The frequency of females, males and juveniles was variable at each sampling point (Fig. 2). An attempt was made to strictly collect adult individuals but given that in some points not enough were found it was necessary to capture juveniles. Although the juvenile condition could imply less bioaccumulation of metals, this pattern did not appear in the analyzes. There was no evidence of umbilical cord and some of the juveniles were shedding their skin. This evidence of growth is an indicator that food intake has been carried out and taking into account that the diet is a route of exposure to metals (Lemaire et al. 2018; Hopkins et al. 2001), it could be concluded that even with few ingested animals metal bioaccumulation takes place.
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 order to compare between organisms, snake tissues and sample points. In the first model, significant differences were obtained for chromium and cadmium concentration, which were higher at point 3 than point 1 (Fig. 5). In the second model lower concentrations were observed near the water discharge zone, particularly in point 3 for lead and chromium (Fig. 7). This may indicate that both the liver and the muscle of snakes are tissues that allow to observe differences along the river. It can be noted as a general trend that metal concentrations are higher in point 1 when analyzing snake tissue.
The absence of an spatial gradient in the concentration of metals, expected to be lower upstream of the discharge of wastewater and higher downstream, may be due to the Bata river being downstream of the Chivor reservoir, that receives sewage from the basin and has dammed waters with high contamination 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 in comparison to some of the other points, which was the general tendency for the second Bayesian linear model (Fig. 7). Similarly, according to conversations with the residents of the municipality, the Chivor dam discharges very high flows in the Bata river, approximately once a year and for several days, and also during periods of long inflows to the reservoir. The sudden increase in the flow would cause the river to be contaminated in its entirety with the dammed waters by transporting different pollutants and eroded material that comes from the dam and the river basin (MAVDT 2005). Additionally, the increase in river flow could drag some organisms downstream, possibly disrupting the expected pattern due to spatial migration. However, it is not possible to stablish the potential source of higher concentration of metals in the river with the current data, given that no predictable results where found at a spatial level.
In addition, since the sampling points are not far from each other, the snakes could migrate from one point to another, perhaps obscuring the expected spatial pattern. Helicops pastazae is a species of aquatic snake little studied, and nothing is known about its movement or migration patterns. In Nerodia sipedon, a neartic 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). Then the possibility that H. pastazae presents migration along the river is possible, and not having relatively fixed home ranges would dilute the expected spatial patterns.
Fish from the Characidae family and H. pastazae aquatic snakes have marked differences in diet, occupying different positions in the food chain. The snakes studied are top predators of this ecosystem and are considered opportunistic predators (Almendáriz et al. 2017). It was expected that predatory snakes had a higher concentration of heavy metals with respect to fish, since there is bioaccumulation along the food chain (Heydari and Riyahi 2015). Even so, there were no significant differences in any metal concentrations between the fish and snake muscle (Fig. 5). It cannot be ruled out that there are different chemical and kinetic dynamics between species, since several researchers have found that different aquatic snakes from the same place of study had different levels of metals in their tissues (Heydari and Riyahi 2015). In fact, a study conducted on a snake of the species Thamnophis sauritus and anura larvae, found that snakes had significantly lower levels of lead and cadmium, compared to larvae, indicating that these metals were not biomagnifying to superior trophic levels (Albrecht et al. 2007). Similarly, it has been established that lead and cadmium concentrations do not increase along the trophic chain in surface water ecosystems, even finding that the levels of these metals were lower in tissues of predatory fish and higher in lower levels of the food chain (Jezierska and Witeska 2006; Kenšová et al. 2010).
Likewise, although there are studies that indicate a generalist diet for the genus Helicops, there are no studies that determine the diet of H. pastazae, except for a study by Almendáriz, Barriga and Rivadeneira (2017), which states that possibly it feeds on fish of the species Hypostomus pyrineusi. Without knowing with certainty the diet of this species of aquatic snake in the study area, it is possible that the prey-predator comparisons bring up an unexpected result.
On the other hand, obtaining statistically equal concentrations between fish and snakes, may indicate that they have some mechanism to get rid of heavy metals. Various studies infer that snakes can excrete metals through different mechanisms, such as cesium through feces, skin shedding, and egg production (Campbell and Campbell 2001). Also, it was found that aquatic snakes of the Nerodia sipedon species can sequester chromium, lead, manganese and mercury in their skin, due to the high concentrations found in comparison with all body tissues, which suggests that through frequent shedding of skin they can excrete metals and decrease the pollutant load (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 (Campbell et al. 2005; Burger et al. 2017).
