Abandoned Uranium Mines (AUM) are the main source of anthropogenic uranium (U) contamination on the Navajo Nation [1]. In parts of the Navajo Nation, AUMs have altered the environment negatively and impacted the lives and health of people since the 1950s [2]. Main routes of U exposure are the ingestion of contaminated drinking water, ingestion of contaminated food stuffs, and inhalation of dust blown particulates, with the liver recognized as a site for U accumulation in the body along with the skeleton and kidney [3]. Previously reported research of characterization of U concentration in sheep, Ovis aries, grazing near AUMs, a decommissioned U milling site, and downstream of a major U tailings pond spill that occurred in 1979 suggest low accumulation in tissues and organs [4,5]. However, little is known about the accumulation of U in sheep liver tissue based on their exposure to different types of AUMs (e.g., open pit, underground). The purpose of this study is to characterize the concentrations in the liver of sheep grazing on or near AUMs on the Navajo Nation. Statistical analysis comparing liver U concentrations between mining and non-mining areas was performed using Kruskal-Wallis and Dunn’s Test. Results suggest U concentration differences were found between the mining and the reference sites. Statistical analyses confirmed that the mining sites concentrations were similar regardless of the type of mining. The findings of this research confirm that U is present at measurable levels in the liver of the sheep.

• Uranium;

• Sheep;

• Navajo Nation;

• Inductively coupled plasma mass spectrometer

Lister A, Credo J, Ingram JC (2024) Determination of Uranium in Sheep (Ovis Aries) Liver by Inductively Coupled Plasma Mass Spectrometry (ICPMS). J Vet Med Res 11(1): 1263.

U: Uranium; ICPMS: Inductively Coupled Plasma Mass Spectrometer; AUM: Abandoned Uranium Mines; US: United States; NAU: Northern Arizona University; SRM: Standard Reference Material; HNO3: Nitric Acid; ppb: parts per billion; R2: Correlation Coefficient; SD: Standard Deviation

Uranium (U) soil concentrations in the United States (US) typically range from two to four parts per million [6], however, in areas that are rich in U ore deposits, soil can have higher concentrations of U. In the US, U concentrations are highest in the southwest (e.g., Arizona, Colorado, Utah, and New Mexico) [6].

Coincidentally, the Navajo Nation is situated on the southwestern part of the US that has higher U concentrations; [7] thus parts of the Navajo Nation have been heavily mined for U from the 1940s through the mid-1980s [8]. The types of mining that occurred on the Navajo Nation were underground and open pit mining [9]. Open pit mining, or surface mining, is used for removing U reserves at a shallow depth, while underground mining is adopted for recovering U deep below the surface [9,10]. The legacy of U mining on the Navajo Nation has left more than 500 abandoned, non-remediated mines and more than 1,000 associated mine features (e.g., rim strips, mine openings, and waste piles) [11].The non-remediated mine and mine feature sites are a problem as they blend into communities and livestock grazing areas on the Navajo Nation [12,13]. People living in communities affected by U mining have expressed concern that their sheep are bioaccumulating U through drinking water and grazing off the land, thus exposing the community to another source of U [14].

Navajo or Diné are sheep-minded people; owning sheep provides a cultural teaching of security (e.g., economic, social, psychological) and a way to bring in a tradition food staple [15-18]. A recent mutton consumption survey of the Cameron community on the Navajo Nation revealed they regularly consume mutton and the vast majority, 92%, feel that mutton is important to traditions [19]. Further, of those surveyed, 73% stated that they consume cooked sheep liver [19].The liver is the largest solid organ in the body performing several vital functions including removal of toxins from the body’s blood supply, glucose maintenance, hemostasis, and is a target for U accumulation [6,20]. Low-level U accumulation in the liver has been observed in ruminant Cervidae and Bovidae from collaborations with local communities in Canada and Arizona, respectably [21-23]. In both instances, the animals collected grazed in areas adjacent to U mine or mill sites. The first Canadian study was conducted in northeastern Saskatchewan, a region with a history of U mining [23]. As the surrounding communities rely on Cervidae (e.g., caribou) as a food source, community members were concerned over mining impacts and U transfer though the lichen-caribouhuman food chain. Liver tissues were collected from 18 caribou (15 females, 3 males). The observed concentrations were low, estimated at 1.9 ppb. The second study in Canada collected tissues from 45 Cervidae (e.g., moose) to assess the health of country foods near uranium mines in northern Saskatchewan [22]. The observed liver concentrations were similar to a second control area which the researchers suggested that the types of soil the moose were grazing may have influenced the concentrations (i.e., natural uranium outcrops vs. mineral-rich shale hills). The findings of the studies above highlight the ability for U to accumulate within the liver at low levels; however, there is a lack of information on the how the different types of U mining (e.g., abandoned open pit and underground mining) can influence the concentrations of U in sheep livers.

