Research Report in support of ASTM D5517 Comparison of the Solubility of Lead in Various Materials and Its Bioavailability

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1 Research Report in support of ASTM D5517 Comparison of the Solubility of Lead in Various Materials and Its Bioavailability Woodhall Stopford Duke University Medical Center Department of Community & Family Medicine Division of Occupational & Environmental Medicine Durham, NC John Turner Angeline Elizabeth Kirby Memorial Health Center Wilkes-Barre, PA Introduction September 5, 2017 A person can come into contact with lead-containing or contaminated materials from work and play. One route of lead absorption is by ingestion. Excess lead absorption has been found in children exposed to soils contaminated with lead from smelters or exposed to house dust contaminated with lead-containing paint. Exposures to soils contaminated with lead ore waste results in a variable degree of risk of absorption: weathering of ores can produce highly bioavailable lead moieties as well as less soluble forms. 1,2,3 Risk of lead absorption after ingestion can be addressed through bioavailability studies using test animals. Factors affecting lead absorption are complex. However, a major factor affecting bioavailability of lead after ingestion appears to be its solubility. Methods to test materials for lead solubility would be useful. This study compares lead solubility of various materials using synthetic gastric juice, followed by extraction with synthetic intestinal juice, with lead bioavailability after subacute dosing of these materials to rats. Materials and Methods Rats were fed mine ores or lead standards for 30, 90 and 180 days at lead level of 100 ppm in feed. Ores were prepared by grinding with a mortar and pestle and by sifting through a 400 mesh screen. The lead oxide and lead sulfide were sifted only. Thus prepared, these materials were then mixed with powdered feed. All reagents used are reagent grade, conforming to the specifications of the Committee on Analytical Reagents of the American Chemical Society. Ore concentrate samples are procured from National Institute for Environmental Health Sciences (NIEHS) and are those used in their 30, 60 and 90 day dosing studies. 4,5 Lead content of these ores ranges from 60-76%. Lead (II) oxide and Lead (II) sulfide are electronics grade (Johnson Matthey/Alpha Aesar) as used in the above NIEHS studies but are tested without further preparation. 32.9% of the lead oxide has a particle size of <10 m while 94.6% of the lead sulfide has a particle size of <10 m. Sieving to 38 m with a 400 mesh screen would not be expected to change the particle distribution of the lead sulfide. Samples

2 are mixed at a 1:10 ratio with lead-free powdered cellulose to simulate feed mixing. For weak acid extractions using Method ASTM D5517 6, a 100 mg sample is shaken with 5 ml of 0.07 N HCl, the ph is then adjusted to with a 2 N solution of HCl and then is shaken continuously at 37 2 o C for 1 h. After allowing to sit at 37 o C. for another hour, the mixture is then filtered through a membrane filter with a pore size of 0.45 m and the eluant tested for lead using flame atomic absorption spectrophotometry 7 with a detection limit of 0.02 g/gm of eluant. To determine the effect of length of weak acid extraction, this extraction regime is compared with the same protocol except that mixed samples are extracted for 6 hours with continuous shaking prior to filtration. Material remaining on filters after a 2 h. extraction is further extracted with 50 ml of a ph 8.4 aqueous salt solution made up to approximate that found in the jejunum in non-fasting man 8 with the following composition: 6.79 g NaCl/L 0.52 g NaCl/L 1.85 g NaHCO3/L 0.11 g CaCl2/L 1.07 g sodium taurocholate/l After sonication for 5 min. in an ultrasonic bath, the sample is then incubated at 37 o C. for 4 h. while continuously shaking and then filtered through a membrane filter with a pore size of 0.45 m. The eluant is analyzed for lead as above. To further judge the effect of amino acids on lead extraction, the synthetic intestinal juice is amended with sulfhydryl amino acids found in the small intestine (based on analyses of ileal fluid) 9,10 and used to test samples with the same procedure as for synthetic intestinal juice above. Sulfhydryl amino acids used in this procedure are: g glutamate/l g histidine/l To further assess the effect of organic acids and enzymes in gastric juice on lead extraction, 0.07N HCl is supplemented with the following, representing organic acids found in gastric juice 11 : 189 mg acetate/l 534 mg citrate/l 174 mg lactate/l 1302 mg malate/l 906 mg pepsin/l (activity units/mg) Extractions are then conducted for 2 h. at 37 o C. following the ASTM D5517 protocol. All amino acids, organic acids and enzymes were obtained from Sigma Chemical Co, St. Louis, MO. The extraction results for each sample are calculated in terms of parts per million or percent of the total lead in each sample that is extracted. Statistical analyses are made using the Quattro Pro 8 (Corel).

