. Development of Broad-Acting Entero-sorbents for Mitigation of Superfund Chemicals and Mixtures during Emergencies and Natural Disasters Meichen Wang, Steven Wheeler* and Timothy Phillips Interdisciplinary Toxicology Program, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4458 *Center for Computational Quantum Chemistry, Department of Chemistry, University of Georgia, Athens, GA 30602

Table 1. Isothermal adsorption of BaP at 26 and 37 Table 2. Qmax, % bound and Log P values for PCP, BaP, lindane, diazinon and aldicarb ABSTRACT RESULTS Temperature Sorbent Qmax Kd ℃ BaP PCP Diazinon Lindane Aldicarb 0.25 Log P = 6.13 Log P = 5.12 Log = 3.81 Log P =3.72 Log P = 1.13 People and animals can be unintentionally exposed to complex mixtures of hazardous chemicals A Figure 3. Langmuir 26 Mont-choline 0.1 9E4 following natural and man-made disasters. A major challenge associated with these plots of PCP on PSM (A) Mont / / 3% / / emergencies at sites of impact is the protection of: 1) vulnerable communities and 0.2 and hydra assay with ℃ Mont-carnitine 0.09 9E4 PSM 0.1 (4.3%) 0.21 (8.3%) 11% 0.53 (22%) 0.4 (12%) neighborhoods, 2) first responders, and 3) those involved in management and cleanup. Of /kg) PSM at 0.1% inclusion immediate concern is the associated mobilization and re-distribution of contaminated sediment

mol and PCP at 2 ppm (B). 0.15 37 Mont-choline 0.14 4.1E4 Mont- 0.09 (3.8%) 0.04 (0.7%) 7% / 0.55 (17%) and soil and its impact on the municipal water supply and food being consumed, increasing the carnitine/choline risk of exposures to hazardous substances. Thus, to minimize human and animal exposures to Mont: Freundlich; ℃ Mont-carnitine 0.09 4.8E4 complex chemical mixtures during disaster events, our laboratory has modified calcium and 0.1 to clay ( to clay PSM: • One goal of this project is to analyze the possible correlations between different chemical sodium montmorillonite clays with the nutrients, L-carnitine and choline at 100% cation Qmax = 0.21; Kd = 2E7. properties and adsorption parameters. This data will help us to predict and tune binding exchange capacity. Based on equilibrium isothermal analysis, we have demonstrated an • The enthalpy was calculated by comparing the individual Kd values at 26 and 37 given performance and delineate mechanisms. 0.05 increased binding capacity (Qmax) by 300% for benzo[a]pyrene (BaP) and selected hydrophobic bound 10 ( ) A = Kd2 pesticides compared to the parent clays. This effect is possibly due to enhanced exposure of ( ) ℃ ℃ −Rln Kd1 9

