes de Especies Silvestres de la Diversidad Biológica con Fines de Investigación Científica No Comercial," certified under the research project PR.6.2018.4967 "Integración de rasgos funcionales y moleculares en estudios evolutivos de comportamiento y fisiología," with mobilization permit P04967S3591_N0004.

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