In this study, the first aim is to characterize the concentrations of U in sheep livers from different U mining areas on the Navajo Nation and compare the results to reference areas in terms of U liver concentrations. The hypothesis is that sheep exposed to U abandoned open pit mining sites will be higher than the abandoned underground U mining site sheep. The rationale is that sheep grazing near abandoned open pit mine features are exposed to more sources of U. These sources include abandoned open pit mining waste piles, unremediated mine features exposed to weathering events (e.g., precipitation, wind), acid mine drainage mechanisms, and mining lands transitioned to livestock grazing areas. In contrast, underground mining is less accessible to grazing animals. The second goal is a non-parametric statistical analysis to determine whether there is a statistical difference among the sample sites.

The following describes the sample collection, sample preparation, chemical analyses, and statistical analyses.

Sample collection

Approval for all procedures done in this study were granted by the Northern Arizona University (NAU) Institutional Animal Care and Use Committee, and by the communities of Cameron and Cove (exposure sites) as well as Eagar and Red Valley (reference sites); both reference sites do not have a history of U mining. The addition of reference sites was included to account for the lack of a Standard Reference Material (SRM) for U accumulation in livestock meat and organs. Samples were collected from Cameron in the summer 2013, and samples were collected from Cove in the summer and fall 2019. Samples were collected from Eagar, AZ in the fall 2015 and spring 2016 and collected from Red Valley in the fall 2021. The communities of Cameron and Cove are located on the Navajo Nation. Cameron is 53 miles northeast of Flagstaff, AZ, and Cove is 237 miles northeast of Flagstaff (Figure 1). The community of Eagar is located 100 miles from the Navajo Nation and the community of Red Valley is 20 miles east of Cove.

Figure 1: Map of the Navajo Nation, sites sampled, and AUM features.

The age ranges of the sheep were: Cameron sheep (N=3) two to six years; Cove sheep (N=6) one to two years; Eager sheep (N=3) one to eight years; Red Valley sheep (N=1) 2 years. The aging of the sheep was conducted by community veterinarians, Drs. Adrienne Ruby and Holly Grams-Johnson.

Sample preparation and analytical methods

All sheep were euthanized using a traditional Navajo butchering technique at the site where the animals were raised in collaboration with the Navajo sheep owners. Livers and other tissue from the euthanized sheep were collected at the time of butchering. Upon collection, tissue specimens were placed in gallon sized freezer bags, placed in an ice chest, and transferred back to NAU (in Flagstaff, Arizona) to be prepared for sample analyses. At each site a chain of custody form was completed to ensure tracking of tissue specimens from each sheep at the time of euthanization.

Liver samples were stored in a freezer at -18°C at NAU until ready for preparation. Preparation procedures included time to air-thaw followed by thinly slicing tissue to be dried for two weeks in the laboratory. Once desiccated, specimens were powdered with a standard kitchen food processor and homogenized in gallon sized freezer bags. Between samples, the food processor was taken apart, soaked in high purity deionized water (18.2 M??cm-1, Thermo Genpure Pro), and each individual part was cleaned with dish soap (Dawn), rinsed with high purity deionized water (18.2 M??cm-1) three times, and set out to dry. Liver samples were weighed to approximately 0.5g aliquots and placed into 25 mL digestion vessels (CEM MARS Xpress PTFE) with 10mL of nitric acid (HNO3) (VWR Aristar Ultra) while allowing 20 minutes of predigest prior to capping. Each digestion run had four digestion blanks, and an even number of unknown liver samples. Loaded liver samples were digested following a “one touch method” using a CEM microwave digestion unit and protocol created by CEM for animal tissue digestion. The specific digestion program increases temperatures to 200 °C over a 15-minute period and then maintains 200 °C for 15 minutes. The vessels are allowed to cool to room temperature, uncapped, filtered using a 0.45 µm filter (PVDF w/PP WHATMAN) with a 15 mL luer-lock syringe (PP/PE, NORM-JET) into a 15 mL centrifuge tube, brought to 15 mL with high purity deionized water (18.2 M??cm-1), and stored in a 4 °C refrigerator until analysis.