PCP water E organophilic siloxane surfaces within the interlayer of amended clays. In further studies, a 0 1 1 = 8 0 0.000005 0.00001 0.000015 0.00002 0.000025 Kd (distribution constant) was derived∆𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻from theT2Langmuir− T1 equation giving ( ) water F processed sorbent material (PSM) with less trace metal concentration was shown to tightly bind 𝑞𝑞 PCP left in solution (mol/L) • Based on the Langmuir and enthalpy equation, H = -55 kJ/mol, H = -45 7 water G multiple environmental compounds including pentachlorophenol (PCP), BaP and pesticides such mont-choline 𝐾𝐾𝐾𝐾 mont-𝑄𝑄𝑄𝑄𝑄𝑄𝑄𝑄carnitine− 𝑞𝑞 𝐶𝐶𝐶𝐶 as lindane, with Qmax values of 0.21, 0.1 and 0.53 mol/kg, respectively. In addition, cultures of Mont PSM Qmax=0.21 kJ/mol. The fact that the enthalpies of Mont-carnitine/choline were much higher than -20 6 water I in vivo • Pentachlorophenol (PCP) is a very persistent high volume industrial solvent. It is used as a kJ/mol, suggests that the adsorption process∆ involves chemisorption∆ . Also, the adult hydra were used as an toxicity indicator to confirm the ability of sorbents to 5 sediment 1 protect against individual chemicals and chemical mixtures from superfund sites. pesticide and wood preservative. PCP has been found as a frequent contaminant in water thermodynamics of the Mont-carnitine/choline reaction with BaP favor tight binding. sediment 5 Computational quantum chemistry models and isotherms are being used to estimate the samples from various Superfund sites. The isothermal analysis in Figure 3 indicated that the 4 0.5 sediment 6 parent material as well as multiple organoclays showed a Freundlich trend, indicating toxins of Toxicity Level 3 thermodynamics of surface-chemical interactions and potential mechanisms of binding. We Figure 6. Langmuir plots sediment HARD anticipate that optimal sorbents developed from this project can eventually be delivered in are partitioning on the sorbent surface rather than tightly binding. PSM markedly increased 0.45 of lindane on PSM. 2 sediment RIV food, snacks, condiments and flavored water, or administered by sachet or capsule during adsorption from a Freundlich trend to a saturable curve that fit the Langmuir model. A 0.4 1 emergencies and natural disasters (Supported by NIEHS SRP P42 ES0277704). binding capacity of 0.21 mol/kg and a high binding affinity of 2E7, suggests PCP binds tightly 0.35 PSM: to PSM surfaces and is not easily dissociated. Qmax = 0.53; Kd = 1.5E5. 0 0.3 0 4 20 28 44 68 92 • The minimum effective con- B 10 0.25 Length of Exposure (Hr) 9 INTRODUCTION centration (MEC) for PCP in 10 hydra assay was 2 µg/mL, 8 0.2 B 2ppm PCP 9 Natural and man-made disasters mobilize environmental chemical contaminants, expose which resulted in 100% 7 0.15 hydra mortality in 92 hr. PSM humans to contaminated soil and threaten the safety of the municipal water and food supply. A 6 0.1 8 resulted in protection with PCP+0.5% Duwamish 1020 major challenge associated with disasters and emergencies is the protection of: 1) vulnerable 5 0.05 7 communities and neighborhoods, 2) first responders, and 3) those involved in management and 0.1% inclusion, whereas no Mont (mol/kg) bound to clay Lindane 4 0 Duwamish 1023 cleanup. We anticipate the inclusion of entero-sorbents in diets as a protective measure to significant protection was 6 shown in parent Mont at 3 PCP+0.1% 0 0.00001 0.00002 0.00003 0.00004 0.00005 minimize unintended exposures to chemical contaminants in food and water. PSM Lindane left in solution (mol/L) 5 Libby 2-2 Multiple classes of organic chemicals have been prioritized as hazardous substances, such as 0.5% inclusion. These find- 2 PSM Qmax=0.53 4 Libby 1-5 polycyclic aromatic hydrocarbons (PAHs), high volume industrial chemicals, and pesticides. To ings indicate that PSM en- 1 • Lindane is one of the most widely detected organochlorine pesticide found in environmental of Toxicity Level develop sorbents for mixtures of commonly found superfund chemicals, we modified parent hanced the ability of parent 0 3 Libby 2-1 Mont to protect against PCP. 0 4 20 28 44 68 92 samples including air, surface water, soil and living organisms. It impacts central nervous, montmorillonite (Mont) with natural nutrients including L-carnitine and choline. These Length of Exposure (Hr) endocrine, immune and reproductive systems and is a probable carcinogen. The results in 2 amended sorbents were reported to be able to adsorb certain hydrophobic herbicides[1]. The Libby 2-5 figure 6 show that PSM is able to increase lindane adsorption with a higher capacity and 1 mechanism of sorbent amendment was to replace the natural inorganic cations with organic 0.1 Figure 4. Langmuir affinity compared to the parent material. cations (carnitine and choline) changing the chemistry of clay surfaces from hydrophilic to 0.09 0 [2] plots of BaP on PSM hydrophobic . Based on our results, the expected molecular models of modified Mont with 3 0 4 20 28 44 68 92 /kg) 0.08 Length of Exposure (Hr) carnitine and choline are shown in Figure 1. and Mont-amended Figure 7. Langmuir plots