3 Results In the 30 day feeding studies 5 bone lead levels ranged from ppm for the controls, ppm for the mine ore concentrates, 4.5 ppm for lead sulfide, 170 ppm for lead oxide, and 270 ppm for lead acetate when each was present at 100 ppm Pb in feed (Table 1). At 60 and 90 days bone lead levels were similar to 30 day values with average bone lead levels ranged from ppm for the controls, ppm for lead sulfide and ppm for mine ore concentrates, each at 100 ppm in the feed. 4 For the lead mine ore concentrates, a 2 hour weak acid extraction using ASTM Method D resulted in extraction of % of available lead vs. 6.8% for lead sulfide, 97.6% for lead oxide and 111% for lead acetate (Table 1). A 6 hour extraction resulted in slightly higher extraction of available lead with values ranging from % for ores, 7.3% for lead sulfide, but no differences for lead acetate and lead oxide: essentially 100% of available lead was leached for these latter two by each extraction method. Further extraction with synthetic intestinal juice resulted in an additional % leaching of the lead from mine ore concentrates and lead sulfide and % from lead acetate and lead oxide. The addition of sulfhydryl amino acids to synthetic intestinal juice resulted in an additional % extraction of lead from mine ore concentrates and lead sulfide and % from lead acetate and lead oxide. The addition of pepsin and organic acids to 0.07N HCl decreased lead release by % from the two mine ore concentrates tested while increasing the amount extracted from lead sulfide by 0.62% (Table 2). Discussion Bone was chosen as a measure of lead bioavailability because it is the major storage site for absorbed lead. Bone lead levels were also used as a measure of bioavailability by Freeman, et al. 12 in their study of the bioavailability of lead form soils contaminated with lead mine wastes. They found that bone lead levels were proportional to the amount of lead fed to the animals. The present investigation compared various physicochemical determinants of solubility of lead ores and lead standards (length of contact, affect of additional extraction with synthetic intestinal juice, and the addition of organic acids and pepsin to synthetic gastric juice) with the bone lead levels found in the 30 day NIEHS feeding study. It appears that the major determinant of lead extractability from the samples tested is acidity. Relative to alkaline synthetic intestinal juice, extraction with ASTM Method D-5517, a weak acid medium, was times more effective as a lead extractant. The addition of sulfhydryl amino acids that are known to bind metals and at concentrations found in the human small intestine increased the efficiency of the synthetic intestinal juice extractant by about two fold. The extraction levels, however, were still appreciably less than that found with dilute hydrocholoric acid extraction alone. The addition of pepsin and organic acids had variable effects on extraction efficiency of dilute acid, decreasing the extraction efficiency of

