Of the DiNEH cohort respondents, those individuals who reported harvesting sheep were recruited for potential participation in the present study. Sheep chosen represented a range of ages , their proximity to mining structures, and a variety of water sources. Three ewes were included in this study. The individual sheep data are compared and reported to reflect an accurate measure of heavy metal uptake in O. aries tissue with respect to the associated forage, water consumption, and their environment. The sheep tissue samples were collected when a sheep rancher harvesting session took place. Three sheep came from two different chapters. From 10 November to 13 December 2012, three ewes were contributed to the study. The O. aries tissue samples were collected in the field immediately after slaughter and included muscle, bone, intestine, lung, liver, kidney, and wool. Upon collection, all samples were placed on dry ice and shipped to the University of New Mexico Analytical Chemistry Laboratory Earth and Planetary Sciences Department for storage and analysis. The 13th cortical rib bone samples were sheared from the proximal, middle, and distal portions and composited together after the removal of excess tissue. The proximal, medial, and distal portions of the small intestine were collected and composited. For lung tissue, the samples were derived from each anatomical lobe and composited. Both kidneys were sampled, and the cortex and medulla were composited separately. Composited muscle samples were from the proximal, medial, and distal portions of the gastrocnemius. Of the wool fiber samples, the area over the neck, middle section, and posterior portions of the animal were sampled and composited. All tissues were representative of 1 g of dried tissue. For coupled organs, the tissues collected from the right side of the sheep were labeled as the sample, and one duplicate was obtained for each tissue type from the left side of the animal. A composited duplicate or replicate was obtained for non-dual type organs . A stainless steel hand auger with a Teflon® coated-core sampler was used to collect the soil samples. To minimize cross contamination, a polyethylene core liner was utilized. Soil samples were obtained from the topsoil and composited. The forage soil samples were obtained by utilizing a topographic soil zone sampling pattern using a random zig-zag pattern.
Soil samples were weighed at 100 g. Physicochemical properties such as temperature, pH, Munsell color, depth, vertical grow rack system and moisture were obtained. The sheep tissue samples were paired with the forage, soil, and water samples. Live plants were removed from the ground soil and handpicked with a latex gloved hand, stored in PE plastic bags, and immediately placed on dry ice. The plant sources were non-cultivated. The plant roots were gently washed with deionized water . The samples were weighed, photographed, bagged, and placed on dry ice for shipment. Due to the collection of sheep tissue and forage late in the harvesting season, non-nascent forage comprised most of the samples. The live plants were placed in a plant press for several weeks with daily press tightening. The dried samples were sent to the UNM Herbarium for identification and archiving. The water samples were collected as a composite grab sample, except for those samples directly collected from a faucet or spigot were collected as first-draw samples. Lab grade PE water bottles were used, the volume of each sample collected was 250 mL. Chemical and physical characteristics data were collected . Nitric acid preservative was added to each water sample, and the sample was immediately placed on dry ice. A duplicate for each sample was obtained. A blank for each sampling session was collected. International Business Machines Statistical Package for Social Sciences for Windows, V. 21 was utilized for statistical analysis. The concentrations of HMs found in sheep tissue, plant, and soil samples are reported in milligrams per kilogram , DW. Heavy metal concentration levels are reported in micrograms per liter for water samples. Percentages, range, mean , standard deviation , and median were used to summarize the data. Independent t tests compared HMs in root versus above ground plant, root soil, and topsoil. Correlations and linear regression tested for associations between sheep tissue and associated water and forage.The field samples arrived on dry ice and were stored in a −20 ◦C freezer until sample preparation and analysis. The organic samples were oven dried at 65 ◦C. Upon sampling the debris were removed from the wool fiber. The wool was washed with 18 mega Ohm water then soaked in dilute HCl. The wool was dried in the oven at 65 ◦C. The samples were prepared by weighing two g dry mass into the digestion tube. Two mL Hydrogen peroxide and five mL HNO3 were added, and the samples were heated gradually up to 95 ◦C and digested for two hours. Next, the digested samples were transferred into 50 mL volume metric flasks and brought to volume using 18 mega ohm water. Three mL of HNO3 was run with each batch of samples. The samples were analyzed using Perkin Elmer NexION 300D ICP/MS by diluting 100 times in glass culture tubes. Mixed standards were prepared using single element standards. The calibration standards range was 5, 10, 25, and 50 µg/L . The Quality Control samples were comprised of Initial Calibration Blank Verification , Initial Calibration Verification , Continuing Calibration Verification , and Matrix Spike , Matrix Spike Duplicate , and Matrix Spike Replicate . The method detection limits are as follows: As 0.3 µg/L, Cd 0.1 µg/L, Mo 0.02 µg/L, Pb 0.008 µg/L, Se 1.3 µg/L, and U 0.008 µg/L.
