Arsenic (As) is the 20th most abundant element on earth. It is found in several forms and compounds in nature, some inorganic and others organic, as a consequence of biotransformation through its interaction with living organisms (Zhang et al. 2022). Some environments have unusually elevated concentrations of As, including various mine sites, coal-burning plants, agricultural areas treated with pesticides, and some marine environments (Kubota et al. 2002; Burger et al. 2009; Haskins et al. 2017). Some freshwater habitats are also in contact with natural deposits of As; dissolution of As from these deposits results in elevated concentrations of the metalloid in water. When As is released in the environment and enters the biogeochemical cycle, it is persistent, changing form only through reactions with other elements and compounds including via biomethylation. More than 300 arsenic species are known, including the organic forms of methylated arsenicals, thiolated arsenicals, arsenosugars, and arsenolipids, along with inorganic forms that include arsenite [As(III)] and arsenate [As(V)] (Eisler 1988; Zhang et al. 2022).

Arsenic is ubiquitous in living tissue where the element is subject to numerous chemical reactions related to metabolism. Despite a widespread distribution in living organisms, As concentrations are usually < 1 mg/kg fresh weight in terrestrial and freshwater flora and fauna (Eisler 1988). The effects of As in organisms are variable as is the relative toxicity of its inorganic oxidation states and organic forms to most organisms in aquatic ecosystems. Inorganic arsenicals are reported to be more toxic than organic arsenicals, and trivalent inorganic forms are more toxic than pentavalent forms. However, the metalloid can be highly toxic and carcinogenic in both inorganic and organic forms. Our understanding of arsenic toxicity at subacute levels in wildlife is limited because of a lack of research on specific symptoms and toxic effects on nonmammalian species (Jamwal et al. 2023). Organisms exposed to As excrete it in several ways through different organs. About 80% of inorganic As and organic metabolites ingested by animals (assumed to be domestic mammals) is absorbed and metabolized in the liver and then excreted through urine and feces, but under chronic exposure, it is deposited in liver, kidney, and skin (Roy et al. 2013). Arsenic concentrations have been reported in many different animal tissues (Fujihara et al. 2003) including liver, muscle, kidney, bone, nails, and blood (Godley et al. 1998; Kubota et al. 2003; Cochran et al. 2018). For vertebrates, several reports have documented notable elevated concentrations of As (e.g., Fujihara et al. 2003; Dovick et al. 2015; Quintela et al. 2019).

Organisms can be exposed to As from water, dust, sediments (both directly from ingestion and indirectly from dermal contact), and food sources (Seltzer and Berry 2005; Foust et al. 2016). Concentrations of As in tissues tend to diminish in food webs moving upward toward top consumers (Kubota et al. 2003; Foust et al. 2016; Zhang et al. 2022) via As transformations within the food web (Maeda et al. 1990; Foust et al. 2016; Ghosh et al. 2022). For example, in sea turtles, As concentrations are orders of magnitude lower compared to primary producers in the same habitat (Kubota et al. 2003).

Arsenic concentrations have been widely studied in various tissues of marine turtles, but less so in freshwater turtles and tortoises (Table 1). In general, As accumulates in turtle muscle (organic forms of As), liver, kidney, and bone, including turtle shells (Berry et al. 2001; Fujihara et al. 2003; Agusa et al. 2008). Organic forms of As such as arsenobetaine have been found in muscle and liver, while other methylated species of As have been reported in the liver and kidneys. Biomethylation represents a detoxification mechanism for inorganic arsenicals in organisms (Eisler 1988). Despite the toxicity of As, no evidence indicates that it is related to mortality in the turtles studied (Saeki et al. 2000; Agusa et al. 2008; Haskins et al. 2017). However, evidence exists of diminished metabolism in organisms with excessive burdens of As (Cochran et al. 2018). Still others suggest that As competes with phosphorus for basic cellular functions (Zhang et al. 2022).

Table 1. Arsenic concentrations reported in turtle tissues from around the world. The list is representative, not exhaustive, and is sorted alphabetically by species, and within species, by arsenic levels, highest to lowest.