Another processed sorbent material (PSM) was developed. PSM is similar to activated carbon in mol 0.07 clays. of diazinon on PSM and Figure 9. Analysis of superfund water and sediment samples using the hydra assay 2.5 • The hydra assay can serve as a toxicity indicator for a single toxin, as well as toxin mixtures. that it contains more surface area and less trace metals than parent material. This is the first 0.06 Mont-carnitine/choline.

clay ( Mont: Freundlich; Water and sediment samples collected from Hurricane Harry in Houston, Libby Montana and report of broad-acting clays that should be safe and effective as toxin binders for human and 0.05 2 animal consumption based on numerous intervention trails in humans and animals (Figure 2). PSM: Mont: Duwamish Washington exhibited different toxicities in the hydra assay. In figure 9, water 0.04 The ultimate goal of this study is to develop broad-acting and tunable sorbents that can be used Qmax = 0.1; Kd = 2E5; Qmax =1.28; Kd = 1E7; samples E and G, and Libby 2-1&5 are more toxic to the hydra compared to other samples. In 1.5 to reduce toxin exposure from complex chemical mixtures in humans and animals. This study 0.03 Mont-carnitine/choline: PSM: ongoing studies, we will determine which sorbents/sorbent mixtures will protect the hydra bound to from these water samples. was designed 1) to investigate the adsorption of representative toxins from different classes of 0.02 Qmax = 0.09; Kd = 9E4. Qmax = 2.4; Kd = 2E8; 1 Mont-carnitine/choline: hazardous chemicals onto the surfaces of nutrient-amended Mont and PSM and, 2) to predict BaP 0.01 their ability to prevent adverse effects of test chemicals using the adult hydra assay. Qmax = 1.35; Kd = 5E8. 0 0.5 CONCLUSIONS 0 0.00001 0.00002 0.00003 0.00004 0.00005 This work indicated that natural nutrients, L-carnitine and choline, significantly increased binding

BaP left in solution (mol/L) (mmol/kg) bound to clay Diazinon 0 PSM Qmax=0.1 Mont Mont-carnitine/choline capacities and efficacy of newly developed clays for benzo[a]pyrene and aldicarb. These amended Figure 1. Computational model of the surfaces of carnitine 0 5E-08 0.0000001 1.5E-07 • Benzo[a]pyrene (BaP) is an environmental pollutant and a carcinogenic component of Diazinon left in solution (mol/L) clays are tunable for diverse toxins based on differences in Qmax, Kd, LogP, enthalpy, solubility, and choline amended montmorillonite (Mont). With the smoked and grilled food. Previous studies have shown that activated carbon is the only Mont PSM Mont-carnitine/choline charge distribution, functional groups, volume, size, etc. permanent positive charge from the quaternary efficient sorbent for BaP adsorption. However, the results in Figure 4 suggest that both PSM • Diazinon is an organophosphate insecticide that is still approved for agricultural use. Diazinon In this work a broad-acting sorbent material was developed using a process that significantly ammonium groups, both carnitine and choline can bind to and Mont-amended clays can increase the binding capacity and affinity compared to parent inhibits acetylcholinesterase (AChE), which hydrolyzes the neurotransmitter acetylcholine enhanced the reactivity of the parent materials. Isothermal analyses, along with the hydra assay the negative surfaces of sorbents. Mont. The amendments replace hydrated cations and neutralize platelet surfaces, exposing (Ach). Isothermal results in figure 7 show that Mont-amended with carnitine/choline and (in vivo), showed that processed sorbent material (PSM) was able to serve as a very effective hydrophobic sites that favor organophilic toxin adsorption. especially PSM both significantly increase binding capacity and affinity for diazinon compared entero-sorbent for prioritized chemicals from various solvents, PAHs, and pesticides. These to parent Mont. included pentachlorophenol, benzo[a]pyrene, lindane, diazinon and aldicarb. This is the first report of a sorbent material (other than activated carbon) with high binding capacity and 0.09 1 Figure 8. Langmuir plots Figure 5. Langmuir plots enthalpy for these chemicals. Importantly, the hydra assay confirmed our in vitro results. 0.08 0.9 of aldicarb on PSM and King father and /kg) of BaP on collapsed In summary, newly developed, broad-acting sorbents can serve as binders to decrease toxin Queen mother Study Monitors Collaborator in Accra Screen 507 Volunteers (following Informed Consent) 0.07 Mont-amended clays. 0.8 Mont-carnitine/choline.