4 from mine ore concentrates while increasing the extraction efficiency from lead sulfide. Extending the extraction time by a factor of 3 increased the amount of lead extracted by 0.5% for lead sulfide and by 0.8-3% for lead ores but there was no effect on lead acetate and lead sulfide which were essentially fully soluble at 2 hours. These findings are similar to that which we found with 6 hour extractions of lead chromate-doped wax crayons (see accompanying Research Report). When compared to an index of lead bioavailability (bone lead) after 30 days of feeding (Table 1), weak acid extraction showed the highest level of extraction for the soluble lead standards (lead oxide and lead acetate) with the greatest bioavailability. In this subacute study, bone lead levels were fold higher with feeding the highly soluble lead standards than after feeding lead sulfide. When looking at weak acid solubility, times as much lead was extracted from lead oxide or lead acetate compared to lead sulfide. When compared to the feeding of mine ore concentrates, bone levels for soluble standards were fold higher while weak acid solubility of lead was 8-19 fold higher. Similar findings were seen with the 60 and 90 day feeding studies. A regression of analysis comparing bone lead to the percentage of lead extracted using ASTM D5517 found an r 2 of 0.96 for the 30 day study, 0.68 for the 60 day study and 0.89 for the 90 day study. The latter two studies involved feeding with the lead sulfide standard and mine ore concentrates only. This study compares favorably with an acute oral dosing of lead ore waste-amended feed by Ruby, et al. 11. In this latter study the authors compared lead absorption at various levels of the intestinal tract with measures of lead solubility. They used a synthetic intestinal juice with a ph, pepsin and organic acid content that was mirrored in this study. In vivo measurements found that the lead ore was primarily solubilized in the stomach with precipitation or sorption of solubilized lead during passage through the small intestine. They felt that their results support the expectation that lead not solubilized in the stomach is not bioaccessible, i.e., not available for absorption. They found that for mine waste lead that had 1/10 th the in vivo solubility of lead acetate, the bioavailable amount of lead (that absorbed) was also 1/10 th that of lead acetate. The amount of lead solubilized in vivo was nearly identical to the amount solubilized with their in vitro gatric juice test. These results support the finding of our study of a relationship between a measure of in vitro lead solubility and lead bioavailability.

5 References 1. Gulson BL, Davis JJ, Mizon KJ, Korsch MJ, Law AJ, Howarth D. Lead bioavailability in the environment of children: blood lead levels in children can be elevated in a mining community. Arch. Environ. Health 49: , Steele MJ, Beck BD, Murphy BL, Strauss HS. Assessing the contribution from lead in mining wastes to blood lead. Reg. Toxicol. Pharmacol. 11: ,, Ruby MV, Davis A, Nicholson A. In situ formation of lead phosphates in soils as a method to immobilize lead. Environ. Sci. Technol. 28: , Dieter MP, Chabra RS, Freeman GB, Liao S, Ryan MJ, Kurtz PJ. Bioaccumulation of tissue lead in F344 rats fed diets with lead sulfide or mine ores. Toxicologist. 14: 157, Dieter MP, Matthews HB, Jeffcoat RA, Moseman RF. Comparison of lead bioavailability in F344 rats fed lead acetate, lead oxide, lead sulfide or lead ore concentrate from Skagway, Alaska. J. Toxicol. Environ. Health 39: 79-93, Standard Test Method for Determining Extractability of Metals from Art Materials, D , Philadelphia, PA: American Society for Testing and Materials. 7. U.S.E.P.A. Test Method SW-846, 7420 Test Method for lead. 8. Davenport HW. Physiology of the Digestive Tract Fuller MF, Milne A, Harris CI, Reid TMS, Keenan R. Amino acid losses in ileostomy fluid on a protein-free diet. Am. J. Clin. Nutr. 59: 70-3, Hill GL. Ileostomy: Surgery, Physiology, and Management. New York: Grune & Stratton, Ruby MV, Davis A, Link TE, Schoof R, Chaney RL, Freeman GB, Bergstrom P. Development of an in vitro screening test to evaluate the in vivo bioaccessibility of ingested mine-waste lead. Environ. Sci. Technol. 27: , Freeman G, Johnson J, Liao S, Schoof R, Bergstrom P. Absolute bioavailability of lead from mining waste soil in rats. Toxicologist 13:301, 1993.

6 Table 1: Bone lead levels (ppm) after feeding standards and ores at 100 ppm lead for 30 days vs. extractable lead levels (%) using weak acid extraction Sample Bone lead at 30 ASTM D h. 0.07N HCl days (ppm) Pb oxide Lead acetate Lead sulfide AL ore MO ore CO ore Table 2: Bone lead levels (ppm) after feeding standards and mine ore concentrates at 100 ppm lead for 30 days vs. additional extractable lead levels after weak acid extraction: 0.07 N HCl plus pepsin and organic acids, synthetic intestinal juice, and synthetic intestinal juice plus amino acids Sample Bone lead at 30 days (ppm) ASTM D5517 (%) Pepsin & organic acids (%) SIJ a (ppm) Pb oxide NA Lead acetate NA Lead sulfide AL ore NA MO ore CO ore a synthetic intestinal juice b synthetic intestinal juice + amino acids SIJ + AA b (ppm)