Comparison between tissues
Significant differences could not be appreciated between the concentration of different metals in the same tissue (Table 1). Even so, different tissues of H. pastazae did bioaccumulate metals differentially (Fig. 7). Cadmium was significantly more concentrated in the liver when compared to muscle, and lead and chromium had significantly higher concentrations in muscle. This corroborates that tissues have different affinity for certain metals, and that not all tissues are useful for evaluating traces of a particular metal. It has been observed that cadmium is accumulated mainly in the kidney and liver of fish (Jezierska and Witeska 2006; Panchanathan and Vattapparumbil 2006). Also, fish muscle is usually the tissue with lower 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, the high concentrations of lead and chromium found in muscle are not explained. There are studies that show that the concentration of cadmium is higher in the liver of fish, while that of lead is homogeneous in the sampled tissues (Zorrilla 2011).
Regarding studies in snakes (Pituophis melanoleucus), it has been found that metals have a greater affinity with the skin, especially lead, because higher concentrations were found with respect to other body tissues (Burger 1992). In contrast, in alligators (Alligator mississippiensis) the liver had the highest concentrations of cadmium, arsenic, manganese, mercury and selenium, and muscle the highest concentrations of lead and chromium (Burger et al. 2000). The highest concentrations of cadmium in liver were found in aquatic snakes (Nerodia fasciata) fed with contaminated and uncontaminated prey (Hopkins et al. 1999), as in Nerodia spp., where chromium and lead were higher in the skin (Burger et al. 2007), which is consistent with other studies conducted in Nerodia sipedon (Campbell et al. 2005). A study in marine snakes (Lapemis curtus) showed no differences in concentration between muscle and liver for lead and cadmium. In the present study, higher concentrations of cadmium were found in the liver compared to muscle, in accordance with other studies in aquatic snakes (Burger et al. 2007; Hopkins et al. 1999; Campbell et al. 2005). Likewise, higher concentrations of lead and chromium were found in the muscle compared to the liver, which is consistent with other studies in aquatic snakes and other reptiles (Burger et al. 2000; Burger et al. 2007; Campbell et al. 2005). The diversity of results in the literature shows that it is difficult to predict the tissue where each type of metal will accumulate.
It has been found in several studies 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 the found in the environment, especially for the cases of copper and cadmium (Jezierska and Witeska 2006). The differential concentration of metals in each tissue could be explained given the differences in metal affinity to sulfhydryl, amino, phosphate, carboxyl and hydroxyl groups, functional groups that can vary between tissues generating different chemical reactions for the formation of an organometallic compound (Zorrilla 2011). Lead has a high capacity for erythrocyte binding and can easily be substituted with divalent cations (Zuluaga, Gallego and Ramírez 2015), so it is possible that it has high affinity with tissues that have high blood flow, as most heavy metals (van der Brink 2004). High affinity of cadmium with kidneys, liver, and bones has been found (Zuluaga, Gallego and Ramírez 2015). Because of these affinity differences, it can be inferred that tissues vary in their value as a bioindicator of different metals.
Snakes as bioindicators
The present study reflects high levels of metals compared to similar studies, especially lead and chromium, found in different tissues of aquatic snakes. There were no differences between the concentrations of metals found in fish and snakes, which shows that aquatic snakes can be a good indicator of 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, a relationship between the levels of heavy metals in the tissues in comparison to the levels of metals in their diet was not found, because, as discussed, it is not known with certainty whether the sampled fish are part of the snake diet. Likewise, several studies have shown that lead and cadmium have lower concentrations in predators than in prey (Jezierska and Witeska 2006; Kenšová et al. 2010). Even so, finding detectable levels of metals in the tissues of snakes, can help us conclude that they are obtaining contaminants from their diet, even though the concentrations are not higher with respect to those of fish. The second requirement is met, since detectable heavy metal levels were observed in the snake tissues, 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), and on the different rates of incorporation and excretion of metals, environmental factors, among others. Regarding the third condition, the present study did not show the expected results with respect to each sampling point in the Bata river, perhaps due to the possibility of snake migration or wastewater discharges, but aquatic snakes did reflect the heavy metal pollution differentially at different sampling points of this river.
Additionally, aquatic snakes are believed to be relatively sedentary, and may be adequate indicators of local contamination (Heinz et al. 1980), although specific migration patterns for H. pastazae are unknown. On the other hand, the abundance of this species of snake in the Bata river and its long life cycle makes them good indicators of contamination at long temporal scales (Burger 1992), and that they can be monitored at different points of the river without threatening their population. Apart from 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, aquatic snakes of the Helicops pastazae species can be useful bioindicators of pollutant accumulation, in this case of heavy metals, at different temporal and spatial scales.