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Contamination of surface water bodies and groundwater aquifers by naturally derived As is common in the western United States (Welch et al. 2000). One such site is Montezuma Well (MW), a natural wetland in central Arizona. MW is model system for studying the fate of As in food webs because it is an isolated ecosystem, physiochemically stable, and As-rich, and has a simple and direct aquatic food web (Foust et al. 2016; Drost et al. 2021b). In addition to other unusual properties of MW (described in more detail below), its water has a significant concentration of geogenic As present primarily as inorganic arsenate [As(V)] (> 100 µg l⁻¹; Foust et al. 2004), 10 times greater than the current EPA maximum contaminant level for arsenic in drinking water (10 µg l⁻¹). This concentration of As is still below concentrations that are acutely toxic to most plants and animals, but greater than concentrations (19–48 µg/l) that have been reported to cause chronic effects in various aquatic organisms (Eisler 1988). Furthermore, MW served as a source of irrigation and drinking water for the prehistoric Sinagua people who inhabited the area from AD 700 to AD 1450, when they abruptly abandoned the area, leaving behind elaborate cliff dwellings that are today protected as part of Montezuma Castle National Monument. One study (Senanayake 2005) suggested that chronic As poisoning may have been one reason that previous inhabitants moved away from the area of the Well.

Our objective was to survey the concentration of As in turtles living in MW, an organism that was not included in the food web analysis of an earlier study (Foust et al. 2016). Omnivorous Sonora mud turtles (Kinosternon sonoriense) are the top predators and dominant biomass in the MW food web. This omnivorous turtle genus has been identified as important to the regulation of ecosystem dynamics in lentic systems (Aresco et al. 2015). Sonora mud turtles were reported to eat the leeches (Senanayake 2005) that were previously considered to be the top predator in the MW food web (Foust et al. 2016). Given that turtles were not included in that earlier study, their role in the As cycle in this simple, direct food chain is as yet undescribed. Using data on the estimated population size of K. sonoriense from 1983 to 2015 (Drost et al. 2021b) and data on body weight (Drost et al. 2021a) we estimated that biomass ranged from 21.4 (SD = 5.3) to 319.9 (SD = 107.0) kg/ha during this time. These are substantial values when compared to other organisms (Congdon et al. 1986; Lovich et al. 2018) and suggest an important role for turtles in the As cycle.

In this preliminary study, we evaluate the As trophic pathways leading to the Sonora mud turtle and compare them to As concentrations reported in other turtles and tortoises. We measured As in water, sediments, and various other organisms that represent different trophic levels of the MW ecosystem. Our results provide only the second analysis of the environmental and biological sources, sinks, and accumulation pathways for arsenic at this unique wetland (Foust et al. 2016), but the first one to include K. sonoriense. Concentrations of As have been evaluated for this species in only 1 previous study, from a low-As environment elsewhere in Arizona, where As concentrations in turtles were less than 1 mg/kg wet weight (King et al. 1996). Numerous plant and algal species have been shown to bioaccumulate As from their environment to concentrations that greatly exceed those in surrounding water and sediment (Chen and Folt 2000; Meharg and Hartley‐Whitaker 2002), including MW (Foust et al. 2016), and we wanted to understand how long-lived turtles at MW accumulate and respond to chronic As exposure.

METHODS

RESULTS

Arsenic Concentrations in Montezuma Well. —

As shown in Table 2, 8 surface water samples were obtained at various locations around MW and had total As concentrations ranging from 108.52 to 123.99 mg/l with a mean of 114.25 mg/l (SD = 5.26). These total As concentrations were consistent with the value of 110 mg/kg reported by Foust et al. (2016). Arsenic in the surface water was present in the pentavalent [As(V)] oxidation state, with no arsenite [As(III)] detected. We obtained 6 submerged sediment samples from the shallow shelf around the perimeter of MW with phosphate extractable As concentrations ranging from 2.71 to 13.20 mg/kg and a mean of 8.99 mg/kg (SD = 3.59). Three submerged aquatic plant samples ranged from 3.52 to 5.38 mg/kg with a mean of 4.37 (SD = 0.94). Five leech samples had As concentrations ranging from 38.07 to 115.09 mg/kg and a mean of 72.18 mg/kg (SD = 29.48). A pair each of damselflies, water scorpions, and amphipods (Table 2) were characterized by As concentrations ranging from 1.83 to 17.01 mg/kg and a mean of 6.35 (SD = 6.34). The values of amphipods were almost an order of magnitude greater than for the 2 arthropods.

Table 2. Arsenic concentrations in Montezuma Well water, biological, and sediment samples. Water concentrations are presented in mg/l, and other samples are mg/kg dry weight. Note there is one sample value (for crayfish) from Beaver Creek, adjacent to Montezuma Well and downstream of the Well outlet.