mol exposures and mitigate the adverse effects of diverse environmental chemicals. The development 0.06 0.7 of PSM has already drawn interest from 3 companies (from the US and China). One of these is 180 Participants Baseline: blood, urine Mont-carnitine/choline: Mont: 0.6 currently in the process of negotiating a license agreement and a supplemental grant for the 0.05 60 60 60 Qmax = 0.09; Kd = 9E4; Freundlich;

to clay ( to clay Phillips laboratory with the Office of Technology Commercialization at TAMU. HD LD PL 0.5 (3.0g NS)(1.5g NS) (Placebo) PSM: Study monitors and colleagues in Ejura 0.04 Collapsed Mont-choline: Qmax = 0.4; Kd = 4E86; Month 1 Blood, urine Qmax = 0.12; Kd = 3E4; 0.4 New HPLC for 0.03

bound Mont-carnitine/choline: biomarkers Collapsed Mont-choline: 0.3 ACKNOWLEDGEMENTS Month 3 Blood, urine 0.02 Qmax = 0.09; Kd = 5E4. Qmax = 0.55; Kd = 2E6. (Monitoring without treatment) 0.2 BaP 0.01 Month 4 Blood, urine 0.1

Aldicarb bound to clay (mol/kg) bound to clay Aldicarb This work was sponsored by NIEHS (Superfund hazardous Substance Research 0 Clay intervention in 0 0.00001 0.00002 0.00003 0.00004 0.00005 0 and Training Program, Project 2), 02-413421-00002. 0 0.000005 0.00001 0.000015 0.00002 0.000025 0.00003 children at 0.75 g/day BaP left in solution (mol/L) (low dose) and 1.5 Aldicarb left in solution (mol/L) Mont-carnitine/choline co Mont-choline co Mont-carnitine Mont PSM Qmax=0.4 Mont-carnitine/choline Qmax=0.55 g/day (high dose) • To investigate the binding mechanism and sites of BaP sorption, amended clay samples were • Aldicarb is a carbamate pesticide and the active substance in the Temik pesticide. Aldicarb is reduced urinary AfM1 REFERENCES excretion by 69 and heated at 200 for 30 min and 800 for 1 hr to collapse the interlayer completely. The a cholinesterase inhibitor which prevents the breakdown of acetylcholine (Ach) at the 78%, respectively, over results from dehydroxylated and heat-collapsed clays showed slight changes in binding synaptic cleft. In the case of severe poisoning, the victim dies of respiratory failure. It is 1. Wang, M., Maki, C. R., and Phillips, T.D. (2017). "Development of High Capacity Enterosorbents the duration of the 2- capacity and affinity,℃ suggesting that ℃BaP binds to edges and basal surfaces instead of the important in potato production, where it is used to control soil-borne nematodes and some for Aflatoxin B1 and Other Hazardous Chemicals." Chemical Research in Toxicology. wk study. collapsed interlayers. Since many toxins bind in the interlayer of clay (not at the edge and/or foliar pests. As shown in Figure 8, both PSM and Mont-carnitine/choline improved binding to 2. Celis, R., et al. (2007). "Selective modification of clay minerals for the adsorption of herbicides widely used in olive groves." J Agric Food Chem 55(16): 6650-6658. Figure 2. Pictures and data from human clinical trials in Ghana and Kenya. Reduction of AfB in basal surfaces), this finding supports the possibility that PSM will sorb BaP and other toxins aldicarb with high Qmax that fit the Langmuir model. Mont-carnitine/choline showed the 1 3. Elmore, S.E., et al. (2014). “Common African cooking processes do not affect the aflatoxin spiked (5–1000 ppb) cornmeal in the presence of UPSN at pH 6 (A) and pH 3.5 (B)[3]. with limited interference. highest Qmax and this was higher than the Qmax for activated carbon. binding efficacy of refined calcium montmorillonite clay.” Food Control 37.