A Thermo X-series II inductively coupled plasma mass spectrometer (ICP-MS) with conical spray chamber (Analytical West), equipped with a c-type nebulizer 1 mL/min (Analytical West), was used for U quantitation. The instrument was tuned prior to analysis; specific instrument and tune settings are available upon request. A calibration curve was obtained with a minimum correlation coefficient (R2) value of 0.997 prior to continuing analysis. U calibration standard concentrations were 0.0, 0.1, 0.25, 0.50, 1.0 ppb, and an internal standard of 0.05 ppb ruthenium was used. The sample concentrations and extraction efficiencies were determined from the U238 to ruthenium isotope 101 (Ru101) mass to charge ratio.

Statistical Analysis

Summary statistics for sheep liver U concentrations were calculated for each sample group including minimum, maximum range, mean, median, and standard deviation (SD). Non-parametric test statistics were calculated due to the small number of samples per group and the non-normal distribution of the results. Specifically, the median sheep liver U concentrations among open pit, underground, and no mining were evaluated for significant difference using the Kruskal-Wallis test. If the test result was significant (p-value <0.05) then a Dunn’s test was conducted to identify pairwise differences between groups [24]. Data were evaluated using R studio (version 2002.07.01) and the following packages (FSA: Ogle DH, Doll JC, Wheeler P, Dinno A (2022). FSA: Fisheries Stock Analysis. R package version 0.9., dplyr version 1.0.7, ggplot2 version 3.3.5, multcompView version 0.1-8) were used in the statistical analysis of the data.

The present study investigated U concentrations in the liver of sheep that grazed in both Navajo communities near abandoned U open pit and underground mining sites as well as reference areas with no history of U mining on the Navajo Nation and off the Navajo Nation. Analytical results indicated the lowest concentration of U in the sheep liver among the four groups ranged from 0.4 (SD = 0.02) to 1.9 (SD = 0.03) ng/g dried weight [Table 1].

Table 1: U concentrations in sheep liver tissues from four sites in Arizona

The liver of the sheep from open pit mining (N=3) had a mean value of 0.7 (SD = 0.3) ng/g dried weight. The underground mining location (N=6) had a mean concentration value of 0.5 (SD = 0.6) ng/g dried weight; however, there was an outlier, sheep six from Cove, had a mean concentration of 2.2 (SD = 0.03) ng/g dried weight, due to the small number of samples analyzed the outlier was not removed from the data set. The reference site on the Navajo Nation, Red Valley (N=1), had the lowest concentration of U in the sheep liver with a mean value of 0.4 (SD = 0.02) ng/g dried weight while the Eagar, AZ reference site had a mean value of 1.9 (SD = 0.03). Boxplots of the results for the median and interquartile range for each group of animals are shown in Figure 2.

Figure 2: (Left) U accumulation in sheep livers from four different communities in Arizona, (Right) Comparison of U accumulation in sheep livers from mining areas to non-mining area.

Overall, the Eagar group U liver concentrations were higher (N=3) compared to the other sites sampled. For additional comparison, the two mining groups were combined to a single group that was compared to the two non-mining locations. The two mining communities had a combined mean concentration of 0.8 (SD = 0.6) ng/g dried weight, and the non-mining locations combined had a mean concentration of 1.5(SD = 0.8) ng/g dried weight.

Results from the Kruskal-Wallis test determined a statistical difference in medians among the four sites (p-value = 2.2 x 10- 16) [24]. Dunn’s test was used to test for stochastic dominance among the four sites following a Kruskal-Wallis test, which determined there was a statistical difference among all the sites and their mean values [24]. Further, results from Dunn’s test determined U accumulation in sheep livers from U mining sites were similar (e.g., open pit, underground). When comparing the reference sites to both mining sites, there was a statistical difference between the mining sites and Eagar but not Red Valley. Results from a Kruskal-Wallis test revealed a difference between the combined mining sites versus the refence sites (p-value = 1.1 x 10-10). A Mann-Whitney U test was used to determine the difference between the aggregated mining group and the reference group [25]. The Mann-Whitney U test showed the aggregated mining group was not similar to the aggregated reference group (1.1 x 10-10).