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Arsenic concentrations in K. sonoriense measured across all tissue types (Table 3) ranged from 0.91 to 26.77 mg/kg with a mean of 7.09 mg/kg (SD = 8.78), not including necropsied turtles sent to Michigan State University that ranged from 0.84 to 9.88 mg/kg (Table 2). Concentrations varied with tissue type and age. Muscle As concentrations ranged from 0.84 to 11.20 mg/kg (n = 6), while values for bone ranged from 0.91 to 2.35 (n = 2) and values for scutes ranged from 17.9 to 26.8 mg/kg (n = 2). A single liver sample measured 2.56 mg/kg, and another bone and scute sample combined measured 2.76 mg/kg. The highest As concentrations we observed (11.2 mg/kg dry weight of muscle and 26.8 mg/kg of scute) were in a male estimated to be 31.5 yrs old. In contrast, an adult female estimated to be 9.3 yrs of age had a concentration of only 0.84 mg/kg dry weight in muscle.

Table 3. Arsenic levels in different tissue types in Sonora mud turtles (Kinosternon sonoriense) from Montezuma Well, Yavapai County, Arizona, in mg/kg dry weight. Code is the individual identifying mark for each turtle (except that UNM = unmarked), Age is calculated based on known long-term recaptures. Straight-line carapace length (CL) is given in mm. Samples processed by Michigan State University, Diagnostic Center for Population and Animal Health are marked with an asterisk.

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Necropsies. —

Two dead specimens were bloated when found in August and November 2015, and the specimen collected in November showed some reddened areas of skin. Neither specimen showed any evidence of recent trauma. The cause of death in both turtles was attributed to emaciation. Virus cultures in both turtles were negative; virus cultures included cultures of the brains for West Nile virus and related arboviruses, and cultures of the livers and kidneys for ranaviruses. The carcasses were considered too decomposed to attempt bacterial cultures. Muscles of male turtle BIN and subadult female JMV contained arsenic levels of 9.88 and 0.84 mg/kg (dry weight), respectively.

DISCUSSION

Arsenic concentrations in turtle tissues have been studied mainly in marine species (Table 1). When As concentration in tissues (sum of all tissues) are compared between marine turtles and nonmarine turtles (tortoises and freshwater turtles), As in the former has a broader range (0.097–165 mg/kg) compared with nonmarine turtles (0.01–10.9 mg/kg). When midranges were calculated from Table 1, freshwater turtles had an order of magnitude lower As concentration than marine species. In clean open marine waters, total arsenic is 0.5–3 μg/l, with a mean of 1.7 μg/l (Neff 2002), while the typical arsenic concentration in unpolluted freshwater ranges from less than 1 to 10 μg/l (Wang et al. 2022).

It is interesting to note that one of the greatest As muscle concentrations we observed (11.2 mg/kg dw) was in a male K. sonoriense estimated to be 31.5 yrs old, while an adult female estimated to be only 9.3 yrs of age had a much lower value (0.84 mg/kg) in muscle. Duration of chronic exposure to As, along with As concentration in the food web, affects the concentration of the metalloid in the tissues of organisms. Long-lived animals such as K. sonoriense, estimated to live more than 40 yrs (Stone et al. 2022), may accumulate significant concentrations of contaminants over time (Rowe 2008). The greatest concentrations (up to 26.8 mg/kg) we observed were in the scutes of 1 of our oldest animals. It is possible that these concentrations may be because of the affinity of As to react with sulfur in the keratin (Alibardi 2005; Mandal 2017) of their scutes over time.

The fate of assimilated arsenic, once it enters the aquatic food web, is not well established. Several reports have documented arsenic biodiminution in the upper trophic levels of freshwater food webs, particularly those that are dominated by fish (Wagemann et al. 1978; Chen and Folt 2000; Mason et al. 2000). Montezuma Well supports a rather unique, but simple and stable, ecosystem. Primary productivity in the MW water column occurs in the form of microphytoplankton (< 5 µm cell diameter), which bloom during spring and summer in the more stable thermal conditions in MW (Boucher et al. 1984). The MW food web also includes a depauperate community of aquatic plants (macrophytes) and mostly endemic invertebrates including predatory leeches, which have among the highest arsenic concentrations, 2810 mg/kg, ever reported for organisms according to Foust et al. (2016). Leeches may accumulate As in the mucus that they secrete as a detoxification mechanism. With their mucus covering washed away, As in leach tissues is only between 45.5 mg/kg in the form of sulfur-coordinated arsenic As(III)-tris-glutathione according to Foust et al. (2016).