A mixed internal standard of Bismuth, Indium, Scandium, and Yttrium was used to match the analyte mass range. Two percent HNO3 was used as a carrier and rinse solution. The elements were analyzed in three modes to minimize interferences; Standard, Dynamic Reaction Cell gas A , and Dynamic Reaction Cell gas B in groups. Upon the completion of analysis, the data were revised, validated, and tabulated, and the concentrations were converted to mg/kg material using instrument corrected concentration readings, sample digest final volume, and sample weight. For each sample, three replicates were measured. The accuracy of the method was verified using the analysis of certified reference materials National Institute of Standards and Technology Standard Reference Material 2709a San Joaqin Soil and NIST SRM 1573a , yielding the following values: Cd: 1.474 ± 0.107 mg/kg and Cd: 0.644 ± 0.089 mg/kg . The precision results were satisfactory with relative standard deviations ranging from 7.3 to 13.8%. The mean age of adult sheep harvesters was 58.67 ± 2.89 years; two of three participants were male. All sheep parts were consumed by the participants for a mean of 52.33 ± 10.78 years. The sheep harvesters reported that 35% of their overall meat intake came from sheep they raised and harvested locally. On average, all the participants reported consuming locally raised sheep once a week. The local harvesters reported other important non-food uses for sheep. All participants reported selling wool,vertical farming companies and two reported using the locally harvested wool to create textiles to sell for income. One of three harvesters reported selling live sheep to market, and two reported selling sheep or lamb cuts to market. All the participants reported sharing sheep meat for free with others. On average, each sheep harvester distributed free meat to two households. Multiple sheep parts were reportedly used for various ceremonial or cultural purposes by all harvesters. The existing literature reports HM levels in kidney tissue, but typically there is no comparison between the kidney medulla and the renal cortex. In this study, the kidney medulla rather than the kidney cortex showed an increased uptake of U, Se, Mo, and As. The renal proximal tubule epithelia are chemically damaged by high acute levels or prolonged low doses of U; the proximal tubules are housed in the renal cortex. The administration of toxic doses of Se demonstrated histopathological changes in the proximal tubules of the sheep. The kidneys maintain Se homeostasis. Renal compromise may cause dysregulation of Se. Our study indicates there may be a difference between HM accumulation in the medulla and the cortex. The renal toxic effects of U and Cd are well supported in the literature. The effects of associated heavy metals on the sheep kidney need further exploration. Meat protein is richer in Se than plants. The literature supports that Se commonly concentrates in the liver and kidneys of animals. Of all sheep organs, elevated Se levels were found in the liver and kidney medulla .In a lamb tissue study, it was reported that Se concentrations in the kidneys were seven to 44 times higher than in other tissue and organs. Similarly, in our study, the medullary levels contained the higher concentrations of Se . The above lamb study reported that leg muscle contained the lowest Se concentrations of the tissues sampled.
We also found that leg muscle contained the lowest Se concentrations in our examination . There is a narrow margin between Se requirement and toxicity. Therefore, taking an accurate measure of food intake containing Se, particularly meat protein, is important. Food processing such as cooking via baking, boiling, and grilling may alter the amount of Se in food. Whether food processing has an additive or minimizing effect on Se concentrations in food is to be determined by research. Adjusting food intake and cooking habits based on various HM measurements and bio-availability may be a plausible intervention once it is informed by research. Elevated levels of Se and Pb were found in sheep wool in the current study. Further, though Th was negligible in all other sheep tissue, it was detected in sheep wool . This finding may indicate that Th may be accumulating across time in sheep wool. Direct dirt and dust aerosol capture and the effects of lanolin may be contributing exposure factors. We did not measure the effects of lanolin in this study. The current study community relies on wool to create textiles. It is common practice to place local plants in hot water to pigment the wool. The wool is handled often by weavers once the wool is removed from the animal, hand-carding the wool, hand-spinning, dyeing, and weaving the textile. The entire process often takes weeks to months, suggesting a potential lengthy human exposure to heavy metals. A considerable amount of time is spent outdoors for such activities, and exposure to various sources of contaminants such as soil, water, and air is a concern. Although this study of three sheep provided interesting insight, future studies should focus on determining the speciation of heavy metals and evaluate which metals have a greater affinity to wool. The majority of the time for this study, heavy metals were found in the greatest amounts in soil > forage roots > above-ground forage parts, respectively. The current study mean Se soil concentrations were equivalent or exceeded the exposed soil and were greater than the control concentrations reported by Dreesen and Cokal. The above-ground forage parts contained the least amount of heavy metals, except for Mo and Cd. The bio-accumulation ratio can partially demonstrate the ability of particular plants to absorb soil heavy metals and transport them to the above ground portions of a plant. The data shows that the uptake of Cd, Mo, and Se by most plants sampled were high under current soil conditions. The highest BF ratios were seen in most forage for Mo , Cd , and Se , which needs further exploration. The high BF ratios seen may indicate a low tolerance of various plants to high concentrations of Mo and Cd. In particular, B. gracilis the most abundant plant, was associated with elevated BF ratios for Cd, Mo, and Se. Generally, in the biota samples there were greater heavy metal concentrations in the plant roots than the above-ground portions, which is consistent with several other plant studies that found that U translocates in greater amounts to the roots than the shoots. Similar to the current study, Soudek et al. reported that U was more localized in the root system. Uranium accumulation was less in grasses than root crops and Brassica spp.. Uranium uptake was found to be 3.9 or 4.5higher in the presence of phosphate deficiency. The micro and macro-nutrients available in soil affected the uptake of Cd in one source.