The frequency of females, males and juveniles was variable 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 evinces that food intake has taken place and considering that it is through diet that the snakes are exposed to metals (Lemaire et al. 2018; Hopkins et al. 2001), it could be concluded that metal bioaccumulation takes place even with just a few ingested animals.
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 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 at point 3 for lead and chromium (Fig. 7). This may indicate that both snake liver and muscle are tissues that can reveal differences in concentrations 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 a spatial gradient in the concentration of metals, expected to be lower upstream of the wastewater discharge point and higher downstream, may be due to the Bata river being located downstream of 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 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). This 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 a spatial level.
In addition, given that the sampling points are not far from each other, the snakes could migrate from one point to another, perhaps obscuring the expected spatial pattern. Not many studies have been conducted on Helicops pastazae, 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), meaning that H. pastazae may well migrate along the river; in turn, the fact that it does not have relatively fixed home ranges would dilute the expected spatial patterns.
Interspecific comparisons
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 in this ecosystem and are considered opportunistic predators (Almendáriz et al. 2017). These predatory snakes were expected to reveal a higher concentration of heavy metals with respect to fish, as bioaccumulation occurs along the food chain (Heydari and Riyahi 2015). Despite this, there were no significant differences in any metal concentrations in the fish and snake muscle (Fig. 5). The existence of different chemical and kinetic dynamics between species cannot be ruled out, as 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 Thamnophis sauritus snake and anura larvae, found that the snakes had significantly lower levels of lead and cadmium than the larvae, indicating that these metals were not biomagnifying to superior trophic levels (Albrecht et al. 2007). It has also been established that lead and cadmium 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 a study by Almendáriz, Barriga and Rivadeneira (2017), which states 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, may mean that prey-predator comparisons result in unexpected outcomes.
On the other hand, obtaining statistically equal concentrations between fish and snakes, may indicate that they have some mechanism to rid themselves 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). 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 (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
No significant differences were found between the concentration of different metals in the same tissue (Table 2). However, different H. pastazae tissues did bioaccumulate metals differentially (Fig. 7). Cadmium was significantly more concentrated in the liver than the 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. For example, cadmium 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 lead and chromium found in muscle. Some studies show cadmium concentrations to be higher in fish liver, while lead 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 with the skin. This is especially true for lead, which was found in higher concentrations in the skin than in other body tissues (Burger 1992). In contrast, in alligators (Alligator mississippiensis) the liver had the highest concentrations of cadmium, arsenic, manganese, mercury, and selenium, while the muscle had 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 involving Nerodia sipedon (Campbell et al. 2005). A study on marine snakes (Lapemis curtus) showed no differences in concentration between muscle and liver for lead and cadmium. In the present study, in accordance with other studies on aquatic snakes (Burger et al. 2007; Hopkins et al. 1999; Campbell et al. 2005) higher concentrations of cadmium were found in the liver than in the muscle. Likewise, higher concentrations of lead and chromium 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. 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.
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 to sulfhydryl, amino, phosphate, carboxyl, and hydroxyl groups, which are functional groups whose concentration 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); thus, it may, as most heavy metals, have a high affinity with tissues with high blood flow (van der Brink 2004). A high affinity has been found between cadmium and the kidneys, liver, and bones (Zuluaga, Gallego and Ramírez 2015). Given these affinity differences, 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 high levels of metals, especially of lead and chromium, 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 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, there appears to be no relationship between the levels of heavy metals in the tissues and the levels of metals in their diet. This is due to the fact that, as discussed, there is no verified information on whether the sampled fish are part of the snake’s diet. Likewise, several studies have shown that the levels of lead and cadmium tend to be lower in predators than in prey (Jezierska and Witeska 2006; Kenšová et al. 2010). Despite this, finding detectable levels of metals in snake tissue, can help us conclude that they absorb contaminants from their diet, even though the concentrations are not higher than those found in fish. 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), and on the different rates of incorporation and excretion of metals, and environmental factors, among others. In terms of the third condition, the present study did not show the expected results with respect to each sampling point in the Bata river. This may be due to the possibility of 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 good indicator of contamination during long temporal scales (Burger 1992), and it they can be monitored at different points of the river without threatening its 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, Helicops pastazae aquatic snakes can be useful bioindicators of heavy metal accumulation at different temporal and spatial scales.