In this study, whole sheep livers were homogenized, dried, powdered, acidified, and analyzed with an ICPMS. U concentrations were measured in nine sheep from Arizona, seven sheep were sampled from the Navajo Nation, and three from a non-Navajo community. The seven sheep that were sampled from the Navajo Nation, three resided in a community with open pit mining (e.g., Cameron), three resided in a community with underground mining (e.g., Cove), and one resided in a community with no mining. The overall lowest concentration was from the community of Red Valley, Red Valley is 20 miles away from the community of Cove and was not mined for U. The overall highest concentration was from a sheep from Cove, Cove is a community that was first mined for U on the Navajo Nation. Comparing the concentration of this study on sheep liver to the Thomas study of U concentrations in moose and caribou, the U concentrations are similar [22,23]. Results suggest the sheep that grazed near abandoned open pit and underground mining sites have similar U concentrations in comparison to each other in this study. This finding does not support our hypothesis that sheep grazing near open pit mining areas will have higher U concentrations in their liver tissues than sheep grazing in proximity to underground mining sites.

Based on interviews with the Cameron community members that provided the sheep for the study, the past histories of the sheep (e.g., grazing habits and health) were unknown to the owners because the sheep were grazed open range. This same limitation was encountered with the Eagar sheep. This unknown history prevents an accurate statement on potential U exposure experienced by the sheep in both locations. However, it is assumed that there is limited exposure of the Eagar sheep to AUM due to the approximate 100-mile distance from U mining activity. The Cove and Red Valley sheep were fitted with geospatial units that tracked their approximate grazing habits for several months; thus, the immediate grazing habits for these sheep were known before the sheep were euthanized. Additionally, one of the Cove owners had his sheep graze near his home and supplemented with hay and grain from the nearby Navajo Agricultural Products Industry, while the other owners from Cove grazed their sheep in the canyons that had mine waste, and in areas away from the canyon that had the mine waste. The Red Valley livestock owners communicated that the sheep for the study lived on a supplemented diet comprised of hay, grain, and water from a regulated water source, and were kept in a corral for most of their life.

In this study, the accumulation of U in sheep livers from four different locations in AZ, three sites located on the Navajo Nation, and one reference site located approximately 100 miles away from the Navajo Nation, is reported. The three sites on the Navajo Nation have the following U mining history: Cameron - open pit mining; Cove - underground mining; Red Valley - no U mining and served as an additional reference site. The accumulation of U in sheep livers was measured by ICPMS. Results suggest that sheep grazing in communities with U mine sites whether they be open pit or underground mining, have lower liver tissue concentrations of U than sheep from areas with no history or U mining. The results of this study suggest that exposure to U by eating sheep liver of animals grazing on or near AUM is no worse than eating sheep liver of animals grazing in non-AUM locations. Comparing the U liver concentrations in the study to other ruminants’ liver studies, the concentration in this study were similar [22,23]. The human health risks of eating locally grown sheep being chronically exposed to U from past U mining remains unknown. Toxicology studies are needed to better understand these risks. Results from this study support the communities’ implications on the consumption, culture, and tradition of Navajo [19-26].The ongoing research from our laboratory will continue to address U accumulation in sheep organs and tissues, a step towards addressing longstanding U mining legacy exposures to Navajo. Future work will lead to a better understanding of exposure, and support interventions that will reduce livestock and human U exposure. The ultimate goal is to improve the lives of the people and their communities on the Navajo Nation.

The authors acknowledge the support of the Cameron, Cove and Leupp communities as well as the Eager rancher. The authors are grateful for the assistance of the Dr. Tommy Rock for facilitating the sheep organ collection, Drs. Adrienne Ruby and Holly Grams-Johnson for their assessment of the sheep, and students in the Ingram laboratory for assistance in sample collection and preparation. The authors are grateful for funding from the NIH-NARCH grant IHS0092-01-00, NIH-NACP grant U54CA14392, the NIH-P50 grant ES026089.

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