The Sonora mud turtle (Kinosternon sonoriense) sits at the top of the MW aquatic food web as an abundant omnivore (Ernst and Lovich 2009). Part of their diverse diet supposedly includes leeches (Senanayake 2005), along with other aquatic invertebrates and opportunistic prey items (Lovich et al. 2010). Leech consumption would expose turtles to As that could accumulate to the high concentrations seen in scutes as our analytical results (Table 3) and Berry et al. (2001) suggest. Arsenic can also accumulate in the keratin of turtle claws (Haskins et al. 2017; Cochran et al. 2018), but we did not sample claws.

Approximately 85% of phosphorus in living vertebrates is contained in bone, and turtles can contain 43% bone by body mass (Coe et al. 1979) due to their bony shell. As such, there is potential for turtles to bioconcentrate As in bone, replacing phosphorus (Akbal et al. 2014). The low concentrations we observed in bone suggest this is not happening to a significant degree with turtles at MW. In contrast, Senanayake (2005) analyzed the ancient bones of animals from museum collections, including K. sonoriense, that were eaten by Native Americans at MW when the site was still occupied (up to about the year 1450). They estimated a median As concentration of 188 mg/kg (range 79.91–365.05 mg/kg) for turtle bones, values about 2 orders of magnitude greater than ours. However, they noted “The sample preparation was carried out to minimize the contamination by surface arsenic. Precautions were taken for this because, in the past, a chemical, containing arsenic may have been used to protect the artifacts from insects.” Given that such treatment is commonplace for museum specimens (Mithander et al. 2017), the concentrations presented should be considered with caution. Indeed, when they analyzed modern turtle bones from the Verde Valley, they found a median As concentration of 2.035 mg/kg (range 1.43–2.64 mg/kg), values almost identical to ours. The high concentrations Senanayake (2005) reported in ancient turtle bones led them to conclude that the Native Americans that lived at Montezuma Well and ate turtles suffered from chronic arsenic poisoning.

The dynamics of As in the MW food web have been studied previously (Foust et al. 2007, 2016), but turtles were not included as the top aquatic predator for most of the invertebrates from the well. Our results differ from the previously documented trend of diminished As moving up the food web from the bottom, with the notable exception of leeches observed in both our study and that of Foust et al. (2016). The concentrations we observed differ from those of Foust et al. in several regards. Only general comparisons and contrasts between our data and theirs are possible because of differences in As extraction techniques, sample collection, and trophic levels used. It is interesting to note that concentrations for turtles are greater than for their arthropod prey species but still much lower than that for leeches. A major difference between the studies is that we did not measure As in plankton and Foust et al. (2016) did not measure As in aquatic vascular plants. The plankton provide food for the amphipods that do not eat vascular plants. The amphipods would seem to be too small for the turtles to eat, but Hulse (1974) noted that K. sonoriense elsewhere in Arizona eats ostracods that are about the same size as amphipods. We did not include water bugs (Belostma bakeri) in our sampling either, which are another potential prey item for mud turtles.

Nevertheless, our results document how the top predator of the MW has relatively low As in its tissues compared to some of the lower trophic levels. In our small sample, turtle scutes were the tissues with the greatest concentrations (Table 3), compared to muscle and bone. Another important finding is that a very old turtle (31.5 yrs old) had the greatest As concentration (27.8 mg/kg in scute) among all samples. This also demonstrates that a turtle in MW could survive at least 31 yrs in the As-rich aquatic habitat. We estimate that some turtles in MW are at least 39 yrs old based on mark-recapture data starting in the 1980s.

We did not identify the organic species of the As present at the site. According to Foust et al. (2016) As(III)-tris-glutathione [(GS)3As(III)] and arsenobetaine are both present in the invertebrate tissues in MW, with the sulfur-coordinated (GS)3As(III) being the only As form in leeches (Foust et al. 2016) and a combination of (GS)3As(III) and aresenobetaine in the insect fauna of the Well. It would be interesting to understand which of these organic forms of As are in K. sonoriense tissues, because turtles likely feed on all aquatic macroinvertebrates and vascular plants in MW as is characteristic of mud turtles (Hulse 1974; Macip-Ríos et al. 2010), ingesting As-laden water as they forage. The Sonora mud turtle populations that inhabit MW and adjacent Wet Beaver Creek have persisted during several decades of research, underscoring the resilience of these turtles under chronic exposure to elevated levels of As. Yet no visible health problems were noted in the MW population, other than the observation of emaciation in 2 necropsied turtles that may or may not have been caused by As exposure.